voyAGEr, a free web interface for the analysis of age-related gene expression alterations in human tissues.

We herein introduce voyAGEr, an online graphical interface to explore age-related gene expression alterations in 49 human tissues. voyAGEr offers a visualisation and statistical toolkit for the finding and functional exploration of sex- and tissue-specific transcriptomic changes with age. In its conception, we developed a novel bioinformatics pipeline leveraging RNA sequencing data, from the GTEx project, encompassing more than 900 individuals. voyAGEr reveals transcriptomic signatures of the known asynchronous ageing between tissues, allowing the observation of tissue-specific age periods of major transcriptional changes, associated with alterations in different biological pathways, cellular composition, and disease conditions. Notably, voyAGEr was created to assist researchers with no expertise in bioinformatics, providing a supportive framework for elaborating, testing and refining their hypotheses on the molecular nature of human ageing and its association with pathologies, thereby also aiding in the discovery of novel therapeutic targets. voyAGEr is freely available at https://compbio.imm.medicina.ulisboa.pt/app/voyAGEr.

betAS: intuitive analysis and visualization of differential alternative splicing using beta distributions.

Next-generation RNA sequencing allows alternative splicing (AS) quantification with unprecedented resolution, with the relative inclusion of an alternative sequence in transcripts being commonly quantified by the proportion of reads supporting it as percent spliced-in (PSI). However, PSI values do not incorporate information about precision, proportional to the respective AS events' read coverage. Beta distributions are suitable to quantify inclusion levels of alternative sequences, using reads supporting their inclusion and exclusion as surrogates for the two distribution shape parameters. Each such beta distribution has the PSI as its mean value and is narrower when the read coverage is higher, facilitating the interpretability of its precision when plotted. We herein introduce a computational pipeline, based on beta distributions accurately modeling PSI values and their precision, to quantitatively and visually compare AS between groups of samples. Our methodology includes a differential splicing significance metric that compromises the magnitude of intergroup differences, the estimation uncertainty in individual samples, and the intragroup variability, being therefore suitable for multiple-group comparisons. To make our approach accessible and clear to both noncomputational and computational biologists, we developed betAS, an interactive web app and user-friendly R package for visual and intuitive differential splicing analysis from read count data.

Mouse Spinal Cord Vascular Transcriptome Analysis Identifies CD9 and MYLIP as Injury-Induced Players.

Traumatic spinal cord injury (SCI) initiates a cascade of cellular events, culminating in irreversible tissue loss and neuroinflammation. After the trauma, the blood vessels are destroyed. The blood-spinal cord barrier (BSCB), a physical barrier between the blood and spinal cord parenchyma, is disrupted, facilitating the infiltration of immune cells, and contributing to a toxic spinal microenvironment, affecting axonal regeneration. Understanding how the vascular constituents of the BSCB respond to injury is crucial to prevent BSCB impairment and to improve spinal cord repair. Here, we focus our attention on the vascular transcriptome at 3- and 7-days post-injury (dpi), during which BSCB is abnormally leaky, to identify potential molecular players that are injury-specific. Using the mouse contusion model, we identified Cd9 and Mylip genes as differentially expressed at 3 and 7 dpi. CD9 and MYLIP expression were injury-induced on vascular cells, endothelial cells and pericytes, at the injury epicentre at 7 dpi, with a spatial expression predominantly at the caudal region of the lesion. These results establish CD9 and MYLIP as two new potential players after SCI, and future studies targeting their expression might bring promising results for spinal cord repair.

Allosteric Antagonist Modulation of TRPV2 by Piperlongumine Impairs Glioblastoma Progression.

