Specialized Tfh cell subsets driving type-1 and type-2 humoral responses in lymphoid tissue.

Effective antibody responses are essential to generate protective humoral immunity. Different inflammatory signals polarize T cells towards appropriate effector phenotypes during an infection or immunization. Th1 and Th2 cells have been associated with the polarization of humoral responses. However, T follicular helper cells (Tfh) have a unique ability to access the B cell follicle and support the germinal center (GC) responses by providing B cell help. We investigated the specialization of Tfh cells induced under type-1 and type-2 conditions. We first studied homogenous Tfh cell populations generated by adoptively transferred TCR-transgenic T cells in mice immunized with type-1 and type-2 adjuvants. Using a machine learning approach, we established a gene expression signature that discriminates Tfh cells polarized towards type-1 and type-2 response, defined as Tfh1 and Tfh2 cells. The distinct signatures of Tfh1 and Tfh2 cells were validated against datasets of Tfh cells induced following lymphocytic choriomeningitis virus (LCMV) or helminth infection. We generated single-cell and spatial transcriptomics datasets to dissect the heterogeneity of Tfh cells and their localization under the two immunizing conditions. Besides a distinct specialization of GC Tfh cells under the two immunizations and in different regions of the lymph nodes, we found a population of Gzmk+ Tfh cells specific for type-1 conditions. In human individuals, we could equally identify CMV-specific Tfh cells that expressed Gzmk. Our results show that Tfh cells acquire a specialized function under distinct types of immune responses and with particular properties within the B cell follicle and the GC.

Analysis of asymptomatic Drosophila models for ALS and SMA reveals convergent impact on functional protein complexes linked to neuro-muscular degeneration.

BACKGROUND: Spinal Muscular Atrophy (SMA) and Amyotrophic Lateral Sclerosis (ALS) share phenotypic and molecular commonalities, including the fact that they can be caused by mutations in ubiquitous proteins involved in RNA metabolism, namely SMN, TDP-43 and FUS. Although this suggests the existence of common disease mechanisms, there is currently no model to explain the resulting motor neuron dysfunction. In this work we generated a parallel set of Drosophila models for adult-onset RNAi and tagged neuronal expression of the fly orthologues of the three human proteins, named Smn, TBPH and Caz, respectively. We profiled nuclear and cytoplasmic bound mRNAs using a RIP-seq approach and characterized the transcriptome of the RNAi models by RNA-seq. To unravel the mechanisms underlying the common functional impact of these proteins on neuronal cells, we devised a computational approach based on the construction of a tissue-specific library of protein functional modules, selected by an overall impact score measuring the estimated extent of perturbation caused by each gene knockdown. RESULTS: Transcriptome analysis revealed that the three proteins do not bind to the same RNA molecules and that only a limited set of functionally unrelated transcripts is commonly affected by their knock-down. However, through our integrative approach we were able to identify a concerted effect on protein functional modules, albeit acting through distinct targets. Most strikingly, functional annotation revealed that these modules are involved in critical cellular pathways for motor neurons, including neuromuscular junction function. Furthermore, selected modules were found to be significantly enriched in orthologues of human neuronal disease genes. CONCLUSIONS: The results presented here show that SMA and ALS disease-associated genes linked to RNA metabolism functionally converge on neuronal protein complexes, providing a new hypothesis to explain the common motor neuron phenotype. The functional modules identified represent promising biomarkers and therapeutic targets, namely given their alteration in asymptomatic settings.

Circulating Tumour Cell Associated MicroRNA Profiles Change during Chemoradiation and Are Predictive of Response in Locally Advanced Rectal Cancer.

