bluuebunny/crossref_metadata_2025_split huggingface dataset:
The CDK12 inhibitor SR-4835 functions as a molecular glue that promotes cyclin K degradation in melanoma
Authors: Houles Thibault , Boucher Jonathan , Lavoie Geneviève , MacLeod Graham , Lin Sichun , Angers Stephane , Roux Philippe P. 
Published: May 2023

SUMMARYCDK12 is a transcriptional cyclin-dependent kinase (CDK) that interacts with cyclin K to regulate different aspects of gene expression. The CDK12-cyclin K complex phosphorylates several substrates, including RNA polymerase II (Pol II), and thereby regulates transcription elongation, RNA splicing, as well as cleavage and polyadenylation. Because of its implication in cancer, including breast cancer and melanoma, multiple pharmacological inhibitors of CDK12 have been identified to date, including THZ531 and SR-4835. While both CDK12 inhibitors affect Poll II phosphorylation, we found that SR-4835 uniquely promotes cyclin K degradation via the proteasome. Using loss-of-function genetic screening, we found that SR-4835 cytotoxicity depends on a functional CUL4-RBX1-DDB1 ubiquitin ligase complex. Consistent with this, we show that DDB1 is required for cyclin K degradation, and that SR-4835 promotes DDB1 interaction with the CDK12-cyclin K complex. Docking studies and structure-activity relationship analyses of SR-4835 revealed the importance of the benzimidazole side-chain in molecular glue activity. Together, our results indicate that SR-4835 acts as a molecular glue that recruits the CDK12-cyclin K complex to the CUL4-RBX1-DDB1 ubiquitin ligase complex to target cyclin K for degradation.
Cell specification and functional interactions in the pig blastocyst inferred from single cell transcriptomics
Authors: Dufour Adrien , Kurilo Cyril , Stöckl Jan B. , Laloë Denis , Bailly Yoann , Manceau Patrick , Martins Frédéric , Turhan Ali G. , Ferchaud Stéphane , Pain Bertrand , Fröhlich Thomas , Foissac Sylvain , Artus Jérôme , Acloque Hervé 
Published: May 2023

AbstractThe early embryonic development of the pig comprises a longin uteropre- and peri-implantation development, which dramatically differs from mouse and human. During this pro-tracted peri-implantation period, an intimate dialogue between the embryo and the uterus is established through a complex series of paracrine signals. It leads to concomitant drastic changes in the embryonic morphology and uterine receptivity to implantation. From day 7 after fertilization, the spherical blastocyst elongates as ovoid, tubular and filamentous wich transforms from a 0.5-1 mm diameter sphere to a 1000 mm long filamentous blastocyst at day 16. At the same time, the inner cell mass moves up to the trophoblast as the Rauber’s layer disappears and evolves as an embryonic disc that is directly exposed to molecules present in the uterine fluids. These drastic changes occurring before implantation are driven and coordinated by interactions between embryonic cells and maternal tissues.To better understand the biology of pig embryos before implantation, we generated a large dataset of single-cell RNAseq at different embryonic stages (early and hatched blastocyst, spheroid and ovoid conceptus) and proteomic datasets from corresponding uterine fluids. These data were cleaned, filtered and represent a total of 34,888 cells. We first characterised the embryonic and extra-embryonic cell populations and their evolution, and identified population-specific markers of the three main populations (epiblast, trophectoderm and hypoblast). Our analysis also confirmed known functions and predicted new biological functions associated with these cell populations.We then inferred gene regulatory networks working on modules of gene regulation (regulon) and selected those that were specifically active in each embryonic population. We confirmed the relevance of the identified regulons by a meta-analysis of two other scRNAseq datasets (porcine and human preimplantation embryos). We then linked these regulons to signalling pathways and biological processes. Our results confirm the molecular specificity and functionality of the three main cell populations and identify novel stage-specific subpopulations. In particular, we discovered two previously unknown subpopulations of the trophectoderm, one characterised by the expression of LRP2, which could represent a subpopulation of progenitor cells, and the other, expressing many pro-apoptotic markers, which could correspond to the cells of the Rauber’s layer. We also provide new insights into the biology of these populations, their reciprocal functional interactions and the molecular dialogue established with the maternal uterine environment.
Genetically engineering endothelial niche in human kidney organoids enables multilineage maturation, vascularization and de novo cell types
Authors: Maggiore Joseph C. , LeGraw Ryan , Przepiorski Aneta , Velazquez Jeremy , Chaney Christopher , Streeter Evan , Silva-Barbosa Anne , Franks Jonathan , Hislop Joshua , Hill Alex , Wu Haojia , Pfister Katherine , Howden Sara E. , Watkins Simon C. , Little Melissa , Humphreys Benjamin D. , Watson Alan , Stolz Donna B. , Kiani Samira , Davidson Alan J. , Carroll Thomas J. , Cleaver Ondine , Sims-Lucas Sunder , Ebrahimkhani Mo R. , Hukriede Neil A. 
Published: May 2023