The use of computational tools to identify biological targets of natural products with anticancer properties and unknown modes of action is gaining momentum. We employed self-organizing maps to deconvolute the phenotypic effects of piperlongumine (PL) and establish a link to modulation of the human transient receptor potential vanilloid 2 (hTRPV2) channel. The structure of the PL-bound full-length rat TRPV2 channel was determined by cryo-EM. PL binds to a transient allosteric pocket responsible for a new mode of anticancer activity against glioblastoma (GBM) in which hTRPV2 is overexpressed. Calcium imaging experiments revealed the importance of Arg539 and Thr522 residues on the antagonistic effect of PL and calcium influx modulation of the TRPV2 channel. Downregulation of hTRPV2 reduces sensitivity to PL and decreases ROS production. Analysis of GBM patient samples associates hTRPV2 overexpression with tumor grade, disease progression, and poor prognosis. Extensive tumor abrogation and long term survival was achieved in two murine models of orthotopic GBM by formulating PL in an implantable scaffold/hydrogel for sustained local therapy. Furthermore, in primary tumor samples derived from GBM patients, we observed a selective reduction of malignant cells in response to PL ex vivo. Our results establish a broadly applicable strategy, leveraging data-motivated research hypotheses for the discovery of novel means tackling cancer.

The splicing factor XAB2 interacts with ERCC1-XPF and XPG for R-loop processing.

RNA splicing, transcription and the DNA damage response are intriguingly linked in mammals but the underlying mechanisms remain poorly understood. Using an in vivo biotinylation tagging approach in mice, we show that the splicing factor XAB2 interacts with the core spliceosome and that it binds to spliceosomal U4 and U6 snRNAs and pre-mRNAs in developing livers. XAB2 depletion leads to aberrant intron retention, R-loop formation and DNA damage in cells. Studies in illudin S-treated cells and Csbm/m developing livers reveal that transcription-blocking DNA lesions trigger the release of XAB2 from all RNA targets tested. Immunoprecipitation studies reveal that XAB2 interacts with ERCC1-XPF and XPG endonucleases outside nucleotide excision repair and that the trimeric protein complex binds RNA:DNA hybrids under conditions that favor the formation of R-loops. Thus, XAB2 functionally links the spliceosomal response to DNA damage with R-loop processing with important ramifications for transcription-coupled DNA repair disorders.

Unraveling Targetable Systemic and Cell-Type-Specific Molecular Phenotypes of Alzheimer's and Parkinson's Brains With Digital Cytometry.

Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most common neurodegenerative disorders worldwide, with age being their major risk factor. The increasing worldwide life expectancy, together with the scarcity of available treatment choices, makes it thus pressing to find the molecular basis of AD and PD so that the causing mechanisms can be targeted. To study these mechanisms, gene expression profiles have been compared between diseased and control brain tissues. However, this approach is limited by mRNA expression profiles derived for brain tissues highly reflecting their degeneration in cellular composition but not necessarily disease-related molecular states. We therefore propose to account for cell type composition when comparing transcriptomes of healthy and diseased brain samples, so that the loss of neurons can be decoupled from pathology-associated molecular effects. This approach allowed us to identify genes and pathways putatively altered systemically and in a cell-type-dependent manner in AD and PD brains. Moreover, using chemical perturbagen data, we computationally identified candidate small molecules for specifically targeting the profiled AD/PD-associated molecular alterations. Our approach therefore not only brings new insights into the disease-specific and common molecular etiologies of AD and PD but also, in these realms, foster the discovery of more specific targets for functional and therapeutic exploration.

NineTeen Complex-subunit Salsa is required for efficient splicing of a subset of introns and dorsal-ventral patterning.

The NineTeen Complex (NTC), also known as pre-mRNA-processing factor 19 (Prp19) complex, regulates distinct spliceosome conformational changes necessary for splicing. During Drosophila midblastula transition, splicing is particularly sensitive to mutations in NTC-subunit Fandango, which suggests differential requirements of NTC during development. We show that NTC-subunit Salsa, the Drosophila ortholog of human RNA helicase Aquarius, is rate-limiting for splicing of a subset of small first introns during oogenesis, including the first intron of gurken Germline depletion of Salsa and splice site mutations within gurken first intron impair both adult female fertility and oocyte dorsal-ventral patterning, due to an abnormal expression of Gurken. Supporting causality, the fertility and dorsal-ventral patterning defects observed after Salsa depletion could be suppressed by the expression of a gurken construct without its first intron. Altogether, our results suggest that one of the key rate-limiting functions of Salsa during oogenesis is to ensure the correct expression and efficient splicing of the first intron of gurken mRNA. Retention of gurken first intron compromises the function of this gene most likely because it undermines the correct structure and function of the transcript 5'UTR.

Expression of receptor activator of NFkB (RANK) drives stemness and resistance to therapy in ER+HER2- breast cancer.

