Gene and lncRNA Profiling of ω3/ω6 Polyunsaturated Fatty Acid-Exposed Human Visceral Adipocytes Uncovers Different Responses in Healthy Lean, Obese and Colorectal Cancer-Affected Individuals.

Colorectal cancer (CRC) is a major life-threatening disease, being the third most common cancer and a leading cause of death worldwide. Enhanced adiposity, particularly visceral fat, is a major risk factor for CRC, and obesity-associated alterations in metabolic, inflammatory and immune profiles in visceral adipose tissue (VAT) strongly contribute to promoting or sustaining intestinal carcinogenesis. The role of diet and nutrition in obesity and CRC has been extensively demonstrated, and AT represents the main place where diet-induced signals are integrated. Among the factors introduced with diet and processed or enriched in AT, ω3/ω6 polyunsaturated fatty acids (PUFAs) are endowed with pro- or anti-inflammatory properties and have been shown to exert either promoting or protective roles in CRC. In this study, we investigated the impact of ex vivo exposure to the ω3 and ω6 PUFAs docosahexaenoic and arachidonic acids on VAT adipocyte whole transcription in healthy lean, obese and CRC-affected individuals. High-throughput sequencing of protein-coding and long non-coding RNAs allowed us to identify specific pathways and regulatory circuits controlled by PUFAs and highlighted an impaired responsiveness of obese and CRC-affected individuals as compared to the strong response observed in healthy lean subjects. This further supports the role of healthy diets and balanced ω3/ω6 PUFA intake in the primary prevention of obesity and cancer.

Improving the Diagnostic Potential of Extracellular miRNAs Coupled to Multiomics Data by Exploiting the Power of Artificial Intelligence.

In recent years, the clinical use of extracellular miRNAs as potential biomarkers of disease has increasingly emerged as a new and powerful tool. Serum, urine, saliva and stool contain miRNAs that can exert regulatory effects not only in surrounding epithelial cells but can also modulate bacterial gene expression, thus acting as a "master regulator" of many biological processes. We think that in order to have a holistic picture of the health status of an individual, we have to consider comprehensively many "omics" data, such as miRNAs profiling form different parts of the body and their interactions with cells and bacteria. Moreover, Artificial Intelligence (AI) and Machine Learning (ML) algorithms coupled to other multiomics data (i.e., big data) could help researchers to classify better the patient's molecular characteristics and drive clinicians to identify personalized therapeutic strategies. Here, we highlight how the integration of "multiomic" data (i.e., miRNAs profiling and microbiota signature) with other omics (i.e., metabolomics, exposomics) analyzed by AI algorithms could improve the diagnostic and prognostic potential of specific biomarkers of disease.

Biomarkers to Monitor Adherence to Gluten-Free Diet by Celiac Disease Patients: Gluten Immunogenic Peptides and Urinary miRNAs.

Celiac disease (CD) is a multifactorial autoimmune enteropathy with a prevalence greater than 1% in the pediatric population. The only therapy for CD patients is a strict gluten-free diet (GFD). Gluten-free food contamination by other cereals during packaging and cooking or accidental ingestion of gluten may cause several intestinal and extraintestinal symptoms in CD patients. Therefore, the monitoring of gluten contamination in food and assessing the level of ingested gluten by analytical biomarkers has been of great interest in recent years. To this aim, small gluten immunogenic peptides (GIPs) obtained by the hydrolysis of gluten and present in urine and feces have been studied as biomarkers of gluten intake and to monitor adherence to GFD by CD patients. More recently, the use of circulating, fecal and urinary miRNAs has emerged as a novel diagnostic tool that can be potentially applied to assess adherence to GFD. Moreover, the presence of GIPs and miRNAs in both feces and urine suggests a similar excretion modality and the possibility of using urinary miRNAs, similarly to GIPs, as potential biomarkers of GFD in CD patients.

Humoral Responses and Ex Vivo IFN-γ Production after Canine Whole Blood Stimulation with Leishmania infantum Antigen or KMP11 Recombinant Protein.

