Miracle-dz/UniFAR-FTUs huggingface dataset:
Resolving the dynamics of photosynthetically produced ROS by high-resolution monitoring of chloroplastic EGSH in Arabidopsis

Plants are naturally subjected to fluctuations in light intensity, causing unbalanced photosynthetic electron fluxes and overproduction of reactive oxygen species (ROS). While high rates of ROS production are harmful, moderate levels play a signaling role, coordinating photosynthetic activity and downstream metabolism. Here, we explore the dynamics of light-dependent oxidant production by high-temporal-resolution monitoring of chloroplastic glutathione redox potential (chl-EGSH) using chloroplast-targeted-roGFP2-expressing Arabidopsis lines, over several days, under dynamic environmental conditions and in correlation with PSII operating efficiency. Peaks in chl-EGSH oxidation during light-darkness transitions, when light harvesting is not balanced with downstream metabolism, were observed. Increasing light intensities triggered a binary oxidation response, with a threshold around the light saturating point, pointing for two regulated oxidative states of the chl-EGSH. These patterns were not affected in npq1 plants which are impaired in non-photochemical quenching. Frequency-dependent oscillations between the two oxidation states were observed under fluctuating light in WT and npq1 plants, but not in pgr5 plants, suggesting a role for PSI photoinhibition in regulation of oxidant production. Remarkably, pgr5 plants showed an increase in chl-EGSH oxidation during the nights following light stresses, linking between day photoinhibition and night redox metabolism. This work provides a comprehensive view on the balance between photosynthesis-dependent ROS production and antioxidant activity during light acclimation.
Human DND1-RRM2 forms a non-canonical domain swapped dimer

Human DND1 (Dead end protein homolog1) is an RNA binding protein. DND1 plays pivotal role in animal development and has been implicated in cancer. DND1 consists of two RNA recognition motifs (RRMs) in tandem and a double stranded RNA binding domain at the C terminal separated by 40 residues flexible linker. The conserved RNP site in the RRM1 domain helps in specific RNA recognition while the RNP sites in RRM2 are not well conserved. DND1 has been reported to be involved in inhibition of microRNA access to target mRNA and it also associate with CCR4-NOT complex that targets mRNA. In order to understand this intriguing contrasting molecular function, we have determined the 2.3 Å resolution crystal structure of the human DND1 RRM2 domain. The structure revealed an interesting non-canonical RRM fold that is maintained by the formation of a domain swapped dimer between β1 and β4 strands across two chains. The domain swapping is attributed by a hinge loop between α2 and β4 that helps in mediating a domain swap forming anti-parallel β sheets. We have delineated the structural basis of stable dimer formation using the residue level dynamics of protein explored by NMR spectroscopy and MD simulations. Our structural and dynamics studies demonstrate the molecular basis for the dimerization of the RRM2 domain and shed light on the possibility for this motif for interaction with other proteins which helps in transcription regulation. Graphical Abstract Highlights First report of a domain swapped dimer formation by an RRM identified through crystal structure determination of the human DND1 RRM2 domain. RRM2 exhibit domain swapped dimerization attributed by hinge loop and disulfide bond formation. Dimer formation is under redox regulation. Major determinants of swapping were identified. DND1 RRM2 is not involved in RNA recognition.
Identification of m6A residues at single-nucleotide resolution using eCLIP and an accessible custom analysis pipeline

