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.

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.

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.

Neuronal regulation of type 2 innate lymphoid cells via neuromedin U.

Group 2 innate lymphoid cells (ILC2s) regulate inflammation, tissue repair and metabolic homeostasis, and are activated by host-derived cytokines and alarmins. Discrete subsets of immune cells integrate nervous system cues, but it remains unclear whether neuron-derived signals control ILC2s. Here we show that neuromedin U (NMU) in mice is a fast and potent regulator of type 2 innate immunity in the context of a functional neuron-ILC2 unit. We found that ILC2s selectively express neuromedin U receptor 1 (Nmur1), and mucosal neurons express NMU. Cell-autonomous activation of ILC2s with NMU resulted in immediate and strong NMUR1-dependent production of innate inflammatory and tissue repair cytokines. NMU controls ILC2s downstream of extracellular signal-regulated kinase and calcium-influx-dependent activation of both calcineurin and nuclear factor of activated T cells (NFAT). NMU treatment in vivo resulted in immediate protective type 2 responses. Accordingly, ILC2-autonomous ablation of Nmur1 led to impaired type 2 responses and poor control of worm infection. Notably, mucosal neurons were found adjacent to ILC2s, and these neurons directly sensed worm products and alarmins to induce NMU and to control innate type 2 cytokines. Our work reveals that neuron-ILC2 cell units confer immediate tissue protection through coordinated neuroimmune sensory responses.

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.

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.

Effectiveness of exercise training on cancer-related fatigue in colorectal cancer survivors: a systematic review and meta-analysis of randomized controlled trials.

PURPOSE: To investigate the effects of exercise training on cancer-related fatigue (CRF) in colorectal cancer survivors. METHODS: Randomized controlled trials published between 1 January 2010 and 19 October 2020, selected through online search conducted in PubMed, Scopus, Web of Science, SPORTDiscus and PEDro databases, were included. Eligible trials compared the effect of exercise training interventions, versus non-exercise controls on CRF, in colorectal cancer survivors, during or after treatment. The methodological quality of individual studies was analysed using the Physiotherapy Evidence Database (PEDro) scale. Standardized mean differences (SMD) that were pooled using random-effects models were included as the effect size. In addition, 95% prediction intervals (PI) were calculated. RESULTS: Six trials involving 330 colorectal cancer patients met the inclusion criteria and presented reasonable to good methodological quality. An overall small-to-moderate effect of exercise training on CRF was found (SMD = - 0.29: 95% CI: [- 0.53; - 0.06]; p = 0.01; PI: [- 0.63; 0.04]; low-quality evidence). Subgroup analysis revealed moderate effects of exercise interventions performed during chemotherapy (SMD = - 0.63; 95% CI: [- 1.06; - 0.21]; p = 0.003) and small, non-significant effects, when exercise training was performed after cancer treatment (SMD = - 0.14; 95% CI: [- 0.43; 0.14]; p = 0.32). Steady improvements were achieved when a combination of aerobic plus resistance exercise was used, in interventions lasting 12 to 24 weeks. CONCLUSION: Exercise training could be regarded as a supportive therapy for the clinical management of CRF in colorectal cancer patients undergoing chemotherapy, but further studies are necessary to clarify the effects of exercise interventions on CRF after cancer treatment.

Latent regulatory potential of human-specific repetitive elements.

At least half of the human genome is derived from repetitive elements, which are often lineage specific and silenced by a variety of genetic and epigenetic mechanisms. Using a transchromosomic mouse strain that transmits an almost complete single copy of human chromosome 21 via the female germline, we show that a heterologous regulatory environment can transcriptionally activate transposon-derived human regulatory regions. In the mouse nucleus, hundreds of locations on human chromosome 21 newly associate with activating histone modifications in both somatic and germline tissues, and influence the gene expression of nearby transcripts. These regions are enriched with primate and human lineage-specific transposable elements, and their activation corresponds to changes in DNA methylation at CpG dinucleotides. This study reveals the latent regulatory potential of the repetitive human genome and illustrates the species specificity of mechanisms that control it.

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.

Expansion of DUB functionality generated by alternative isoforms - USP35, a case study.