The role of RANKL-RANK pathway in progesterone-driven mammary carcinogenesis and triple negative breast cancer tumorigenesis has been well characterized. However, and despite evidences of the existence of RANK-positive hormone receptor (HR)-positive breast tumors, the implication of RANK expression in HR-positive breast cancers has not been addressed before. Here, we report that RANK pathway affects the expression of cell cycle regulators and decreases sensitivity to fulvestrant of estrogen receptor (ER)-positive (ER+)/HER2- breast cancer cells, MCF-7 and T47D. Moreover, RANK overexpressing cells had a staminal and mesenchymal phenotype, with decreased proliferation rate and decreased susceptibility to chemotherapy, but were more invasive in vivo. In silico analysis of the transcriptome of human breast tumors, confirmed the association between RANK expression and stem cell and mesenchymal markers in ER+HER2- tumors. Importantly, exposure of ER+HER2- cells to continuous RANK pathway activation by exogenous RANKL, in vitro and in vivo, induced a negative feedback effect, independent of RANK levels, leading to the downregulation of HR and increased resistance to hormone therapy. These results suggest that ER+HER2- RANK-positive cells may constitute an important reservoir of slow cycling, therapy-resistance cancer cells; and that RANK pathway activation is deleterious in all ER+HER2- breast cancer cells, independently of RANK levels.

IgE to epitopes of Ara h 2 enhance the diagnostic accuracy of Ara h 2-specific IgE.

BACKGROUND: Understanding the discrepancy between IgE sensitization and allergic reactions to peanut could facilitate diagnosis and lead to novel means of treating peanut allergy. OBJECTIVE: To identify differences in IgE and IgG4 binding to peanut peptides between peanut-allergic (PA) and peanut-sensitized but tolerant (PS) children. METHODS: PA (n = 56), PS (n = 42) and nonsensitized nonallergic (NA, n = 10) patients were studied. Synthetic overlapping 15-mer peptides of peanut allergens (Ara h 1-11) were spotted onto microarray slides, and patients' samples were tested for IgE and IgG4 binding using immunofluorescence. IgE and IgG4 levels to selected peptides were quantified using ImmunoCAP. Diagnostic model comparisons were performed using likelihood-ratio tests between each specified nominal logistic regression models. RESULTS: Seven peptides on Ara h 1, Ara h 2, and Ara h 3 were bound more by IgE of PA compared to PS patients on the microarray. IgE binding to one peptide on Ara h 5 and IgG4 binding to one Ara h 9 peptide were greater in PS than in PA patients. Using ImmunoCAP, IgE to the Ara h 2 peptides enhanced the diagnostic accuracy of Ara h 2-specific IgE. Ratios of IgG4/IgE to 4 out of the 7 peptides were higher in PS than in PA subjects. CONCLUSIONS: Ara h 2 peptide-specific IgE added diagnostic value to Ara h 2-specific IgE. Ability of peptide-specific IgG4 to surmount their IgE counterpart seems to be important in established peanut tolerance.

Author Correction: Light-entrained and brain-tuned circadian circuits regulate ILC3s and gut homeostasis.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Androgen-regulated transcription of ESRP2 drives alternative splicing patterns in prostate cancer.

Prostate is the most frequent cancer in men. Prostate cancer progression is driven by androgen steroid hormones, and delayed by androgen deprivation therapy (ADT). Androgens control transcription by stimulating androgen receptor (AR) activity, yet also control pre-mRNA splicing through less clear mechanisms. Here we find androgens regulate splicing through AR-mediated transcriptional control of the epithelial-specific splicing regulator ESRP2. Both ESRP2 and its close paralog ESRP1 are highly expressed in primary prostate cancer. Androgen stimulation induces splicing switches in many endogenous ESRP2-controlled mRNA isoforms, including splicing switches correlating with disease progression. ESRP2 expression in clinical prostate cancer is repressed by ADT, which may thus inadvertently dampen epithelial splice programmes. Supporting this, treatment with the AR antagonist bicalutamide (Casodex) induced mesenchymal splicing patterns of genes including FLNB and CTNND1. Our data reveals a new mechanism of splicing control in prostate cancer with important implications for disease progression.