Methylation at the N6 position of adenosine (m6A) is one of the most abundant RNA modifications found in eukaryotes, however accurate detection of specific m6A nucleotides within transcripts has been historically challenging due to m6A and unmodified adenosine having virtually indistinguishable chemical properties. While previous strategies such as methyl-RNA immunoprecipitation and sequencing (MeRIP-Seq) have relied on m6A-specific antibodies to isolate RNA fragments containing the modification, these methods do not allow for precise identification of individual m6A residues. More recently, modified cross-linking and immunoprecipitation (CLIP) based approaches that rely on inducing specific mutations during reverse transcription via UV crosslinking of the anti-m6A antibody to methylated RNA have been employed to overcome this limitation. However, the most utilized version of this approach, miCLIP, can be technically challenging to use for achieving high-complexity libraries. Here we present an improved methodology that yields high library complexity and allows for the straightforward identification of individual m6A residues with reliable confidence metrics. Based on enhanced CLIP (eCLIP), our m6A-eCLIP (meCLIP) approach couples the improvements of eCLIP with the inclusion of an input sample and an easy-to-use computational pipeline to allow for precise calling of m6A sites at true single nucleotide resolution. As the effort to accurately identify m6As in an efficient and straightforward way intensifies, this method is a valuable tool for investigators interested in unraveling the m6A epitranscriptome.
Single-molecule study of the dynamic organization of Vascular Endothelial Growth Factor Receptor 2 on the endothelial cell surface

There is growing evidence that receptors exhibit dynamic, nanoscale organization on the cell surface, which plays an important role in their signaling function. Vascular Endothelial Growth Factor (VEGF) Receptor 2 (VEGFR-2) is a critical Receptor Tyrosine Kinase in endothelial cells, where it initiates signaling cascades that promote angiogenesis. Little is known about the nanoscale organization of VEGFR-2 on the endothelial cell surface and how it influences VEGF binding and subsequent signaling. To fill this gap in our knowledge, we performed live-cell single-molecule imaging of endogenous VEGFR-2 in primary endothelial cells, followed by a pipeline of automated image analysis and multiscale data analysis to quantitatively characterize and extract relationships between VEGFR-2 mobility, interactions, phosphorylation and ligand binding. We found that surface VEGFR-2 in unstimulated cells can be mobile or immobile/confined, both of which can be monomeric or non-monomeric, with a complex interplay between mobility and inter-receptor interactions. The immobile/confined mode depends on VEGFR-2 phosphorylation, even in unstimulated cells, and is enriched further in the presence of VEGF. Co-imaging of VEGFR-2 and labeled VEGF allowed us to test two models of VEGFR-2 activation, namely VEGF-induced dimerization versus VEGF binding to pre-existing dimers. We found evidence for both models, with stronger evidence for binding and stabilizing pre-existing dimers. Our study is the first to characterize the dynamic nanoscale organization of VEGFR-2 in its native plasma membrane environment; further investigations of the mechanisms that regulate it and its role in VEGFR-2 signaling will be critical for a more complete understanding of angiogenic signaling. Significance Statement Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2) is a critical receptor on the surface of endothelial cells (which line the blood vessels). It promotes the formation of new blood vessels in response to its ligand VEGF. Currently we know little about the organization and dynamics of this receptor on the cell surface, with multiple proposed models regarding its activation mechanism by VEGF. To fill this knowledge gap, we conducted a quantitative single-molecule imaging study of endogenous VEGFR-2 in primary endothelial cells, revealing the dynamic nanoscale organization of VEGFR-2 in its native environment. Our study also addresses the interplay between this organization, ligand binding and receptor activation, shedding light on the cell biophysical properties of this developmentally important receptor.
Generation of excitatory and inhibitory neurons from common progenitors via Notch signaling in the cerebellum

Brain neurons arise from relatively few progenitors capable of giving rise to an enormous diversity of neuronal types. Nonetheless, a cardinal feature of mammalian brain neurogenesis in both the cortex and the cerebellum is that excitatory neurons and inhibitory neurons derive from separate, spatially segregated, progenitors. Whether bi-potential progenitors with an intrinsic capacity to generate both excitatory and inhibitory lineages exist and how such a fate decision may be regulated is unknown. Using cerebellar development as a model, we discover that individual embryonic cerebellar progenitors give rise to both inhibitory and excitatory lineages. We find that gradations of Notch activity levels determine the fates of the progenitors and their daughters. Daughters with the highest levels of Notch activity retain the progenitor fate. Daughters with intermediate levels of Notch activity become fate restricted to generate inhibitory neurons, while daughters with very low levels of Notch signaling adopt the excitatory fate. Therefore, Notch mediated binary cell fate choice is a mechanism for regulating the ratio of excitatory to inhibitory neurons from common progenitors. Graphical summary
Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection (IMPACT) System for Rapid Viral DNA Sensing