AbstractVascularization plays a critical role in organ maturation and cell type development. Drug discovery, organ mimicry, and ultimately transplantation in a clinical setting thereby hinges on achieving robust vascularization ofin vitroengineered organs. Here, focusing on human kidney organoids, we overcome this hurdle by combining an inducibleETS translocation variant 2(ETV2) human induced pluripotent stem cell (iPSC) line, which directs endothelial fate, with a non-transgenic iPSC line in suspension organoid culture. The resulting human kidney organoids show extensive vascularization by endothelial cells with an identity most closely related to endogenous kidney endothelia. Vascularized organoids also show increased maturation of nephron structures including more mature podocytes with improved marker expression, foot process interdigitation, an associated fenestrated endothelium, and the presence of renin+cells. The creation of an engineered vascular niche capable of improving kidney organoid maturation and cell type complexity is a significant step forward in the path to clinical translation. Furthermore, this approach is orthogonal to native tissue differentiation paths, hence readily adaptable to other organoid systems and thus has the potential for a broad impact on basic and translational organoid studies.Translational StatementDeveloping therapies for patients with kidney diseases relies on a morphologically and physiologically representativein vitromodel. Human kidney organoids are an attractive model to recapitulate kidney physiology, however, they are limited by the absence of a vascular network and mature cell populations. In this work, we have generated a genetically inducible endothelial niche that, when combined with an established kidney organoid protocol, induces the maturation of a robust endothelial cell network, induces a more mature podocyte population, and induces the emergence a functional renin population. This advance significantly increases the clinical relevance of human kidney organoids for etiological studies of kidney disease and future regenerative medicine strategies.Graphical AbstractGenetically engineered endothelial niche induces mature cell populations in human kidney organoids
A GPCR screening in human keratinocytes identifies that the metabolite receptor HCAR3 controls epithelial proliferation, migration, and cellular respiration
Authors: Pilar Pedro M. , Lund Katherine , Kang Sun Woo Sophie , Chen Ting , Stuelten Christina H. , Porat-Shliom Natalie , Iglesias-Bartolome Ramiro 
Published: May 2023

AbstractEpithelial cells in the skin and other tissues rely on signals from their environment to maintain homeostasis and respond to injury, and G protein-coupled receptors (GPCRs) play a critical role in this communication. A better understanding of the GPCRs expressed in epithelial cells will contribute to understanding the relationship between cells and their niche and could lead to developing new therapies to modulate cell fate. This study used human primary keratinocytes as a model to investigate the specific GPCRs regulating epithelial cell proliferation and differentiation. We identified three key receptors, hydroxycarboxylic acid-receptor 3 (HCAR3), leukotriene B4-receptor 1 (LTB4R), and G Protein-Coupled Receptor 137 (GPR137) and found that knockdown of these receptors led to changes in numerous gene networks that are important for maintaining cell identity and promoting proliferation while inhibiting differentiation. Our study also revealed that the metabolite receptor HCAR3 regulates keratinocyte migration and cellular metabolism. Knockdown of HCAR3 led to reduced keratinocyte migration and respiration, which could be attributed to altered metabolite use and aberrant mitochondrial morphology caused by the absence of the receptor. This study contributes to understanding the complex interplay between GPCR signaling and epithelial cell fate decisions.
The role of subicular VIP-expressing interneurons on seizure dynamics in the intrahippocampal kainic acid model of temporal lobe epilepsy
Authors: Rahimi Sadegh , Salami Pariya , Matulewicz Pawel , Schmuck Armin , Bukovac Anneliese , Ramos-Prats Arnau , Tasan Ramon Osman , Drexel Meinrad 
Published: May 2023