Protein ubiquitylation is a dynamic post-translational modification that can be reversed by deubiquitylating enzymes (DUBs). It is unclear how the small number (∼100) of DUBs present in mammalian cells regulate the thousands of different ubiquitylation events. Here, we analysed annotated transcripts of human DUBs and found ∼300 ribosome-associated transcripts annotated as protein coding, which thus increases the total number of DUBs. By using USP35, a poorly studied DUB, as a case study, we provide evidence that alternative isoforms contribute to the functional expansion of DUBs. We show that there are two different USP35 isoforms that localise to different intracellular compartments and have distinct functions. Our results reveal that isoform 1 is an anti-apoptotic factor that inhibits staurosporine- and TNF-related apoptosis-inducing ligand (TRAIL; also known as TNFSF10)-induced apoptosis. In contrast, USP35 isoform 2 is an integral membrane protein of the endoplasmic reticulum (ER) that is also present at lipid droplets. Manipulations of isoform 2 levels cause rapid ER stress, likely through deregulation of lipid homeostasis, and lead to cell death. Our work highlights how alternative isoforms provide functional expansion of DUBs and sets directions for future research.This article has an associated First Person interview with the first author of the paper.

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.

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.

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.

MBNL proteins repress ES-cell-specific alternative splicing and reprogramming.

Previous investigations of the core gene regulatory circuitry that controls the pluripotency of embryonic stem (ES) cells have largely focused on the roles of transcription, chromatin and non-coding RNA regulators. Alternative splicing represents a widely acting mode of gene regulation, yet its role in regulating ES-cell pluripotency and differentiation is poorly understood. Here we identify the muscleblind-like RNA binding proteins, MBNL1 and MBNL2, as conserved and direct negative regulators of a large program of cassette exon alternative splicing events that are differentially regulated between ES cells and other cell types. Knockdown of MBNL proteins in differentiated cells causes switching to an ES-cell-like alternative splicing pattern for approximately half of these events, whereas overexpression of MBNL proteins in ES cells promotes differentiated-cell-like alternative splicing patterns. Among the MBNL-regulated events is an ES-cell-specific alternative splicing switch in the forkhead family transcription factor FOXP1 that controls pluripotency. Consistent with a central and negative regulatory role for MBNL proteins in pluripotency, their knockdown significantly enhances the expression of key pluripotency genes and the formation of induced pluripotent stem cells during somatic cell reprogramming.

The Kinematic Chain of Arm Elevation Is Impaired in Patients with Chronic Obstructive Pulmonary Disease.

Patients with chronic obstructive pulmonary disease (COPD) often complain about difficulties in performing activities with their arms above shoulders height. These difficulties have been associated with increased cardiorespiratory demand and altered lung mechanics; however, musculoskeletal-related mechanisms may also contribute to constrain the mechanics of the upper body quadrant, increasing the effort to perform the activities. This exploratory research aimed to assess potential changes in the kinematic chain of arm elevation in patients with COPD. A secondary analysis from a cross-sectional exploratory case-control and prediction study was conducted in 15 patients with COPD (2 females) and 15 controls (8 females) matched for age and body mass index. The sagittal alignment and active range of motion (ROM) of the head, thoracic spine and shoulder complex were measured, using a computer software, in digital lateral photographs obtained in three different testing positions: arms at rest, arms at 90° of shoulder flexion and full arm elevation. From rest to full arm elevation, both groups moved from a more flexed to a less flexed or more upright thoracic spine position (∼7°, p < 0.001, 0.419 < ηp2 <0.767). However, the COPD group showed significantly less shoulder flexion (∼12°, p = 0.007, d = 1.05) and thoracic spine extension (∼6°, p = 0.015, ηp2 = 0.139) ROM than the control group in the full arm elevation position. These findings suggest that this population may show changes in the kinematic chain of arm elevation that possibly contribute to arm movement-related complains and limited performance in their daily living.

Widespread intron retention in mammals functionally tunes transcriptomes.

Alternative splicing (AS) of precursor RNAs is responsible for greatly expanding the regulatory and functional capacity of eukaryotic genomes. Of the different classes of AS, intron retention (IR) is the least well understood. In plants and unicellular eukaryotes, IR is the most common form of AS, whereas in animals, it is thought to represent the least prevalent form. Using high-coverage poly(A)(+) RNA-seq data, we observe that IR is surprisingly frequent in mammals, affecting transcripts from as many as three-quarters of multiexonic genes. A highly correlated set of cis features comprising an "IR code" reliably discriminates retained from constitutively spliced introns. We show that IR acts widely to reduce the levels of transcripts that are less or not required for the physiology of the cell or tissue type in which they are detected. This "transcriptome tuning" function of IR acts through both nonsense-mediated mRNA decay and nuclear sequestration and turnover of IR transcripts. We further show that IR is linked to a cross-talk mechanism involving localized stalling of RNA polymerase II (Pol II) and reduced availability of spliceosomal components. Collectively, the results implicate a global checkpoint-type mechanism whereby reduced recruitment of splicing components coupled to Pol II pausing underlies widespread IR-mediated suppression of inappropriately expressed transcripts.