Locally advanced rectal cancer (LARC) has traditionally been treated with trimodality therapy consisting of neoadjuvant radiation +/- chemotherapy, surgery, and adjuvant chemotherapy. There is currently a clinical need for biomarkers to predict treatment response and outcomes, especially during neoadjuvant therapy. Liquid biopsies in the form of circulating tumour cells (CTCs) and circulating nucleic acids in particular microRNAs (miRNA) are novel, the latter also being highly stable and clinically relevant regulators of disease. We studied a prospective cohort of 52 patients with LARC, and obtained samples at baseline, during treatment, and post-treatment. We enumerated CTCs during chemoradiation at these three time-points, using the IsofluxTM (Fluxion Biosciences Inc., Alameda, CA, USA) CTC Isolation and detection platform. We then subjected the isolated CTCs to miRNA expression analyses, using a panel of 106 miRNA candidates. We identified CTCs in 73% of patients at baseline; numbers fell and miRNA expression profiles also changed during treatment. Between baseline and during treatment (week 3) time-points, three microRNAs (hsa-miR-95, hsa-miR-10a, and hsa-miR-16-1*) were highly differentially expressed. Importantly, hsa-miR-19b-3p and hsa-miR-483-5p were found to correlate with good response to treatment. The latter (hsa-miR-483-5p) was also found to be differentially expressed between good responders and poor responders. These miRNAs represent potential predictive biomarkers, and thus a potential miRNA-based treatment strategy. In this study, we demonstrate that CTCs are present and can be isolated in the non-metastatic early-stage cancer setting, and their associated miRNA profiles can potentially be utilized to predict treatment response.

Small non-coding RNAs encoded by RNA viruses: old controversies and new lessons from the COVID-19 pandemic.

The recurring outbreaks caused by emerging RNA viruses have fostered an increased interest in the research of the mechanisms that regulate viral life cycles and the pathological outcomes associated with infections. Although interactions at the protein level are well-studied, interactions mediated by RNA molecules are less explored. RNA viruses can encode small non-coding RNAs molecules (sncRNAs), including viral miRNAs (v-miRNAs), that play important roles in modulating host immune responses and viral replication by targeting viral or host transcripts. Starting from the analysis of public databases compiling the known repertoire of viral ncRNA molecules and the evolution of publications and research interests on this topic in the wake of the COVID-19 pandemic, we provide an updated view on the current knowledge on viral sncRNAs, with a focus on v-miRNAs encoded by RNA viruses, and their mechanisms of action. We also discuss the potential of these molecules as diagnostic and prognostic biomarkers for viral infections and the development of antiviral therapies targeting v-miRNAs. This review emphasizes the importance of continued research efforts to characterize sncRNAs encoded by RNA viruses, identifies the most relevant pitfalls in the study of these molecules, and highlights the paradigm changes that have occurred in the last few years regarding their biogenesis, prevalence and functional relevance in the context of host-pathogen interactions.

DIS3L2 knockdown impairs key oncogenic properties of colorectal cancer cells via the mTOR signaling pathway.

DIS3L2 degrades different types of RNAs in an exosome-independent manner including mRNAs and several types of non-coding RNAs. DIS3L2-mediated degradation is preceded by the addition of nontemplated uridines at the 3'end of its targets by the terminal uridylyl transferases 4 and 7. Most of the literature that concerns DIS3L2 characterizes its involvement in several RNA degradation pathways, however, there is some evidence that its dysregulated activity may contribute to cancer development. In the present study, we characterize the role of DIS3L2 in human colorectal cancer (CRC). Using the public RNA datasets from The Cancer Genome Atlas (TCGA), we found higher DIS3L2 mRNA levels in CRC tissues versus normal colonic samples as well as worse prognosis in patients with high DIS3L2 expression. In addition, our RNA deep-sequencing data revealed that knockdown (KD) of DIS3L2 induces a strong transcriptomic disturbance in SW480 CRC cells. Moreover, gene ontology (GO) analysis of significant upregulated transcripts displays enrichment in mRNAs encoding proteins involved in cell cycle regulation and cancer-related pathways, which guided us to evaluate which specific hallmarks of cancer are differentially regulated by DIS3L2. To do so, we employed four CRC cell lines (HCT116, SW480, Caco-2 and HT-29) differing in their mutational background and oncogenicity. We demonstrate that depletion of DIS3L2 results in reduced cell viability of highly oncogenic SW480 and HCT116 CRC cells, but had little or no impact in the more differentiated Caco-2 and HT-29 cells. Remarkably, the mTOR signaling pathway, crucial for cell survival and growth, is downregulated after DIS3L2 KD, whereas AZGP1, an mTOR pathway inhibitor, is upregulated. Furthermore, our results indicate that depletion of DIS3L2 disturbs metastasis-associated properties, such as cell migration and invasion, only in highly oncogenic CRC cells. Our work reveals for the first time a role for DIS3L2 in sustaining CRC cell proliferation and provides evidence that this ribonuclease is required to support the viability and invasive behavior of dedifferentiated CRC cells.