A fully Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection (IMPACT) system is developed for viral DNA detection. This powerful system is patterned with high-aspect ratio micropillars to enhance reporter probe binding. After surface modification and probe immobilization, CRISPR Cas12a/crRNA complex is injected into the fully enclosed system. With the presence of double-stranded DNA target, the CRISPR enzyme is activated and non-specifically cleaves the ssDNA reporters initially immobilized on the micropillars. This collateral cleavage releases fluorescence dyes into the assay, and the intensity is linearly proportional to the target DNA concentration ranging from 0.1 to 10 nM. Importantly, this system does not rely on traditional dye-quencher labeled probe thus eliminating the fluorescence background presented in the assay. Furthermore, our one-step detection protocol is performed at isothermal conditions (37°C) without using complicated and time-consuming off-chip probe hybridization and denaturation. This miniaturized and fully packed IMPACT chip demonstrates rapid, sensitive, and simple nucleic acid detection and is an ideal candidate for the next generation molecular diagnostic platform for point-of-care (POC) applications, responding to emerging and deadly pathogen outbreaks.
Sox2 controls neural stem cell self-renewal through a Fos-centered gene regulatory network

The Sox2 transcription factor is necessary for the long-term self-renewal of neural stem cells (NSC). Its mechanism of action is still poorly defined. To identify molecules regulated by Sox2, and acting in mouse NSC maintenance, we transduced, into Sox2-deleted NSC, genes whose expression is strongly downregulated following Sox2 loss (Fos, Jun, Egr2), individually or in combination. Fos alone rescued long-term proliferation, as shown by in vitro cell growth and clonal analysis. Further, pharmacological inhibition of the FOS/JUN AP1 complex binding to its targets, decreased cell proliferation and expression of the putative target Suppressor of cytokine signaling 3 (Socs3). Additionally, Fos requirement for efficient long-term proliferation was demonstrated by the reduction of NSC clones capable of long-term expansion following CRISPR/Cas9-mediated Fos inactivation. Previous work showed that the Socs3 gene is strongly downregulated following Sox2 deletion, and its reexpression by lentiviral transduction rescues long-term NSC proliferation. Fos appears to be an upstream regulator of Socs3, possibly together with Jun and Egr2; indeed, Sox2 reexpression in Sox2-deleted NSC progressively activates both Fos and Socs3 expression; in turn, Fos transduction activates Socs3 expression. Based on available SOX2 ChIPseq and ChIA-PET data, we propose a model whereby Sox2 is a direct activator of both Socs3 and Fos, as well as possibly Jun and Egr2; further, we provide direct evidence for FOS and JUN binding on Socs3 promoter, suggesting direct transcriptional regulation. These results provide the basis for developing a model of a network of interactions, regulating critical effectors of NSC proliferation and long-term maintenance. Significance statement Proliferation and maintenance of NSC are essential during normal brain development, and, postnatally, for the maintenance of hippocampal function and memory until advanced age. Little is known about the molecular mechanisms that maintain the critical aspects of NSC biology (quiescence and proliferation) in postnatal age. Our work provides a methodology, transduction of genes deregulated following Sox2 deletion, that allows to test many candidate genes for their ability to sustain NSC proliferation. In principle, this may have interesting implications for identifying targets for pharmacological manipulations.
Variability in mitochondrial import, mitochondrial health and mtDNA copy number using Type II and Type V CRISPR effectors