The effect of Leishmania infantum soluble antigen (LSA) and recombinant Kinetoplastid Membrane Protein 11 (rKMP11) on the induction of ex vivo specific IFN-γ (n = 69) and antibody responses (n = 108) was determined in dogs. All dogs were tested for serological response to both antigens and divided into Group 1: healthy (Asturias, Spain, n = 26), Group 2: sick (n = 46), Group 3: healthy Ibizan hounds (Mallorca, Spain, n = 22) and Group 4: healthy (Bari, Italy, n = 14). Antibody levels were higher for LSA when compared to rKMP11 (p = 0.001). Ibizan hounds were all seronegative to rKMP11 and 18% were low seropositive to LSA. Sick dogs presented higher antibody response to both antigens compared to the rest of the groups (p < 0.0001). All groups showed higher IFN-γ levels after LSA compared to rKMP11 responses (p < 0.05). The highest response to LSA was found in Ibizan hounds (p < 0.05). IFN-γ to LSA and rKMP11 stimulation was observed in 34% and in 2.8% of the sick dogs, respectively. Here, we demonstrated that anti-rKMP11 antibodies are mainly present in dogs with moderate to severe disease. Furthermore, cellular immune response measured by specific ex vivo IFN-γ production was more intense to LSA than stimulated to rKMP11.

Circulating microRNAs as novel non-invasive biomarkers of paediatric celiac disease and adherence to gluten-free diet.

BACKGROUND: Celiac Disease (CD) is a multifactorial autoimmune enteropathy (with a prevalence of approximately 1% worldwide) that exhibits a wide spectrum of clinical, serological and histological manifestations. For the diagnosis of paediatric CD, the gold standard is the combination of serological tests (with high TGA-IgA values greater than 10 times the upper limit of normal) and duodenal biopsy (with a positive TGA-IgA but low titer). Therefore, a diagnostic test that totally excludes an invasive approach has not been discovered so far and the discovery of novel biological markers would represent an undoubted advantage for the diagnosis of CD and prognostic evaluation. MicroRNAs (miRNAs), small non-coding RNAs (18-22 nucleotides) that regulate gene expression at post-transcriptional level and play important roles in many biological processes, represent a novel class of potential disease biomarkers. Their presence in biological fluids (i.e., serum, plasma, saliva, urine) provides the opportunity to employ circulating miRNAs as novel non-invasive biomarkers. METHODS: In our prospective observational study, we examined the expression of circulating miRNAs in a cohort of CD patients (both at diagnosis and on gluten-free diet, respectively referred as CD and GFD) compared to healthy controls. By small RNA-Seq we discovered a set of circulating miRNAs that were further validated by qPCR with specific assays. FINDINGS: We found that out of the 13 miRNAs able to discriminate the three groups (i.e., CD, GFD and controls), three of them, namely miR-192-5p, miR-215-5p and miR-125b-5p (alone or in combination), were able to discriminate these three groups with high accuracy and specificity. INTERPRETATION: Our conclusions emphasize that these circulating miRNAs can be employed not only for the diagnosis of CD patients with a low TGA-IgA titer but also to monitor the adherence to a gluten-free diet by CD patients. In conclusion, we suggest the use of the circulating miRNAs identified in this work as a novel diagnostic and follow-up tool for paediatric CD. FUNDING: This work was supported by Fondazione Celiachia Onlus (FC) Grant n° 018/FC/2013 and by Italian Ministry of Health (Ricerca Corrente).

Tumor cell invasion into Matrigel: optimized protocol for RNA extraction.

3D models are increasingly used to study mechanisms driving tumor progression and mimicking in vitro processes such as invasion and migration. However, there is a need to establish more protocols based on 3D culture systems that allow for downstream molecular biology investigations. Materials & methods: Here we present a method for optimal RNA extraction from highly aggressive primary glioma cells invading into Matrigel. The method has been established by comparing previously reported protocols, available commercial kits and optimizing specific steps for matrix dissociation, RNA separation and purification. Results and conclusion: The protocol allows RNA extraction from cells embedded into Matrigel, with optimal yield, purity and integrity suitable for subsequent sequencing analysis of both high and low molecular weight RNA.

Potential use of noncoding RNAs and innovative therapeutic strategies to target the 5'UTR of SARS-CoV-2.