A New Perspective for Vineyard Terroir Identity: Looking for Microbial Indicator Species by Long Read Nanopore Sequencing.

Grapevine is one of the most important fruit crops worldwide, being Portugal one of the top wine producers. It is well established that wine sensory characteristics from a particular region are defined by the physiological responses of the grapevine to its environment and thus, the concept of terroir in viticulture was established. Among all the factors that contribute to terroir definition, soil microorganisms play a major role from nutrient recycling to a drastic influence on plant fitness (growth and protection) and of course wine production. Soil microbiome from four different terroirs in Quinta dos Murças vineyard was analysed through long-read Oxford Nanopore sequencing. We have developed an analytical pipeline that allows the identification of function, ecologies, and indicator species based on long read sequencing data. The Douro vineyard was used as a case study, and we were able to establish microbiome signatures of each terroir.

Human Satellite 1A analysis provides evidence of pericentromeric transcription.

BACKGROUND: Pericentromeric regions of human chromosomes are composed of tandem-repeated and highly organized sequences named satellite DNAs. Human classical satellite DNAs are classified into three families named HSat1, HSat2, and HSat3, which have historically posed a challenge for the assembly of the human reference genome where they are misrepresented due to their repetitive nature. Although being known for a long time as the most AT-rich fraction of the human genome, classical satellite HSat1A has been disregarded in genomic and transcriptional studies, falling behind other human satellites in terms of functional knowledge. Here, we aim to characterize and provide an understanding on the biological relevance of HSat1A. RESULTS: The path followed herein trails with HSat1A isolation and cloning, followed by in silico analysis. Monomer copy number and expression data was obtained in a wide variety of human cell lines, with greatly varying profiles in tumoral/non-tumoral samples. HSat1A was mapped in human chromosomes and applied in in situ transcriptional assays. Additionally, it was possible to observe the nuclear organization of HSat1A transcripts and further characterize them by 3' RACE-Seq. Size-varying polyadenylated HSat1A transcripts were detected, which possibly accounts for the intricate regulation of alternative polyadenylation. CONCLUSION: As far as we know, this work pioneers HSat1A transcription studies. With the emergence of new human genome assemblies, acrocentric pericentromeres are becoming relevant characters in disease and other biological contexts. HSat1A sequences and associated noncoding RNAs will most certainly prove significant in the future of HSat research.

Mutation-class dependent signatures outweigh disease-associated processes in cystic fibrosis cells.

BACKGROUND: The phenotypic heterogeneity observed in Cystic Fibrosis (CF) patients suggests the involvement of other genes, besides CFTR. Here, we combined transcriptome and proteome analysis to understand the global gene expression patterns associated with five prototypical CFTR mutations. RESULTS: Evaluation of differentially expressed genes and proteins unveiled common and mutation-specific changes revealing functional signatures that are much more associated with the specific molecular defects associated with each mutation than to the CFTR loss-of-function phenotype. The combination of both datasets revealed that mutation-specific detected translated-transcripts (Dtt) have a high level of consistency. CONCLUSIONS: This is the first combined transcriptomic and proteomic study focusing on prototypical CFTR mutations. Analysis of Dtt provides novel insight into the pathophysiology of CF, and the mechanisms through which each mutation class causes disease and will likely contribute to the identification of new therapeutic targets and/or biomarkers for CF.

Virulence-related metabolism is activated in Botrytis cinerea mostly in the interaction with tolerant green grapes that remain largely unaffected in contrast with susceptible green grapes.