Current methodologies for targeting the mitochondrial genome for basic research and/or therapeutic strategy development in mitochondrial diseases are restricted by practical limitations and technical inflexibility. The development of a functional molecular toolbox for CRISPR-mediated mitochondrial genome editing is therefore desirable, as this could enable precise targeting of mtDNA haplotypes using the precision and tuneability of CRISPR enzymes; however, published reports of “MitoCRISPR” systems have, to date, lacked reproducibility and independent corroboration. Here, we have explored the requirements for a functional MitoCRISPR system in human cells by engineering several versions of CRISPR nucleases, including the use of alternative mitochondrial protein targeting sequences and smaller paralogues, and the application of gRNA modifications that reportedly induce mitochondrial import. We demonstrate varied mitochondrial targeting efficiencies and influences on mitochondrial dynamics/function of different CRISPR nucleases, with Lachnospiraceae bacterium ND2006 (Lb) Cas12a being better targeted and tolerated than Cas9 variants. We also provide evidence of Cas9 gRNA association with mitochondria in HeLa cells and isolated yeast mitochondria, even in the absence of a targeting RNA aptamer. Finally, we present evidence linking mitochondrial-targeted LbCas12a/crRNA with increased mtDNA copy number dependent upon DNA binding and cleavage activity. We discuss reproducibility issues and the future steps necessary if MitoCRISPR is to be realised.
Proliferation and Survival of T-cell Acute Lymphoblastic Leukaemia Depends on mTOR-regulated Glutamine Uptake and EAAT1-dependent Conversion of Glutamine to Aspartate and Nucleotides

T-cell acute lymphoblastic leukaemia (T-ALL) is a cancer of the immune system. Approximately 20% of paediatric and 50% of adult T-ALL patients relapse and die from the disease. To improve patient outcome new drugs are needed. With the aim to identify new therapeutic targets, we integrated transcriptomics and metabolomics data, including live-cell NMR-spectroscopy, of cell lines and patient samples. We found that T-ALL cells have limited energy availability, resulting in active AMPK-signalling and reduced autophagy. Despite this, mTOR kinase remains active and essential for glutamine-uptake that fuels rapid proliferation. Glutamine fuels aspartate synthesis and together they supply three nitrogen atoms in purines and all atoms but one carbon in pyrimidine rings. We show that EAAT1, a glutamate-aspartate transporter normally only expressed in the CNS, is crucial for glutamine conversion to nucleotides and that T-ALL cell proliferation depends on EAAT1 function, identifying it as a target for T-ALL treatment. Finally, we performed anin silicoscreen and identified a novel EAAT1-specific allosteric inhibitor.
The microRNA, miR-133b, functions to slow Duchenne muscular dystrophy pathogenesis

Duchenne muscular dystrophy (DMD) is characterized by progressive degeneration of skeletal muscles. To date, there are no treatments available to slow or prevent the disease. Hence, it remains essential to identify molecular factors that promote muscle biogenesis since they could serve as therapeutic targets for treating DMD. While the muscle enriched microRNA, miR-133b, has been implicated in the biogenesis of muscle fibers, its role in DMD remains unknown. To assess the role of miR-133b in DMD-affected skeletal muscles, we genetically ablated miR-133b in the mdx mouse model of DMD. In the absence of miR-133b, the tibialis anterior muscle of juvenile and adult mdx mice is populated by small muscle fibers with centralized nuclei, exhibits increased fibrosis, and thickened interstitial space. Additional analysis revealed that loss of miR-133b exacerbates DMD-pathogenesis partly by altering the number of satellite cells and levels of protein-encoding genes, including previously identified miR-133b targets as well as genes involved in cell proliferation and fibrosis. Altogether, our data demonstrate that skeletal muscles utilize miR-133b to mitigate the deleterious effects of DMD.
Engineered IL-10 variants elicit potent immuno-modulatory activities at therapeutic low ligand doses

