Large-scale analysis of Drosophila core promoter function using synthetic promoters

The core promoter plays a central role in setting metazoan gene expression levels, but how exactly it “computes” expression remains poorly understood. To dissect its function, we carried out a comprehensive structure–function analysis in Drosophila. First, we performed a genome-wide bioinformatic analysis, providing an improved picture of the sequence motifs architecture. We then measured synthetic promoters’ activities of ~3,000 mutational variants with and without an external stimulus (hormonal activation), at large scale and with high accuracy using robotics and a dual luciferase reporter assay. We observed a strong impact on activity of the different types of mutations, including knockout of individual sequence motifs and motif combinations, variations of motif strength, nucleosome positioning, and flanking sequences. A linear combination of the individual motif features largely accounts for the combinatorial effects on core promoter activity. These findings shed new light on the quantitative assessment of gene expression in metazoans

Reversibility in Chemical Reactions

open access bookIn this chapter we give an overview of techniques for the modelling and reasoning about reversibility of systems, including outof- causal-order reversibility, as it appears in chemical reactions. We consider the autoprotolysis of water reaction, and model it with the Calculus of Covalent Bonding, the Bonding Calculus, and Reversing Petri Nets. This exercise demonstrates that the formalisms, developed for expressing advanced forms of reversibility, are able to model autoprotolysis of water very accurately. Characteristics and expressiveness of the three formalisms are discussed and illustrated

The PRINTS database: a fine-grained protein sequence annotation and analysis resource—its status in 2012

The PRINTS database, now in its 21st year, houses a collection of diagnostic protein family ‘fingerprints’. Fingerprints are groups of conserved motifs, evident in multiple sequence alignments, whose unique inter-relationships provide distinctive signatures for particular protein families and structural/functional domains. As such, they may be used to assign uncharacterized sequences to known families, and hence to infer tentative functional, structural and/or evolutionary relationships. The February 2012 release (version 42.0) includes 2156 fingerprints, encoding 12 444 individual motifs, covering a range of globular and membrane proteins, modular polypeptides and so on. Here, we report the current status of the database, and introduce a number of recent developments that help both to render a variety of our annotation and analysis tools easier to use and to make them more widely available

Immobilized fibrinogen activates human platelets through GPVI

GPVI, a major platelet activation receptor for collagen and fibrin, is considered as a particularly promising safe antithrombotic target. In this study, we show that human GPVI signals upon platelet adhesion to fibrinogen. Full spreading of human platelets on fibrinogen is abolished in platelets from GPVI-deficient patients suggesting that fibrinogen activates platelets through GPVI. While mouse platelets fail to spread on fibrinogen, human-GPVI-transgenic mouse platelets show full spreading and increased Ca2+ signalling through the tyrosine kinase Syk. Direct binding of fibrinogen to human GPVI was shown by surface plasmon resonance and by increased adhesion of human GPVI-transfected Rbl-2H3 cells to fibrinogen relative to mock-transfected cells. Blockade of human GPVI with the Fab of the monoclonal antibody 9O12 impairs platelet aggregation on preformed platelet aggregates in flowing blood independent of collagen and fibrin exposure. These results demonstrate that human GPVI binds to immobilized fibrinogen and show that this contributes to platelet spreading and platelet aggregation under flow

First demonstration of 3D optical readout of a TPC using a single photon sensitive Timepix3 based camera

The ARIADNE project is developing innovative optical readout technologies for two-phase liquid Argon time projection chambers (LArTPCs). Optical readout presents an exciting alternative to the current paradigm of charge readout. Optical readout is simple, scalable and cost effective. This paper presents first demonstration of 3D optical readout of TPC, using CF4 gas as a proof of principle. Both cosmic rays and an Americium-241 alpha source have been imaged in 100 mbar CF4. A single-photon sensitive camera was developed by combining a Timepix3 (TPX3) based camera with an image intensifier. When a pixel of TPX3 is hit, a packet containing all information about the hit is produced. This packet contains the x,y coordinates of the pixel, time of arrival (ToA) and time over threshold (ToT) information. The z position of the hit in the TPC is determined by combining drift velocity with ToA information. 3D event reconstruction is performed by combining the pixels x,y location with this calculated z position. Calorimetry is performed using time over threshold, a measure of the intensity of the hit