Light-entrained and brain-tuned circadian circuits regulate ILC3s and gut homeostasis.

Group 3 innate lymphoid cells (ILC3s) are major regulators of inflammation, infection, microbiota composition and metabolism1. ILC3s and neuronal cells have been shown to interact at discrete mucosal locations to steer mucosal defence2,3. Nevertheless, it is unclear whether neuroimmune circuits operate at an organismal level, integrating extrinsic environmental signals to orchestrate ILC3 responses. Here we show that light-entrained and brain-tuned circadian circuits regulate enteric ILC3s, intestinal homeostasis, gut defence and host lipid metabolism in mice. We found that enteric ILC3s display circadian expression of clock genes and ILC3-related transcription factors. ILC3-autonomous ablation of the circadian regulator Arntl led to disrupted gut ILC3 homeostasis, impaired epithelial reactivity, a deregulated microbiome, increased susceptibility to bowel infection and disrupted lipid metabolism. Loss of ILC3-intrinsic Arntl shaped the gut 'postcode receptors' of ILC3s. Strikingly, light-dark cycles, feeding rhythms and microbial cues differentially regulated ILC3 clocks, with light signals being the major entraining cues of ILC3s. Accordingly, surgically or genetically induced deregulation of brain rhythmicity led to disrupted circadian ILC3 oscillations, a deregulated microbiome and altered lipid metabolism. Our work reveals a circadian circuitry that translates environmental light cues into enteric ILC3s, shaping intestinal health, metabolism and organismal homeostasis.

Dissecting celastrol with machine learning to unveil dark pharmacology.

By coalescing bespoke machine learning and bioinformatics analyses with cell-based assays, we unveil the pharmacology of celastrol. Celastrol is a direct modulator of the progesterone and cannabinoid receptors, and its effects correlate with the antiproliferative activity. We demonstrate how in silico methods may drive systems biology studies for natural products.

Natural product-drug conjugates for modulation of TRPV1-expressing tumors.

We report the design, synthesis and biological evaluation of natural product-drug conjugates for treatment of prostate cancers over-expressing the transient receptor potential vanilloid 1 (TRPV1) channel. We validate the relevance of TRPV1 as a target in prostate cancer patients by using a bioinformatics approach and provide proof-of-concept for the drug delivery strategy through bioorthogonal chemistry and stability assays under simulated physiological conditions. In cell-based assays, the constructs displayed modest activity. Moreover, we serendipitously discover that a stoichiometric combination of a TRPV1 agonist with a small, positively charged cytotoxic may provide new research avenues in personalized medicines for prostate cancer.

Pan-cancer association of a centrosome amplification gene expression signature with genomic alterations and clinical outcome.

Centrosome amplification (CA) is a common feature of human tumours and a promising target for cancer therapy. However, CA's pan-cancer prevalence, molecular role in tumourigenesis and therapeutic value in the clinical setting are still largely unexplored. Here, we used a transcriptomic signature (CA20) to characterise the landscape of CA-associated gene expression in 9,721 tumours from The Cancer Genome Atlas (TCGA). CA20 is upregulated in cancer and associated with distinct clinical and molecular features of breast cancer, consistently with our experimental CA quantification in patient samples. Moreover, we show that CA20 upregulation is positively associated with genomic instability, alteration of specific chromosomal arms and C>T mutations, and we propose novel molecular players associated with CA in cancer. Finally, high CA20 is associated with poor prognosis and, by integrating drug sensitivity with drug perturbation profiles in cell lines, we identify candidate compounds for selectively targeting cancer cells exhibiting transcriptomic evidence for CA.

psichomics: graphical application for alternative splicing quantification and analysis.

Alternative pre-mRNA splicing generates functionally distinct transcripts from the same gene and is involved in the control of multiple cellular processes, with its dysregulation being associated with a variety of pathologies. The advent of next-generation sequencing has enabled global studies of alternative splicing in different physiological and disease contexts. However, current bioinformatics tools for alternative splicing analysis from RNA-seq data are not user-friendly, disregard available exon-exon junction quantification or have limited downstream analysis features. To overcome such limitations, we have developed psichomics, an R package with an intuitive graphical interface for alternative splicing quantification and downstream dimensionality reduction, differential splicing and gene expression and survival analyses based on The Cancer Genome Atlas, the Genotype-Tissue Expression project, the Sequence Read Archive project and user-provided data. These integrative analyses can also incorporate clinical and molecular sample-associated features. We successfully used psichomics in a laptop to reveal alternative splicing signatures specific to stage I breast cancer and associated novel putative prognostic factors.