After the increasing number of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections all over the world, researchers and clinicians are struggling to find a vaccine or innovative therapeutic strategies to treat this viral infection. The severe acute respiratory syndrome coronavirus infection that occurred in 2002, Middle East respiratory syndrome (MERS) and other more common infectious diseases such as hepatitis C virus, led to the discovery of many RNA-based drugs. Among them, siRNAs and antisense locked nucleic acids have been demonstrated to have effective antiviral effects both in animal models and humans. Owing to the high genomic homology of SARS-CoV-2 and severe acute respiratory syndrome coronavirus (80-82%) the use of these molecules could be employed successfully also to target this emerging coronavirus. Trying to translate this approach to treat COVID-19, we analyzed the common structural features of viral 5'UTR regions that can be targeted by noncoding RNAs and we also identified miRNAs binding sites suitable for designing RNA-based drugs to be employed successfully against SARS-CoV-2.

Integrated Transcriptome Analysis of Human Visceral Adipocytes Unravels Dysregulated microRNA-Long Non-coding RNA-mRNA Networks in Obesity and Colorectal Cancer.

Obesity, and the obesity-associated inflammation, represents a major risk factor for the development of chronic diseases, including colorectal cancer (CRC). Dysfunctional visceral adipose tissue (AT) is now recognized as key player in obesity-associated morbidities, although the biological processes underpinning the increased CRC risk in obese subjects are still a matter of debate. Recent findings have pointed to specific alterations in the expression pattern of non-coding RNAs (ncRNAs), such as microRNAs (miRNAs), and long non-coding RNAs (lncRNAs), as mechanisms underlying dysfunctional adipocyte phenotype in obesity. Nevertheless, the regulatory networks and interrelated processes relevant for adipocyte functions, that may contribute to a tumor-promoting microenvironment, are poorly known yet. To this end, based on RNA sequencing data, we identified lncRNAs and miRNAs, which are aberrantly expressed in visceral adipocytes from obese and CRC subjects, as compared to healthy lean control, and validated a panel of modulated ncRNAs by real-time qPCR. Furthermore, by combining the differentially expressed lncRNA and miRNA profiles with the transcriptome analysis dataset of adipocytes from lean and obese subjects affected or not by CRC, lncRNA-miRNA-mRNA adipocyte networks were defined for obese and CRC subjects. This analysis highlighted several ncRNAs modulation that are common to both obesity and CRC or unique of each disorder. Functional enrichment analysis of network-related mRNA targets, revealed dysregulated pathways associated with metabolic processes, lipid and energy metabolism, inflammation, and cancer. Moreover, adipocytes from obese subjects affected by CRC exhibited a higher complexity, in terms of number of genes, lncRNAs, miRNAs, and biological processes found to be dysregulated, providing evidence that the transcriptional and post-transcriptional program of adipocytes from CRC patients is deeply affected by obesity. Overall, this study adds further evidence for a central role of visceral adipocyte dysfunctions in the obesity-cancer relationship.

Total serum IgD from healthy and sick dogs with leishmaniosis.

BACKGROUND: Canine leishmaniosis (CanL) due to Leishmania infantum is characterized by the development of both cellular and humoral immune responses. The dysfunction of T cell-mediated immunity leads to a lack of proliferation of T cells in response to Leishmania antigens with the consequence of parasite dissemination that seems to be related to a T cell exhaustion mediated by regulatory B cells expressing immunoglobulin D (IgD). The aim of this study was to determine and compare the total serum IgD in dogs with clinical leishmaniosis and in clinically healthy dogs. RESULTS: A total of 147 dog sera were studied. All dogs were tested for L. infantum-specific antibodies by quantitative ELISA. Interferon-gamma (IFN-γ) production was also determined by sandwich ELISA after blood stimulation with L. infantum soluble antigen (LSA) or concanavalin A (ConA). The quantification of total IgD was performed using a human IgD sandwich ELISA quantification set. Dogs were classified in three different groups. Group 1 included 40 clinically healthy non-infected dogs, all serologically negative to L. infantum-specific antibodies and non-producers of IFN-γ upon LSA stimulation. Group 2 included 63 clinically healthy infected dogs that were LSA IFN-γ producers (n = 61) and/or IFN-γ non-producers (n = 2) as well as negative to medium seropositive to L. infantum antigen. Finally, Group 3 included 44 dogs with clinical leishmaniosis (IFN-γ producers, n = 23; and IFN-γ non-producers, n = 21) that were negative to highly positive to L. infantum-specific antibodies. No significant differences were observed when the total IgD concentration was compared within groups. Additionally, total IgD of sick IFN-γ producers and IFN-γ non-producers was not significantly different. Finally, total IgD concentration was not statistically related to demographic parameters such as age, sex and breed. CONCLUSIONS: The results of this study demonstrated that there were no differences between groups in total serum IgD. Total serum IgD does not appear to be a marker of disease in CanL.

