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Unlocking the secrets of SARS-CoV-2 nsp3 by combining experiments with AlphaFold2 domain prediction
No full textNonstructural protein 3 (nsp3) is crucial for SARS-CoV-2 infection. It is the largest protein of the virus with roughly 2000 residues, and a major drug target. However, because of its size, disordered regions, and transmembrane domains, the atomic structure of the whole protein has not yet been established. Only 10 out of its 16 domains were individually determined in experiments. Here, we demonstrate how structural bioinformatics, AI-based fold prediction, and traditional experiments complement each other and can shed light on the makeup of this important protein, both in SARS-CoV-2 and in related viruses. Our method can be generalized for other multidomain proteins. Our prediction-based approach reveals a previously undescribed folded domain, which we could confirm experimentally. Our research also suggests a potential function of the domain Y1: this domain may be involved in the assembly of nsp3, nsp4, and nsp6 into the hexameric pore, which was discovered by electron tomography and exports RNA into the cytosol. The Y1 hexamer, however, could not be expressed on its own. We revise domain segmentation and nomenclature of nsp3 domains
ShineGAL4 drivers for tissue and cell-type specific optogenetics in Drosophila.
No full textAn optogenetic split-GAL4 system, ShineGAL4, allows genes to be manipulated with unprecedented spatiotemporal precision. Here, we convert a panel of 14 GAL4 drivers widely used in Drosophila research into their ShineGAL4 counterparts. Homology assisted CRISPR knock-in (HACK) is used to replace GAL4 with the GAL4 DNA binding domain fused to a Magnet photoswitch. We show that the resulting ShineGAL4 drivers enable gene expression to be rapidly induced by light specifically in fat body, muscles, enterocytes, oenocytes, Malpighian tubules, neurons, neuroblast lineages, glial subtypes or in all glia. We also develop an optogenetic cassette for photoactivation of GAL4 in 'silent' FLP-out clones. This panel of optogenetic tools will enable precise spatiotemporal control of gene expression in a wide range of different Drosophila tissues and cell-types
A functional overlap between actively transcribed genes and chromatin insulator elements.
No full textThe mammalian genome is organised into large topologically associating domains (TADs) and smaller sub-TADs or enhancer-promoter loops, which may contribute to the regulation of gene expression. These dynamic structures arise, at least partly, via cohesin-mediated loop extrusion delimited by insulator elements. By studying the structure and function of the alpha-globin locus during erythroid differentiation, we have previously shown that the juxtaposition of the enhancers and promoters during this process partly depends on cohesin-mediated loop extrusion, which appears to be delimited by 12 largely convergently orientated CTCF boundary elements. To define the downstream boundary of the sub-TAD, we removed four CTCF sites in informative combinations. This showed that rather than CTCF insulators, it is the transcriptionally active alpha-globin gene that defines the downstream boundary of the sub-TAD. Further, insertion of actively transcribed fragments of the α-globin gene between the enhancers and native genes leads to a reduction in native α-globin expression and accumulation of cohesin at the insertion site. This highlights an overlap in the functional role of the fundamental elements of the genome
Triply responsive control of ion transport with an artificial channel creates a switchable AND to OR logic gate.
No full textIon channels are ubiquitous in Nature, performing complex and essential tasks in our bodies. Synthetic chemists have begun to understand how to form artificial channels, which hold great promise as components in artificial cells, and in synthetic biology more widely. Future generations of these systems will be critical in the treatment of channelopathies. Despite advances in synthetic ion transporters, the current generation cannot approach the selectivity and controllability of the biological ion channels they seek to emulate, and multimodal control over activity remains hard to achieve. Herein, we present a synthetic ion channel whose activity can be controlled by three orthogonal stimuli (light, pH, guest/ligand), based on a pillar[5]arene functionalized with photoswitchable ortho-tetrafluoroazobenzene moieties. We demonstrate excellent control over ten photoswitches (E-to-Z 82%, Z-to-E quant.). We show that the most active Z-isomer forms dimeric ion channels in membranes. Single molecule planar bilayer conductance studies show high and low conductance states dependent on irradiation wavelength. We demonstrate that this activity can be modulated over 170-fold by controlling pH, irradiation, and guest addition. We use these three stimuli to design a pH switchable AND to OR logic gate system, creating a powerful addition to the canon of synthetic ion channels
Pigmented birthmarks and spinal neurofibromas in KRAS mosaicism—not to be confused with NF1
No full textWe report a child presenting with pigmentary skin lesions and spinal neurofibromas who was diagnosed molecularly with KRAS mosaicism. We review the previous literature of two cases of congenital skin lesions and neurofibromas and spinal nerve root hypertrophy caused by KRAS variants and highlight this presentation as an important differential diagnosis for neurofibromatosis
Apicortin defines the Plasmodium apical conoid body but is dispensable for the parasite life cycle.
No full textApicomplexan parasites such as Plasmodium spp. and Toxoplasma gondii possess unique tubulin-based structures, including subpellicular microtubules and apical polar rings, which are essential for parasite motility, host cell invasion, and replication. Apicortin, a microtubule-associated protein, contains a doublecortin (DC) domain and a partial tubulin polymerization-promoting protein (TPPP) domain, both implicated in microtubule binding and stabilization. How tubulin-based structures are maintained is poorly understood, but it may involve Apicortin, so far found only in apicomplexans and the placozoan Trichoplax adhaerens Here, we investigated the location and function of Apicortin in Plasmodium berghei Live-cell imaging of a transgenic parasite line expressing GFP-tagged Apicortin showed its location at the apical end of invasive parasite stages within the mosquito vector. Super-resolution and expansion microscopy revealed that Apicortin forms a distinct ringlike structure in the apical complex region at the apical end. However, deletion of the Apicortin gene had no effect on parasite development, indicating that this protein is not essential. This suggests that there may be redundancy or compensatory functions in the mechanisms that stabilize the apical complex
Autologous human iPSC-derived alveolus-on-chip reveals early pathological events of Mycobacterium tuberculosis infection.