AbstractThe subiculum, a key output region of the hippocampus, is increasingly recognized as playing a crucial role in seizure initiation and spread. The subiculum consists of glutamatergic pyramidal cells, which show alterations in intrinsic excitability in the course of epilepsy, and multiple types of GABAergic interneurons, which exhibit varying characteristics in epilepsy. In this study, we aimed to assess the role of the vasoactive intestinal peptide interneurons (VIP-INs) of the ventral subiculum in the pathophysiology of temporal lobe epilepsy. We observed that an anatomically restricted inhibition of VIP-INs of the ventral subiculum was sufficient to reduce seizures in the intrahippocampal kainic acid model of epilepsy, changing the circadian rhythm of seizures, emphasizing the critical role of this small cell population in modulating TLE. As we expected, permanent unilateral or bilateral silencing of VIP-INs of the ventral subiculum in non-epileptic animals did not induce seizures or epileptiform activity. Interestingly, transient activation of VIP-INs of the ventral subiculum was enough to increase the frequency of seizures in the acute seizure model. Our results offer new perspectives on the crucial involvement of VIP-INs of the ventral subiculum in the pathophysiology of TLE. Given the observed predominant disinhibitory role of the VIP-INs input in subicular microcircuits, modifications of this input could be considered in the development of therapeutic strategies to improve seizure control.
Kit Ligand and Kit receptor tyrosine kinase sustain synaptic inhibition of Purkinje Cells
Authors: Zaman Tariq , Vogt Daniel , Prokop Jeremy , Alsabia Qusai Abdulkhaliq , Simms Gabriel , Stafford April , Luikart Bryan W. , Williams Michael R. 
Published: May 2023

SummaryThe cell-type specific expression of ligand/receptor and cell-adhesion molecules is a fundamental mechanism through which neurons regulate connectivity. Here we determine a functional relevance of the long-established mutually exclusive expression of the receptor tyrosine kinase Kit and the trans-membrane protein Kit Ligand by discrete populations of neurons in the mammalian brain. Kit is enriched in molecular layer interneurons (MLIs) of the cerebellar cortex (i.e., stellate and basket cells), while cerebellar Kit Ligand is selectively expressed by a target of their inhibition, Purkinje cells (PCs). Byin vivogenetic manipulation spanning embryonic development through adulthood, we demonstrate that PC Kit Ligand and MLI Kit are required for, and capable of driving changes in, inhibition of PCs. Collectively, these works in mice demonstrate that the Kit Ligand/Kit receptor dyad sustains mammalian central synapse function and suggest a rationale for the affiliation of Kit mutation with neurodevelopmental disorders.Visual Abstract
Adaptation to sorbic acid in low sugar promotes resistance of spoilage yeasts to the preservative
Authors: Harvey Harry J. , Hendry Alex C. , Chirico Marcella , Archer David B. , Avery Simon V. 
Published: May 2023

AbstractThe weak acid sorbic acid is a common preservative used in soft drink beverages to control microbial spoilage. Consumers and industry are increasingly transitioning to low-sugar food formulations, but potential impacts of reduced-sugar on preservative efficacy are barely characterised. In this study, we report enhanced sorbic acid resistance of spoilage yeasts in low-glucose conditions. We had anticipated that low glucose may induce respiratory metabolism, previously shown to be targeted by sorbic acid. However, a shift from respiratory to fermentative metabolism was correlated with the sorbic acid resistance in low glucose. Fermentation-deficient yeast species did not show the low-glucose resistance phenotype. Phenotypes observed for certain yeast deletion strains suggested roles for glucose signalling and repression pathways in the sorbic acid resistance at low glucose. This low- glucose induced sorbic acid resistance was alleviated by supplementing yeast cultures with succinic acid, a metabolic intermediate of respiratory metabolism (and a food-safe additive) that promoted respiration. The results indicate that metabolic adaptation of spoilage yeasts promotes sorbic acid resistance at low glucose, providing new insight into potential spoilage, and preservation, of foodstuffs as both food producers and consumers move towards a reduced-sugar landscape.
Stochastic RNA editing of the Complexin C-terminus within single neurons regulates neurotransmitter release
Authors: Brija Elizabeth A. , Guan Zhuo , Jetti Suresh K. , Littleton J. Troy 
Published: May 2023