Comparative branching-time semantics for Markov chains

This paper presents various semantics in the branching-time spectrum of discrete-time and continuous-time Markov chains (DTMCs and CTMCs).\ud Strong and weak bisimulation equivalence and simulation pre-orders are covered and are logically characterised in terms of the temporal logics PCTL (Probabilistic Computation Tree Logic) and CSL (Continuous Stochastic Logic). Apart from presenting various existing branching-time relations in a uniform manner, this paper presents the following new results: (i) strong simulation for CTMCs, (ii) weak simulation for CTMCs and DTMCs, (iii) logical characterizations thereof (including weak bisimulation for DTMCs), (iv) a relation between weak bisimulation and weak simulation equivalence, and (v) various connections between equivalences and pre-orders in the continuous- and discrete-time setting. The results are summarized in a branching-time spectrum for DTMCs and CTMCs elucidating their semantics as well as their relationship

Nebulized Recombinant Tissue Plasminogen Activator (rt-PA) for Acute COVID-19-Induced Respiratory Failure: An Exploratory Proof-of-Concept Trial

Acute lung injury in COVID-19 results in diffuse alveolar damage with disruption of the alveolar-capillary barrier, coagulation activation, alveolar fibrin deposition and pulmonary capillary thrombi. Nebulized recombinant tissue plasminogen activator (rt-PA) has the potential to facilitate localized thrombolysis in the alveolar compartment and improve oxygenation. In this proof-of-concept safety study, adults with COVID-19-induced respiratory failure and a <300 mmHg PaO2/FiO2 (P/F) ratio requiring invasive mechanical ventilation (IMV) or non-invasive respiratory support (NIRS) received nebulized rt-PA in two cohorts (C1 and C2), alongside standard of care, between 23 April–30 July 2020 and 21 January–19 February 2021, respectively. Matched historical controls (MHC; n = 18) were used in C1 to explore efficacy. Safety co-primary endpoints were treatment-related bleeds and <1.0–1.5 g/L fibrinogen reduction. A variable dosing strategy with clinical efficacy endpoint and minimal safety concerns was determined in C1 for use in C2; patients were stratified by ventilation type to receive 40–60 mg rt-PA daily for ≤14 days. Nine patients in C1 (IMV, 6/9; NIRS, 3/9) and 26 in C2 (IMV, 12/26; NIRS, 14/26) received nebulized rt-PA for a mean (SD) of 6.7 (4.6) and 9.1(4.6) days, respectively. Four bleeds (one severe, three mild) in three patients were considered treatment related. There were no significant fibrinogen reductions. Greater improvements in mean P/F ratio from baseline to study end were observed in C1 compared with MHC (C1; 154 to 299 vs. MHC; 154 to 212). In C2, there was no difference in the baseline P/F ratio of NIRS and IMV patients. However, a larger improvement in the P/F ratio occurred in NIRS patients (NIRS; 126 to 240 vs. IMV; 120 to 188) and fewer treatment days were required (NIRS; 7.86 vs. IMV; 10.5). Nebulized rt-PA appears to be well-tolerated, with a trend towards improved oxygenation, particularly in the NIRS group. Randomized clinical trials are required to demonstrate the clinical effect significance and magnitude

A non-coding RNA balancing act: miR-346-induced DNA damage is limited by the long non-coding RNA NORAD in prostate cancer