Transcriptome Profiles of Human Visceral Adipocytes in Obesity and Colorectal Cancer Unravel the Effects of Body Mass Index and Polyunsaturated Fatty Acids on Genes and Biological Processes Related to Tumorigenesis.

Obesity, a low-grade inflammatory condition, represents a major risk factor for the development of several pathologies including colorectal cancer (CRC). Although the adipose tissue inflammatory state is now recognized as a key player in obesity-associated morbidities, the underlying biological processes are complex and not yet precisely defined. To this end, we analyzed transcriptome profiles of human visceral adipocytes from lean and obese subjects affected or not by CRC by RNA sequencing (n = 6 subjects/category), and validated selected modulated genes by real-time qPCR. We report that obesity and CRC, conditions characterized by the common denominator of inflammation, promote changes in the transcriptional program of adipocytes mostly involving pathways and biological processes linked to extracellular matrix remodeling, and metabolism of pyruvate, lipids and glucose. Interestingly, although the transcriptome of adipocytes shows several alterations that are common to both disorders, some modifications are unique under obesity (e.g., pathways associated with inflammation) and CRC (e.g., TGFß signaling and extracellular matrix remodeling) and are influenced by the body mass index (e.g., processes related to cell adhesion, angiogenesis, as well as metabolism). Indeed, cancer-induced transcriptional program is deeply affected by obesity, with adipocytes from obese individuals exhibiting a more complex response to the tumor. We also report that in vitro exposure of adipocytes to ω3 and ω6 polyunsaturated fatty acids (PUFA) endowed with either anti- or pro-inflammatory properties, respectively, modulates the expression of genes involved in processes potentially relevant to carcinogenesis, as assessed by real-time qPCR. All together our results suggest that genes involved in pyruvate, glucose and lipid metabolism, fibrosis and inflammation are central in the transcriptional reprogramming of adipocytes occurring in obese and CRC-affected individuals, as well as in their response to PUFA exposure. Moreover, our results indicate that the transcriptional program of adipocytes is strongly influenced by the BMI status in CRC subjects. The dysregulation of these interrelated processes relevant for adipocyte functions may contribute to create more favorable conditions to tumor establishment or favor tumor progression, thus linking obesity and colorectal cancer.

Circulating microRNAs and Bioinformatics Tools to Discover Novel Diagnostic Biomarkers of Pediatric Diseases.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the post-transcriptional level. Current studies have shown that miRNAs are also present in extracellular spaces, packaged into various membrane-bound vesicles, or associated with RNA-binding proteins. Circulating miRNAs are highly stable and can act as intercellular messengers to affect many physiological processes. MicroRNAs circulating in body fluids have generated strong interest in their potential use as clinical biomarkers. In fact, their remarkable stability and the relative ease of detection make circulating miRNAs ideal tools for rapid and non-invasive diagnosis. This review summarizes recent insights about the origin, functions and diagnostic potential of extracellular miRNAs by especially focusing on pediatric diseases in order to explore the feasibility of alternative sampling sources for the development of non-invasive pediatric diagnostics. We will also discuss specific bioinformatics tools and databases for circulating miRNAs focused on the identification and discovery of novel diagnostic biomarkers of pediatric diseases.

Intestinal and Circulating MicroRNAs in Coeliac Disease.

MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression at the post-transcriptional level and play a key role in the pathogenesis of autoimmune and gastrointestinal diseases. Previous studies have revealed that miRNAs are dysregulated in intestinal biopsies of patients affected by coeliac disease (CD). Combined bioinformatics analyses of miRNA expression profiles and mRNA target genes as classified by Gene Ontology, are powerful tools to investigate the functional role of miRNAs in coeliac disease. However, little is still known about the function of circulating miRNAs, their expression level compared to tissue miRNAs, and whether the mechanisms of post-transcriptional regulation are the same of tissue miRNAs. In any case, if we assume that a cell-cell communication process has to occur, and that circulating miRNAs are delivered to recipient cells, we can derive useful information by performing target predictions. Interestingly, all of the mRNA targets of dysregulated miRNAs reported in the literature (i.e., miR-31-5p, miR-192, miR-194, miR-449a and miR-638) belong to several important biological processes, such as Wnt signaling, cell proliferation and differentiation, and adherens junction pathways. Although we think that these predictions have to be necessarily confirmed by "wet-lab" data, the miRNAs dysregulated during the development of CD could be potentially involved in the pathogenesis of coeliac disease and their correlation with circulating miRNAs offers new possibilities to use them as disease biomarkers.