AbstractNeurotransmitter release requires assembly of the SNARE complex fusion machinery, with multiple SNARE-binding proteins regulating this process to control when and where synaptic vesicle fusion occurs. Complexin (Cpx) controls spontaneous and evoked neurotransmitter release by modulating SNARE complex zippering. Although the central SNARE-binding helix is essential, post-translational modifications to Cpx’s C-terminal membrane-binding amphipathic helix modulate its activity. Here we demonstrate that RNA editing of the Cpx C-terminus regulates its ability to clamp SNARE-mediated fusion and alters presynaptic output. RNA editing of Cpx within single neurons is stochastic, generating up to eight edit variants that fine-tune neurotransmitter release by changing the subcellular localization and clamping properties of the protein. Similar editing rules for other synaptic genes were observed, indicating stochastic editing at single adenosines and across multiple mRNAs can generate unique synaptic proteomes within the same population of neurons to fine-tune presynaptic output.
Isolation of Biologically Active Extracellular Vesicles-Associated AAVs for Gene Delivery to the Brain by Size Exclusion Chromatography
Authors: Henriques Carina , Lopes Miguel M. , Albuquerque Patrícia , Rufino-Ramos David , Gaspar Laetitia S. , Lobo Diana , Leandro Kevin , Silva Ana Carolina , Baganha Rafael , Duarte Sónia , Maguire Casey A. , Santana Magda , Almeida Luís Pereira de , Nobre Rui Jorge 
Published: May 2023

AbstractExtracellular vesicles-associated adeno-associated viral vectors (EV-AAVs) emerged as a new opportunity for non-invasive gene therapy targeting the central nervous system (CNS). However, in previous reports, only AAV serotypes with known ability to cross the blood-brain barrier (BBB) have been used for EV-AAV production and testing through non-invasive strategies. In this work, we aimed at optimizing a size exclusion chromatography (SEC) protocol for the production and isolation of natural and biologically active brain-targeting EV-AAVs, that could be applied to any AAV serotype and further used for non-invasive gene delivery to the CNS. We performed a comparison between SEC and differential ultracentrifugation (UC) isolation protocols in terms of yield, contaminants, and transgene expression efficiency. We found that SEC allows a higher recovery of EV-AAVs, free of cell contaminating proteins and with lesssoloAAVs than UC. Remarkably, SEC-purified EV-AAVs also showed to be more potent at transgene expression thansoloAAVs in neuronal cell lines. EV-AAVs exhibited the ability to cross the BBB in neonatal mice upon intravenous administration. In conclusion, SEC-purified brain-targeting EV-AAVs show to be a promising gene delivery vector for therapy of brain disorders.Graphical AbstractDuring the production of AAV vectors, a small percentage of AAVs is secreted in association with extracellular vesicles, named “EV-AAVs”. EV-AAVs can be efficiently isolated by size exclusion chromatography (SEC). When intravenously injected in mice, brain targeting EV-AAVs can cross the blood brain barrier (BBB) and transduce neuronal cells.
Multimodal Deep Learning Model Unveils Behavioral Dynamics of V1 Activity in Freely Moving Mice
Authors: Xu Aiwen , Hou Yuchen , Niell Cristopher M. , Beyeler Michael 
Published: May 2023

AbstractDespite their immense success as a model of macaque visual cortex, deep convolutional neural networks (CNNs) have struggled to predict activity in visual cortex of the mouse, which is thought to be strongly dependent on the animal’s behavioral state. Furthermore, most computational models focus on predicting neural responses to static images presented under head fixation, which are dramatically different from the dynamic, continuous visual stimuli that arise during movement in the real world. Consequently, it is still unknown how natural visual input and different behavioral variables may integrate over time to generate responses in primary visual cortex (V1). To address this, we introduce a multimodal recurrent neural network that integrates gaze-contingent visual input with behavioral and temporal dynamics to explain V1 activity in freely moving mice. We show that the model achieves state-of-the-art predictions of V1 activity during free exploration and demonstrate the importance of each component in an extensive ablation study. Analyzing our model using maximally activating stimuli and saliency maps, we reveal new insights into cortical function, including the prevalence of mixed selectivity for behavioral variables in mouse V1. In summary, our model offers a comprehensive deep-learning framework for exploring the computational principles underlying V1 neurons in freely-moving animals engaged in natural behavior.
The Geometric Basis of Epithelial Convergent Extension
Authors: Brauns Fridtjof , Claussen Nikolas H. , Lefebvre Matthew F. , Wieschaus Eric F. , Shraiman Boris I. 
Published: May 2023