Botrytis cinerea is responsible for the gray mold disease, severely affecting Vitis vinifera grapevine and hundreds of other economically important crops. However, many mechanisms of this fruit-pathogen interaction remain unknown. The combined analysis of the transcriptome and metabolome of green fruits infected with B. cinerea from susceptible and tolerant genotypes was never performed in any fleshy fruit, mostly because green fruits are widely accepted to be resistant to this fungus. In this work, peppercorn-sized fruits were infected in the field or mock-treated, and berries were collected at green (EL32) stage from a susceptible (Trincadeira) and a tolerant (Syrah) variety. RNAseq and GC-MS data suggested that Syrah exhibited a pre-activated/basal defense relying on specific signaling pathways, hormonal regulation, namely jasmonate and ethylene metabolisms, and linked to phenylpropanoid metabolism. In addition, putative defensive metabolites such as shikimic, ursolic/ oleanolic, and trans-4-hydroxy cinnamic acids, and epigallocatechin were more abundant in Syrah than Trincadeira before infection. On the other hand, Trincadeira underwent relevant metabolic reprogramming upon infection but was unable to contain disease progression. RNA-seq analysis of the fungus in planta revealed an opposite scenario with higher gene expression activity within B. cinerea during infection of the tolerant cultivar and less activity in infected Trincadeira berries. The results suggested an activated virulence state during interaction with the tolerant cultivar without visible disease symptoms. Together, this study brings novel insights related to early infection strategies of B. cinerea and the green berry defense against necrotrophic fungi.

Biological interacting units identified in human protein networks reveal tissue-functional diversification and its impact on disease.

Protein-protein interactions (PPI) play an essential role in the biological processes that occur in the cell. Therefore, the dissection of PPI networks becomes decisive to model functional coordination and predict pathological de-regulation. Cellular networks are dynamic and proteins display varying roles depending on the tissue-interactomic context. Thus, the use of centrality measures in individual proteins fall short to dissect the functional properties of the cell. For this reason, there is a need for more comprehensive, relational, and context-specific ways to analyze the multiple actions of proteins in different cells and identify specific functional assemblies within global biomolecular networks. Under this framework, we define Biological Interacting units (BioInt-U) as groups of proteins that interact physically and are enriched in a common Gene Ontology. A search strategy was applied on 33 tissue-specific (TS) PPI networks to generate BioInt libraries associated with each particular human tissue. The cross-tissue comparison showed that housekeeping assemblies incorporate different proteins and exhibit distinct network properties depending on the tissue. Furthermore, disease genes (DGs) of tissue-associated pathologies preferentially accumulate in units in the expected tissues, which in turn were more central in the TS networks. Overall, the study reveals a tissue-specific functional diversification based on the identification of specific protein units and suggests vulnerabilities specific of each tissue network, which can be applied to refine protein-disease association methods.

Therapeutic Metabolic Reprograming Using microRNAs: From Cancer to HIV Infection.

MicroRNAs (miRNAs) are crucial regulators of cellular processes, including metabolism. Attempts to use miRNAs as therapeutic agents are being explored in several areas, including the control of cancer progression. Recent evidence suggests fine tuning miRNA activity to reprogram tumor cell metabolism has enormous potential as an alternative treatment option. Indeed, cancer growth is known to be linked to profound metabolic changes. Likewise, the emerging field of immunometabolism is leading to a refined understanding of how immune cell proliferation and function is governed by glucose homeostasis. Different immune cell types are now known to have unique metabolic signatures that switch in response to a changing environment. T-cell subsets exhibit distinct metabolic profiles which underlie their alternative differentiation and phenotypic functions. Recent evidence shows that the susceptibility of CD4+ T-cells to HIV infection is intimately linked to their metabolic activity, with many of the metabolic features of HIV-1-infected cells resembling those found in tumor cells. In this review, we discuss the use of miRNA modulation to achieve metabolic reprogramming for cancer therapy and explore the idea that the same approach may serve as an effective mechanism to restrict HIV replication and eliminate infected cells.