Interleukin-10 is a dimeric cytokine with both immune-suppressive and immune-stimulatory activities. Despite its immuno-modulatory potential, IL-10-based therapies have shown only marginal benefits in the clinic. Here we have explored whether the stability of the IL-10-receptor complex contributes to IL-10 immuno-modulatory potency. For that, we have generated an IL-10 mutant with greatly enhanced affinity for its IL-10Rβ receptor via yeast surface display. The affinity enhanced IL-10 variants recruited IL-10Rβ more efficiently into active cell surface signaling complexes than the wild-type cytokine and triggered more potent STAT1 and STAT3 activation in human monocytes and CD8 T cells. This in turn led to more robust induction of IL-10-mediated gene expression programs at a wide range of ligand concentrations in both human cell subsets. IL-10 regulated genes are involved in monocyte energy homeostasis, migration and trafficking, and genes involved in CD8 T cell exhaustion. Interestingly, at non-saturating doses, IL-10 lost key components of its gene-expression program, which may explain its lack of efficacy in clinical settings. Remarkably, our engineered IL-10 variant exhibited a more robust bioactivity profile than IL-10 wt at all the doses tested in monocytes and CD8 T cells. Moreover, CAR-modified T cells expanded with the engineered IL-10 variant displayed superior cytolytic activity than those expanded with IL-10 wt. Our study provides unique insights into how IL-10-receptor complex stability fine-tunes IL-10 biology, and opens new opportunities to revitalize failed IL-10 therapies.
Correlations of genotype with climate parameters suggest Caenorhabditis elegans niche preferences

Species inhabit a variety of environmental niches, and the adaptation to a particular niche is often controlled by genetic factors, including gene-by-environment interactions. The genes that vary in order to regulate the ability to colonize a niche are often difficult to identify, especially in the context of complex ecological systems and in experimentally uncontrolled natural environments. Quantitative genetic approaches provide an opportunity to investigate correlations between genetic factors and environmental parameters that might define a niche. Previously, we have shown how a collection of 208 whole-genome sequenced wild Caenorhabditis elegans can facilitate association mapping approaches. To correlate climate parameters with the variation found in this collection of wild strains, we used geographic data to exhaustively curate daily weather measurements in short-term (three month), middle-term (one year), and long-term (three year) durations surrounding the data of strain isolation. These climate parameters were then used as quantitative traits in the mapping approaches. We identified 10 QTL underlying variation in three traits: elevation, relative humidity, and average temperature. We then performed statistical analyses to further narrow the genomic interval of interest to identify gene candidates with variants potentially underlying phenotypic differences. Additionally, we performed two-strain competition assays at high and low temperatures to validate a QTL for temperature preference and found suggestive evidence that genotypes might be adapted to particular temperatures. 100-word summary for G3 Quantitative genetic approaches provide an opportunity to investigate correlations between genetic factors and environmental parameters that might define a niche, but these genes are difficult to identify, especially in the context of complex ecological systems. Here, we used a collection of 152 sequenced wild Caenorhabditis elegans to correlate climate parameters with the variation found in this collection of wild strains. We identified 10 QTL in five traits, including elevation, relative humidity, and temperature. Additionally, we performed competition assays to validate a QTL for temperature preference and found suggestive evidence that genotypes might be adapted to particular temperatures.
Embodied virtual reality for the study of real-world motor learning

Motor-learning literature focuses on simple laboratory-tasks due to their controlled manner and the ease to apply manipulations to induce learning and adaptation. Recently, we introduced a billiards paradigm and demonstrated the feasibility of real-world-neuroscience using wearables for naturalistic full-body motion-tracking and mobile-brain-imaging. Here we developed an embodied virtual-reality (VR) environment to our real-world billiards paradigm, which allows to control the visual feedback for this complex real-world task, while maintaining sense of embodiment. The setup was validated by comparing real-world ball trajectories with the trajectories of the virtual balls, calculated by the physics engine. We then ran our learning protocol in the embodied VR. Subjects played billiard shots when they held the physical cue and hit a physical ball on the table while seeing it all in VR. We found comparable learning trends in the embodied VR to those we previously reported in the physical real-world task. Embodied VR can be used for learning real-world tasks in a highly controlled environment which enables applying visual manipulations, common in laboratory-tasks and rehabilitation, to a real-world full-body task. Embodied VR enables to manipulate feedback and apply perturbations to isolate and assess interactions between specific motor-learning components, thus enabling addressing the current questions of motor-learning in real-world tasks. Such a setup can be used for rehabilitation, where VR is gaining popularity but the transfer to the real-world is currently limited, presumably, due to the lack of embodiment.