AbstractShape changes of epithelia during animal development, such as convergent extension, are achieved through concerted mechanical activity of individual cells. While much is known about the corresponding large scale tissue flow and its genetic drivers, fundamental questions regarding local control of contractile activity on cellular scale and its embryo-scale coordination remain open. To address these questions, we develop a quantitative, model-based analysis framework to relate cell geometry to local tension in recently obtained timelapse imaging data of gastrulatingDrosophilaembryos. This analysis provides a systematic decomposition of cell shape changes and T1-rearrangements into internally driven, active, and externally driven, passive, contributions. Our analysis provides evidence that germ band extension is driven by active T1 processes that self-organize through positive feedback acting on tensions. More generally, our findings suggest that epithelial convergent extension results from controlled transformation of internal force balance geometry which combines the effects of bottom-up local self-organization with the top-down, embryo-scale regulation by gene expression.
Uncovering structural features that control substrate specificity in a Lactobacillus chlorogenic acid esterase
Authors: Omori Kellie K. , Okumura Tracie L. S. , Carl Nathaniel B. , Dinn Brianna T. , Ly Destiny , Sacapano Kylie N. , Tajii Allie , Owens Cedric P. 
Published: May 2023

AbstractThe structural determinants of chlorogenic acid esterase (CE) substrate specificity are poorly understood. Here, we establish how aLactobacillus helveticusCE selects for its substrate, chlorogenic acid (CGA). We determine that a Lys residue in an extended loop over the active site imparts substrate specificity by hydrogen bonding to CGA. Mutation of the Lys residue abolishes CGA specificity. Comparison with other bacterial CEs reveals that the extended loop is not conserved. However, the hydrogen bonding functionality to CGA is preserved thanks to other residues. Structural comparison with ferulic acid esterases (FAEs), a related enzyme class, shows that CEs feature a more restricted active site, reflecting the fact that they hydrolyze smaller substrates compared to FAEs.
Citrate cross-feeding betweenPseudomonas aerguinosagenotypes supportslasRmutant fitness
Authors: Mould Dallas L. , Finger Carson E. , Botelho Nico , Stuut Stacie E. , Hogan Deborah A. 
Published: May 2023

ABSTRACTAcross the tree of life, clonal populations—from cancer to chronic bacterial infections — frequently give rise to subpopulations with different metabolic phenotypes. Metabolic exchange or cross-feeding between subpopulations can have profound effects on both cell phenotypes and population-level behavior. InPseudomonas aeruginosa, subpopulations with loss-of-function mutations in thelasRgene are common. Though LasR is often described for its role in density-dependent virulence factor expression, interactions between genotypes suggest potential metabolic differences. The specific metabolic pathways and regulatory genetics enabling such interactions were previously undescribed. Here, we performed an unbiased metabolomics analysis that revealed broad differences in intracellular metabolomes, including higher levels of intracellular citrate in LasR- strains. We found that while both strains secreted citrate, only LasR- strains, consumed citrate in rich media. Elevated activity of the CbrAB two component system which relieves carbon catabolite repression enabled citrate uptake. Within mixed genotype communities, we found that the citrate responsive two component system TctED and its gene targets OpdH (porin) and TctABC (transporter) required for citrate uptake were induced and required for enhanced RhlR signalling and virulence factor expression in LasR- strains. Enhanced citrate uptake by LasR- strains eliminates differences in RhlR activity between LasR+ and LasR- strains thereby circumventing the sensitivity of LasR- strains to quorum sensing controlled exoproducts. Citrate cross feeding also induces pyocyanin production in LasR- strains co-cultured withStaphylococcus aureus, another species known to secrete biologically-active concentrations of citrate. Metabolite cross feeding may play unrecognized roles in competitive fitness and virulence outcomes when different cell types are together.IMPORTANCECross-feeding can change community composition, structure and function. Though cross-feeding has predominantly focused on interactions between species, here we unravel a cross-feeding mechanism between frequently co-observed isolate genotypes ofPseudomonas aeruginosa. Here we illustrate an example of how such clonally-derived metabolic diversity enables intraspecies cross-feeding. Citrate, a metabolite released by many cells includingP. aeruginosa, was differentially consumed between genotypes, and this cross-feeding induced virulence factor expression and fitness in genotypes associated with worse disease.