Network Approaches to Study Endogenous RNA Competition and Its Impact on Tissue-Specific microRNA Functions.

microRNAs are small non-coding RNAs that play a key role in regulating gene expression. These molecules exert their function through sequence complementarity with microRNA responsive elements and are typically located in the 3' untranslated region of mRNAs, negatively regulating expression. Even though the relevant role of miRNA-dependent regulation is broadly recognized, the principles governing their ability to lead to specific functional outcomes in distinct cell types are still not well understood. In recent years, an intriguing hypothesis proposed that miRNA-responsive elements act as communication links between different RNA species, making the investigation of microRNA function even more complex than previously thought. The competing endogenous RNA hypothesis suggests the presence of a new level of regulation, whereby a specific RNA transcript can indirectly influence the abundance of other transcripts by limiting the availability of a common miRNA, acting as a "molecular sponge". Since this idea has been proposed, several studies have tried to pinpoint the interaction networks that have been established between different RNA species and whether they contribute to normal cell function and disease. The focus of this review is to highlight recent developments and achievements made towards the process of characterizing competing endogenous RNA networks and their role in cellular function.

Developmental bifurcation of human T follicular regulatory cells.

Germinal centers (GCs) are anatomic structures where B cells undergo affinity maturation, leading to production of high-affinity antibodies. The balance between T follicular helper (TFH) and regulatory (TFR) cells is critical for adequate control of GC responses. The study of human TFH and TFR cell development has been hampered because of the lack of in vitro assays reproducing in vivo biology, along with difficult access to healthy human lymphoid tissues. We used a single-cell transcriptomics approach to study the maturation of TFH and TFR cells isolated from human blood, iliac lymph nodes (LNs), and tonsils. As independent tissues have distinct proportions of follicular T cells in different maturation states, we leveraged the heterogeneity to reconstruct the maturation trajectory for human TFH and TFR cells. We found that the dominant maturation of TFR cells follows a bifurcated trajectory from precursor Treg cells, with one arm of the bifurcation leading to blood TFR cells and the other leading to the most mature GC TFR cells. Overall, our data provide a comprehensive resource for the transcriptomics of different follicular T cell populations and their dynamic relationship across different tissues.

Genomic Tackling of Human Satellite DNA: Breaking Barriers through Time.

(Peri)centromeric repetitive sequences and, more specifically, satellite DNA (satDNA) sequences, constitute a major human genomic component. SatDNA sequences can vary on a large number of features, including nucleotide composition, complexity, and abundance. Several satDNA families have been identified and characterized in the human genome through time, albeit at different speeds. Human satDNA families present a high degree of sub-variability, leading to the definition of various subfamilies with different organization and clustered localization. Evolution of satDNA analysis has enabled the progressive characterization of satDNA features. Despite recent advances in the sequencing of centromeric arrays, comprehensive genomic studies to assess their variability are still required to provide accurate and proportional representation of satDNA (peri)centromeric/acrocentric short arm sequences. Approaches combining multiple techniques have been successfully applied and seem to be the path to follow for generating integrated knowledge in the promising field of human satDNA biology.

Fasciola hepatica hijacks host macrophage miRNA machinery to modulate early innate immune responses.

Fasciola hepatica, a global worm parasite of humans and their livestock, regulates host innate immune responses within hours of infection. Host macrophages, essential to the first-line defence mechanisms, are quickly restricted in their ability to initiate a classic protective pro-inflammatory immune response. We found that macrophages from infected animals are enriched with parasite-derived micro(mi)RNAs. The most abundant of these miRNAs, fhe-miR-125b, is released by the parasite via exosomes and is homologous to a mammalian miRNA, hsa-miR-125b, that is known to regulate the activation of pro-inflammatory M1 macrophages. We show that the parasite fhe-miR-125b loads onto the mammalian Argonaut protein (Ago-2) within macrophages during infection and, therefore, propose that it mimics host miR-125b to negatively regulate the production of inflammatory cytokines. The hijacking of the miRNA machinery controlling innate cell function could be a fundamental mechanism by which worm parasites disarm the early immune responses of their host to ensure successful infection.

IsomiR_Window: a system for analyzing small-RNA-seq data in an integrative and user-friendly manner.