Intrarater Agreement of Elbow Extension Range of Motion in the Upper Limb Neurodynamic Test 1 Using a Smartphone Application.

OBJECTIVE: To estimate the intrarater agreement of the Compass application of a smartphone in the assessment of elbow extension range of motion (EE-ROM) at pain onset and maximum tolerable point during the Upper Limb Neurodynamic Test 1 (ULNT1). DESIGN: Within-day intrarater agreement study. SETTING: Private and university clinical settings. PARTICIPANTS: Volunteers (N=41; 21 men; age, 31.34±13.27y; height, 1.67±0.07m; body mass, 70.53±12.37kg) recruited from the community, with no symptoms or musculoskeletal abnormalities in their upper body quadrant and no regional or systemic nerve dysfunction. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Ninety-five percent limits of agreement (LOA), standard error of the measurement, and minimal detectable change at the 95% confidence level (MDC95) of EE-ROM at pain onset and maximum tolerable point during the ULNT1. RESULTS: Standard error of the measurement and MDC95 were relatively high on both sides when considering the onset of pain (standard error of the measurement, 6.6°-6.8°; MDC95, 18.4°-18.8°). Better results were found for the maximum tolerable point (standard error of the measurement, 4.2°-4.8°; MDC95, 11.7°-13.2°). The 95% LOA showed a similar trend. CONCLUSIONS: Smartphone measurements showed relatively wide agreement parameters of elbow extension during the ULNT1. These results are, nevertheless, comparable with previous studies using goniometric assessment when considering maximal pain tolerance. Further research is needed before the possible widespread use of the smartphone in neurodynamic assessment.

Trypanosoma brucei histone H1 inhibits RNA polymerase I transcription and is important for parasite fitness in vivo.

Trypanosoma brucei is a unicellular parasite that causes sleeping sickness in humans. Most of its transcription is constitutive and driven by RNA polymerase II. RNA polymerase I (Pol I) transcribes not only ribosomal RNA genes, but also protein-encoding genes, including variant surface glycoproteins (VSGs) and procyclins. In T. brucei, histone H1 (H1) is required for VSG silencing and chromatin condensation. However, whether H1 has a genome-wide role in transcription is unknown. Here, using RNA sequencing we show that H1 depletion changes the expression of a specific cohort of genes. Interestingly, the predominant effect is partial loss of silencing of Pol I loci, such as VSG and procyclin genes. Labelling of nascent transcripts with 4-thiouridine showed that H1 depletion does not alter the level of labelled Pol II transcripts. In contrast, the levels of 4sU-labelled Pol I transcripts were increased by two- to sixfold, suggesting that H1 preferentially blocks transcription at Pol I loci. Finally, we observed that parasites depleted of H1 grow almost normally in culture but they have a reduced fitness in mice, suggesting that H1 is important for host-pathogen interactions.

Kinematic comparison and description of the 3-dimensional shoulder kinematics of 2 shoulder rotation tests.

OBJECTIVES: The purpose of this study was to compare shoulder external rotation range of motion (ROM) during the hand-behind-neck (HBN) test and a standard shoulder external rotation test and to describe the 3-dimensional scapular motion during the HBN test. METHODS: An electromagnetic tracking device was used to assess the dominant shoulder of 14 healthy participants while performing active full ROM in a standard shoulder external rotation test in an elevated position (EREP) and in the HBN test. The humeral and scapular 3-dimensional positions at the end of EREP and HBN were compared using a paired-sample t test. A correlation analysis was performed between humeral and scapular angles to assess the contribution of scapular motion to the full shoulder ROM during the HBN test. RESULTS: No significant differences were found between the HBN test and the EREP at the end-range of the glenohumeral external rotation (HBN: 15.6° ± 6.3° vs EREP: 23.4° ± 4.7°; P = .08) and on scapular internal-external rotation (HBN test: 21.2° ± 6.3° vs EREP: 15.6° ± 1.8°; P = .23). Significant differences were found in scapular upward rotation (HBN: 21.2° ± 6.3° vs EREP: 15.6° ± 1.8°; P < .01) and scapular spinal tilt (HBN: -0.4° ± 2.3° vs EREP: 8.1° ± 2.1°; P < .01). There was a positive correlation between the humeral angles and scapular internal and posterior spinal tilt angles with the HBN test. CONCLUSIONS: The results of the present study showed that, in young asymptomatic participants with no known shoulder pathology, the end-range of shoulder rotation was similar in the HBN test and in a standard shoulder rotation test. During the HBN test, the scapula assumed a more internal and anterior spinal tilted position at the end-range of active shoulder external rotation. These results suggest that the HBN test may be used to assess the end-range of glenohumeral external rotation.