Direct and Intestinal Epithelial Cell-Mediated Effects of TLR8 Triggering on Human Dendritic Cells, CD14+CD16+ Monocytes and γδ T Lymphocytes.

Toll-like receptor (TLR)7/8 plays a crucial role in host recognition/response to viruses and its mucosal expression directly correlates with intestinal inflammation. The aim of this study was to investigate the role of TLR7/8 stimulation of intestinal epithelium in shaping the phenotype and functions of innate immunity cell subsets, and to define direct and/or epithelial cell-mediated mechanisms of the TLR7/8 agonist R848 immunomodulatory activity. We describe novel, TLR8-mediated, pro- and anti-inflammatory effects of R848 on ex vivo cultured human blood monocytes and γδ T lymphocytes, either induced by direct immune cell stimulation or mediated by intestinal epithelial cells (IEC). Apical stimulation with R848 led to its transport across normal polarized epithelial cell monolayer and resulted in the inhibition of monocyte differentiation toward immunostimulatory dendritic cells and Th1 type response. Furthermore, γδ T lymphocyte activation was promoted following direct exposure of these cells to the agonist. Conversely, a selective enrichment of the CD14+CD16+ monocyte subpopulation was observed, which required a CCL2-mediated inflammatory response of normal epithelial cells to R848. Of note, a TLR-mediated activation of control γδ T lymphocytes was promoted by inflamed intestinal epithelium from active Crohn's disease patients. This study unravels a novel regulatory mechanism linking the activation of the TLR8 pathway in IEC to the monocyte-mediated inflammatory response, and highlights the capacity of the TLR7/8 agonist R848 to directly enhance the activation of γδ T lymphocytes. Overall these results expand the range of cell targets and immune responses controlled by TLR8 triggering that may contribute to the antiviral response, to chronic inflammation, as well as to the adjuvant activity of TLR8 agonists, highlighting the role of intestinal epithelium microenvironment in shaping TLR agonist-induced responses.

Circulating microRNA Profiles as Liquid Biopsies for the Characterization and Diagnosis of Fibromyalgia Syndrome.

This work was aimed at investigating the circulating microRNA (miRNA) profiles in serum and saliva of patients affected by fibromyalgia syndrome (FM), correlating their expression values with clinical and clinimetric parameters and to suggest a mathematical model for the diagnosis of FM. A number of 14 FM patients and sex- and age-matched controls were enrolled in our study. The expression of a panel of 179 miRNAs was evaluated by qPCR. Statistical analyses were performed in order to obtain a mathematical linear model, which could be employed as a supporting tool in the diagnosis of FM. Bioinformatics analysis on miRNA targets were performed to obtain the relevant biological processes related to FM syndrome and to characterize in details the disease. Six miRNAs were found downregulated in FM patients compared to controls. Five of these miRNAs have been included in a linear predictive model that reached a very high sensitivity (100 %) and a high specificity (83.3 %). Moreover, miR-320b displayed a significant negative correlation (r = -0.608 and p = 0.036) with ZSDS score. Finally, several biological processes related to brain function/development and muscular functions were found potentially implicated in FM syndrome. Our study suggests that the study of circulating miRNA profiles coupled to statistical and bioinformatics analyses is a useful tool to better characterize the FM syndrome and to propose a preliminary model for its diagnosis.

Structural Features of the ATP-Binding Cassette (ABC) Transporter ABCA3.

In this review we reported and discussed the structural features of the ATP-Binding Cassette (ABC) transporter ABCA3 and how the use of bioinformatics tools could help researchers to obtain a reliable structural model of this important transporter. In fact, a model of ABCA3 is still lacking and no crystallographic structures (of the transporter or of its orthologues) are available. With the advent of next generation sequencing, many disease-causing mutations have been discovered and many more will be found in the future. In the last few years, ABCA3 mutations have been reported to have important pediatric implications. Thus, clinicians need a reliable structure to locate relevant mutations of this transporter and make genotype/phenotype correlations of patients affected by ABCA3-related diseases. In conclusion, we strongly believe that the model preliminarily generated by these novel bioinformatics tools could be the starting point to obtain more refined models of the ABCA3 transporter.