BACKGROUND: IsomiRs are miRNA variants that vary in length and/or sequence when compared to their canonical forms. These variants display differences in length and/or sequence, including additions or deletions of one or more nucleotides (nts) at the 5' and/or 3' end, internal editings or untemplated 3' end additions. Most available tools for small RNA-seq data analysis do not allow the identification of isomiRs and often require advanced knowledge of bioinformatics. To overcome this, we have developed IsomiR Window, a platform that supports the systematic identification, quantification and functional exploration of isomiR expression in small RNA-seq datasets, accessible to users with no computational skills. METHODS: IsomiR Window enables the discovery of isomiRs and identification of all annotated non-coding RNAs in RNA-seq datasets from animals and plants. It comprises two main components: the IsomiR Window pipeline for data processing; and the IsomiR Window Browser interface. It integrates over ten third-party softwares for the analysis of small-RNA-seq data and holds a new algorithm that allows the detection of all possible types of isomiRs. These include 3' and 5'end isomiRs, 3' end tailings, isomiRs with single nucleotide polymorphisms (SNPs) or potential RNA editings, as well as all possible fuzzy combinations. IsomiR Window includes all required databases for analysis and annotation, and is freely distributed as a Linux virtual machine, including all required software. RESULTS: IsomiR Window processes several datasets in an automated manner, without restrictions of input file size. It generates high quality interactive figures and tables which can be exported into different formats. The performance of isomiR detection and quantification was assessed using simulated small-RNA-seq data. For correctly mapped reads, it identified different types of isomiRs with high confidence and 100% accuracy. The analysis of a small RNA-seq data from Basal Cell Carcinomas (BCCs) using isomiR Window confirmed that miR-183-5p is up-regulated in Nodular BCCs, but revealed that this effect was predominantly due to a novel 5'end variant. This variant displays a different seed region motif and 1756 isoform-exclusive mRNA targets that are significantly associated with disease pathways, underscoring the biological relevance of isomiR-focused analysis. IsomiR Window is available at https://isomir.fc.ul.pt/ .

Machine learning modelling of blood lipid biomarkers in familial hypercholesterolaemia versus polygenic/environmental dyslipidaemia.

Familial hypercholesterolaemia increases circulating LDL-C levels and leads to premature cardiovascular disease when undiagnosed or untreated. Current guidelines support genetic testing in patients complying with clinical diagnostic criteria and cascade screening of their family members. However, most of hyperlipidaemic subjects do not present pathogenic variants in the known disease genes, and most likely suffer from polygenic hypercholesterolaemia, which translates into a relatively low yield of genetic screening programs. This study aims to identify new biomarkers and develop new approaches to improve the identification of individuals carrying monogenic causative variants. Using a machine-learning approach in a paediatric dataset of individuals, tested for disease causative genes and with an extended lipid profile, we developed new models able to classify familial hypercholesterolaemia patients with a much higher specificity than currently used methods. The best performing models incorporated parameters absent from the most common FH clinical criteria, namely apoB/apoA-I, TG/apoB and LDL1. These parameters were found to contribute to an improved identification of monogenic individuals. Furthermore, models using only TC and LDL-C levels presented a higher specificity of classification when compared to simple cut-offs. Our results can be applied towards the improvement of the yield of genetic screening programs and corresponding costs.

Decoding the Role of Satellite DNA in Genome Architecture and Plasticity-An Evolutionary and Clinical Affair.

Repetitive DNA is a major organizational component of eukaryotic genomes, being intrinsically related with their architecture and evolution. Tandemly repeated satellite DNAs (satDNAs) can be found clustered in specific heterochromatin-rich chromosomal regions, building vital structures like functional centromeres and also dispersed within euchromatin. Interestingly, despite their association to critical chromosomal structures, satDNAs are widely variable among species due to their high turnover rates. This dynamic behavior has been associated with genome plasticity and chromosome rearrangements, leading to the reshaping of genomes. Here we present the current knowledge regarding satDNAs in the light of new genomic technologies, and the challenges in the study of these sequences. Furthermore, we discuss how these sequences, together with other repeats, influence genome architecture, impacting its evolution and association with disease.