Background: miR‑346 was identified as an activator of Androgen Receptor (AR) signalling that associates with DNA damage response (DDR)‑linked transcripts in prostate cancer (PC). We sought to delineate the impact of miR‑346 on DNA damage, and its potential as a therapeutic agent. Methods: RNA‑IP, RNA‑seq, RNA‑ISH, DNA fibre assays, in vivo xenograft studies and bioinformatics approaches were used alongside a novel method for amplification‑free, single nucleotide‑resolution genome‑wide mapping of DNA breaks (INDUCE‑seq). Results: miR‑346 induces rapid and extensive DNA damage in PC cells ‑ the first report of microRNA‑induced DNA damage. Mechanistically, this is achieved through transcriptional hyperactivation, R‑loop formation and replication stress, leading to checkpoint activation and cell cycle arrest. miR‑346 also interacts with genome‑protective lncRNA NORAD to disrupt its interaction with PUM2, leading to PUM2 stabilisation and its increased turnover of DNA damage response (DDR) transcripts. Confirming clinical relevance, NORAD expression and activity strongly correlate with poor PC clinical outcomes and increased DDR in biopsy RNA‑seq studies. In contrast, miR‑346 is associated with improved PC survival. INDUCE‑seq reveals that miR‑346‑induced DSBs occur preferentially at binding sites of the most highly‑transcriptionally active transcription factors in PC cells, including c‑Myc, FOXA1, HOXB13, NKX3.1, and importantly, AR, resulting in target transcript downregulation. Further, RNA‑seq reveals widespread miR‑346 and shNORAD dysregulation of DNA damage, replication and cell cycle processes. NORAD drives target‑directed miR decay (TDMD) of miR‑346 as a novel genome protection mechanism: NORAD silencing increases mature miR‑346 levels by several thousand‑fold, and WT but not TDMD‑mutant NORAD rescues miR‑346‑induced DNA damage. Importantly, miR‑346 sensitises PC cells to DNA‑damaging drugs including PARP inhibitor and chemotherapy, and induces tumour regression as a monotherapy in vivo, indicating that targeting miR‑346:NORAD balance is a valid therapeutic strategy

Post-Covid-19 Irritable Bowel Syndrome

Objectives The long-term consequences of COVID-19 infection on the gastrointestinal tract remain unclear. Here, we aimed to evaluate the prevalence of gastrointestinal symptoms and post-COVID-19 disorders of gut-brain interaction after hospitalisation for SARS-CoV-2 infection. Design GI-COVID-19 is a prospective, multicentre, controlled study. Patients with and without COVID-19 diagnosis were evaluated on hospital admission and after 1, 6 and 12 months post hospitalisation. Gastrointestinal symptoms, anxiety and depression were assessed using validated questionnaires. Results The study included 2183 hospitalised patients. The primary analysis included a total of 883 patients (614 patients with COVID-19 and 269 controls) due to the exclusion of patients with pre-existing gastrointestinal symptoms and/or surgery. At enrolment, gastrointestinal symptoms were more frequent among patients with COVID-19 than in the control group (59.3% vs 39.7%, p < 0.001). At the 12-month follow-up, constipation and hard stools were significantly more prevalent in controls than in patients with COVID-19 (16% vs 9.6%, p=0.019 and 17.7% vs 10.9%, p=0.011, respectively). Compared with controls, patients with COVID-19 reported higher rates of irritable bowel syndrome (IBS) according to Rome IV criteria: 0.5% versus 3.2%, p=0.045. Factors significantly associated with IBS diagnosis included history of allergies, chronic intake of proton pump inhibitors and presence of dyspnoea. At the 6-month follow-up, the rate of patients with COVID-19 fulfilling the criteria for depression was higher than among controls. Conclusion Compared with controls, hospitalised patients with COVID-19 had fewer problems of constipation and hard stools at 12 months after acute infection. Patients with COVID-19 had significantly higher rates of IBS than controls

Plasticity of the Muscle Stem Cell Microenvironment

Satellite cells (SCs) are adult muscle stem cells capable of repairing damaged and creating new muscle tissue throughout life. Their functionality is tightly controlled by a microenvironment composed of a wide variety of factors, such as numerous secreted molecules and different cell types, including blood vessels, oxygen, hormones, motor neurons, immune cells, cytokines, fibroblasts, growth factors, myofibers, myofiber metabolism, the extracellular matrix and tissue stiffness. This complex niche controls SC biology-quiescence, activation, proliferation, differentiation or renewal and return to quiescence. In this review, we attempt to give a brief overview of the most important players in the niche and their mutual interaction with SCs. We address the importance of the niche to SC behavior under physiological and pathological conditions, and finally survey the significance of an artificial niche both for basic and translational research purposes