Androgen-dependent alternative mRNA isoform expression in prostate cancer cells.

Background: Androgen steroid hormones are key drivers of prostate cancer. Previous work has shown that androgens can drive the expression of alternative mRNA isoforms as well as transcriptional changes in prostate cancer cells. Yet to what extent androgens control alternative mRNA isoforms and how these are expressed and differentially regulated in prostate tumours is unknown. Methods: Here we have used RNA-Seq data to globally identify alternative mRNA isoform expression under androgen control in prostate cancer cells, and profiled the expression of these mRNA isoforms in clinical tissue. Results: Our data indicate androgens primarily switch mRNA isoforms through alternative promoter selection. We detected 73 androgen regulated alternative transcription events, including utilisation of 56 androgen-dependent alternative promoters, 13 androgen-regulated alternative splicing events, and selection of 4 androgen-regulated alternative 3' mRNA ends. 64 of these events are novel to this study, and 26 involve previously unannotated isoforms. We validated androgen dependent regulation of 17 alternative isoforms by quantitative PCR in an independent sample set. Some of the identified mRNA isoforms are in genes already implicated in prostate cancer (including LIG4, FDFT1 and RELAXIN), or in genes important in other cancers (e.g. NUP93 and MAT2A). Importantly, analysis of transcriptome data from 497 tumour samples in the TGCA prostate adenocarcinoma (PRAD) cohort identified 13 mRNA isoforms (including TPD52, TACC2 and NDUFV3) that are differentially regulated in localised prostate cancer relative to normal tissue, and 3 ( OSBPL1A, CLK3 and TSC22D3) which change significantly with Gleason grade and  tumour stage. Conclusions: Our findings dramatically increase the number of known androgen regulated isoforms in prostate cancer, and indicate a highly complex response to androgens in prostate cancer cells that could be clinically important.

Decoding a cancer-relevant splicing decision in the RON proto-oncogene using high-throughput mutagenesis.

Mutations causing aberrant splicing are frequently implicated in human diseases including cancer. Here, we establish a high-throughput screen of randomly mutated minigenes to decode the cis-regulatory landscape that determines alternative splicing of exon 11 in the proto-oncogene MST1R (RON). Mathematical modelling of splicing kinetics enables us to identify more than 1000 mutations affecting RON exon 11 skipping, which corresponds to the pathological isoform RON∆165. Importantly, the effects correlate with RON alternative splicing in cancer patients bearing the same mutations. Moreover, we highlight heterogeneous nuclear ribonucleoprotein H (HNRNPH) as a key regulator of RON splicing in healthy tissues and cancer. Using iCLIP and synergy analysis, we pinpoint the functionally most relevant HNRNPH binding sites and demonstrate how cooperative HNRNPH binding facilitates a splicing switch of RON exon 11. Our results thereby offer insights into splicing regulation and the impact of mutations on alternative splicing in cancer.

PTBP1-Mediated Alternative Splicing Regulates the Inflammatory Secretome and the Pro-tumorigenic Effects of Senescent Cells.

Oncogene-induced senescence is a potent tumor-suppressive response. Paradoxically, senescence also induces an inflammatory secretome that promotes carcinogenesis and age-related pathologies. Consequently, the senescence-associated secretory phenotype (SASP) is a potential therapeutic target. Here, we describe an RNAi screen for SASP regulators. We identified 50 druggable targets whose knockdown suppresses the inflammatory secretome and differentially affects other SASP components. Among the screen candidates was PTBP1. PTBP1 regulates the alternative splicing of genes involved in intracellular trafficking, such as EXOC7, to control the SASP. Inhibition of PTBP1 prevents the pro-tumorigenic effects of the SASP and impairs immune surveillance without increasing the risk of tumorigenesis. In conclusion, our study identifies SASP inhibition as a powerful and safe therapy against inflammation-driven cancer.