No full textImmunocompetent and experimentally accessible alveolar systems to study human respiratory diseases are lacking. Here, we developed a single-donor human induced pluripotent stem cell-derived lung-on-chip (iLoC) containing type II and I alveolar epithelial cells, vascular endothelial cells, and macrophages in a microfluidic device that mimic lung three-dimensional mechanical stretching and air-liquid interface. Imaging and single-cell RNA sequencing analysis revealed that the iLoC recapitulated cellular profiles present in the human distal lung. Infection of the iLoC with the human pathogen Mycobacterium tuberculosis (Mtb) showed that both macrophages and epithelial cells were infected but not permissive to bacterial replication. Stochastically, large macrophage clusters containing necrotic macrophages supporting Mtb replication were observed. A genetically engineered autophagy-deficient iLoC revealed that after Mtb infection, macrophage necrosis was higher upon ATG14 deficiency without bacterial replication. Together, we report an autologous, genetically tractable human alveolar model to study lung diseases and therapies
Influenza surveillance in the Central African Republic from 2015 to 2018 to inform vaccination and treatment strategies.
No full textBACKGROUND: Surveillance of influenza infections and virus characterisation are essential to guide prevention strategies. In the Central African Republic (CAR), data on influenza viruses are patchy and poorly documented. OBJECTIVE: To study the clinical, seasonal, genetic and phenotypic characteristics of influenza viruses circulating in the CAR population. MATERIALS AND METHODS: From January 2015 to December 2018, the presence of influenza A and B viruses in patients presenting with influenza-like illness (ILI) symptoms or severe acute respiratory infections (SARI) was investigated by RT-qPCR. Influenza genetic diversity was evaluated by phylogenetic analyses, and antigenic properties were investigated by haemagglutination inhibition assays, whereas reduced susceptibility to neuraminidase inhibitors was assessed through the presence of known genetic markers and neuraminidase assay. The relationship between patients' clinical characteristics and infection status was investigated using statistical analyses. RESULTS: Over the surveillance period influenza viruses were detected in 9.7% of samples (n = 6134), with the highest intensity of circulation occurring in 2016 (15.8%), attributed mainly to A(H3N2). Periods of increased influenza transmission (June to October) generally coincided with rainy seasons; however, variations in terms of monthly distribution of cases between years were evident. Hospitalisation rates (SARI) were most frequent in infants (0-11 months, 37.9%) and young children (1-4 years, 24.8%), whereas influenza prevalences were highest in the 15-49 (12.0%) and ≥ 50 (15.2%) years old categories. A new A(H1N1)pdm09 6B.1 hemagglutinin subgroup characterised by amino acid substitutions S84N, S162N and I216T was detected in 2015, with associated antigenic drift, and subsequently, two of these viruses showed highly reduced inhibition by oseltamivir. CONCLUSION: This study showcases the value of local influenza sentinel networks to specifically inform vaccination strategies and highlights the need for improved strain characterisation in tropical regions
The effects of multimedia education tools on patients' health knowledge.
No full textBACKGROUND: Delivering health education and information to people and communities to increase their knowledge is an essential part of shared healthcare decision making. However, not all written materials are easily understood and thus multimedia interventions may be an effective alternative. AIMS: This systematic review aimed to identify quantitative evidence related to the effectiveness of multimedia education tools versus standard of care or written educational materials on adult patients' knowledge and understanding of health information. METHODS: The authors conducted a systematic review following JBI methodological guidance. The Medline, Embase and Emcare databases were searched in February 2024. Some 15 publications were identified for inclusion, 10 with animations and five with videos of people. RESULTS: The results were synthesised into three topics: (i) the effectiveness of multimedia interventions; (ii) the components of the multimedia interventions; and (iii) health literacy and language. Overall, data from 73.3% (n=11) of the studies indicated that a multimedia approach to information delivery could lead to an increase in knowledge and understanding. CONCLUSIONS: Multimedia interventions can be an effective tool for delivering health information, but special attention is required to health literacy, language barriers and the needs of populations from low- or middle-income countries, particularly where the ability to speak or write English may be limited. IMPLICATIONS FOR PRACTICE: Community nurses can develop and use multimedia education tools during home visits, group health education sessions, or supporting self-care activities for people with long-term conditions for improved patient outcomes
Quantitative Assessment of Clearing Protocol Efficacy for Multi-Dimensional Microscopy
No full textLight scattering properties of biological tissue often limit imaging depth and resolution when acquiring images; optical tissue clearing significantly reduces light scattering, boosting the signal-to-noise ratio and providing crucial spatial information for deep imaging. Here, we present a user-friendly, open-source analytical pipeline for assessing tissue clearing efficacy, and illustrate our pipeline with a simple proof of concept. This pipeline provides a standard framework for the quantitative analysis of clearing protocols, allowing life science researchers to select an optimal protocol for their specific needs.Poster presented as part of the Crick BioImage Analysis Symposium 2025.Permission has been given by authors to upload to Crick Figshare. Copyright remains with the original authors.</p