Sialylation of N-linked glycans influences the immunomodulatory effects of IgM on T cells.

Human serum IgM Abs are composed of heavily glycosylated polymers with five glycosylation sites on the µ (heavy) chain and one glycosylation site on the J chain. In contrast to IgG glycans, which are vital for a number of biological functions, virtually nothing is known about structure-function relationships of IgM glycans. Natural IgM is the earliest Ig produced and recognizes multiple Ags with low affinity, whereas immune IgM is induced by Ag exposure and is characterized by a higher Ag specificity. Natural anti-lymphocyte IgM is present in the serum of healthy individuals and increases in inflammatory conditions. It is able to inhibit T cell activation, but the underlying molecular mechanism is not understood. In this study, to our knowledge, we show for the first time that sialylated N-linked glycans induce the internalization of IgM by T cells, which in turn causes severe inhibition of T cell responses. The absence of sialic acid residues abolishes these inhibitory activities, showing a key role of sialylated N-glycans in inducing the IgM-mediated immune suppression.

Rat mir-155 generated from the lncRNA Bic is 'hidden' in the alternate genomic assembly and reveals the existence of novel mammalian miRNAs and clusters.

MicroRNAs (miRNAs) are a class of small noncoding RNAs acting as post-transcriptional gene expression regulators in many physiological and pathological conditions. During the last few years, many novel mammalian miRNAs have been predicted experimentally with bioinformatics approaches and validated by next-generation sequencing. Although these strategies have prompted the discovery of several miRNAs, the total number of these genes still seems larger. Here, by exploiting the species conservation of human, mouse, and rat hairpin miRNAs, we discovered a novel rat microRNA, mir-155. We found that mature miR-155 is overexpressed in rat spleen myeloid cells treated with LPS, similarly to humans and mice. Rat mir-155 is annotated only on the alternate genome, suggesting the presence of other "hidden" miRNAs on this assembly. Therefore, we comprehensively extended the homology search also to mice and humans, finally validating 34 novel mammalian miRNAs (two in humans, five in mice, and up to 27 in rats). Surprisingly, 15 of these novel miRNAs (one for mice and 14 for rats) were found only on the alternate and not on the reference genomic assembly. To date, our findings indicate that the choice of genomic assembly, when mapping small RNA reads, is an important option that should be carefully considered, at least for these animal models. Finally, the discovery of these novel mammalian miRNA genes may contribute to a better understanding of already acquired experimental data, thereby paving the way to still unexplored investigations and to unraveling the function of miRNAs in disease models.

Long non-coding RNAs and p53 regulation.

The advent of novel and high-throughput sequencing (next generation) technologies allowed for the sequencing of the genome at an unprecedented depth. The majority of transcribed RNAs have been classified as non-coding RNAs. Among them, long non-coding RNAs (lncRNAs) are emerging as important regulators in many biological processes. Here, we discuss the role of those lncRNAs which are under the control of p53 or that are able to regulate its activity, due to the central role of p53 pathway in many conditions. We also briefly discussed the emerging need of having novel strategies and computational tools to completely unravel the multifaceted roles of lncRNAs and to pave the way to the development of novel diagnostic and therapeutic applications based on these peculiar molecules.

Bioinformatics tools and novel challenges in long non-coding RNAs (lncRNAs) functional analysis.

The advent of next generation sequencing revealed that a fraction of transcribed RNAs (short and long RNAs) is non-coding. Long non-coding RNAs (lncRNAs) have a crucial role in regulating gene expression and in epigenetics (chromatin and histones remodeling). LncRNAs may have different roles: gene activators (signaling), repressors (decoy), cis and trans gene expression regulators (guides) and chromatin modificators (scaffolds) without the need to be mutually exclusive. LncRNAs are also implicated in a number of diseases. The huge amount of inhomogeneous data produced so far poses several bioinformatics challenges spanning from the simple annotation to the more complex functional annotation. In this review, we report and discuss several bioinformatics resources freely available and dealing with the study of lncRNAs. To our knowledge, this is the first review summarizing all the available bioinformatics resources on lncRNAs appeared in the literature after the completion of the human genome project. Therefore, the aim of this review is to provide a little guide for biologists and bioinformaticians looking for dedicated resources, public repositories and other tools for lncRNAs functional analysis.