bluuebunny/crossref_metadata_2025_split huggingface dataset:
Culture of pluripotent stem cells in microscale droplets modulates differentiation and tissue patterning towards organoids on chip
Authors: Vertti-Quintero Nadia , Delahousse Clara , Aristov Andrey , Traboulsi Tatiana , Cossec Jack-Christophe , Baroud Charles N. , Sart Sébastien 
Published: October 2024

1SummaryThe differentiation of pluripotent stem cells (PSCs) and their self-organization into organoids are influenced by cell-cell interactions mediated by contacts and secreted molecules. These interactions are enhanced in microfluidic droplets due to confinement and small culture volumes. However, a comprehensive study on the culture of PSCs within droplets and the impact of this microenvironment has yet to be conducted. In this study, we present a droplet platform for the 3D culture of PSCs at various stages of cellular commitment. We demonstrate PSC differentiation into the three germ layers and the feasibility of organoid formation within droplets. Our findings reveal that culturing PSCs in confined volumes regulates cell fate decisions, promoting tissue patterning in gastruloids through the sequential induction of growth and migration of distinct differentiated cell populations, and facilitating the self-organization of cardiac organoids. This technological approach provides unique insights into the intrinsic factors regulating tissue self-patterningin vitro.2Highlights and eTOC blurbHighlightsDroplet microfluidics allows expansion and supports the pluripotency of 3D aggregates of PSCs.Droplet microfluidics supports and regulates spontaneous differentiation into embryoid bodies.Droplet promotes tissue patterning in gastruloids through the sequential induction of growth and migration of mesoderm followed by ectoderm.Perfused microfluidic droplets support long term culture and derivation of organoids on chip.Vertti-Quinteroet al. introduces a microfluidic droplet platform for the 3D culture of pluripotent stem cells (PSCs) at various differentiation stages. The format supports the long term 3D culture and the differentiation of PSCs -either spontaneous or directed-. This “microscale culture” can regulate PSCs fate decision, while promoting tissue pattering -as demonstrated in gastruloids polarization- and allowing self-organization towards cardioids formation.
The Mag-Click-Capture-Release Technology for Selective Capture and Release of Hepatocyte-Derived Extracellular Vesicles as Biomarkers for Liver Disease
Authors: Booijink Richell , Mentink Anouk , Jansen Larissa , Mentink Sven , van Rein Bo , Geraets Lieke , Scholten Jorinde , Brusse Maureen , Fu Siyu , Boonstra Andre , Bansal Ruchi 
Published: October 2024

AbstractChronic liver diseases, such as liver cirrhosis and hepatocellular carcinoma (HCC), present major global health challenges, often diagnosed late. Circulating extracellular vesicles (EVs), which carry disease-specific biomolecular cargo, is emerging as an early diagnostic and prognostic biomarker for several diseases including cancer. However, current EV purification methods including ultracentrifugation and size exclusion chromatography present several limitations. Here, we present the Mag-Click-Capture-Release Technology for selective capture and release of EVs that combinesmagnetic beads, trans-cyclooctene (TCO) and tetrazine (Tz)clickchemistry, immuno(antibody)-basedcaptureand disulfide-drivenreleaseof EVs. Importantly, the Mag-Click-Capture-Release Technology is customizable, whereby using specific antibodies conjugated to TCO antibodies, different EV subtypes can be selectively captured and released for further analysis. With our Mag-Click-Capture-Release Technology, we successfully isolated hepatocyte-derived EVs from human serum with good recovery, high specificity and purity when compared with standard ultracentrifugation. Validation in serum samples obtained from cirrhosis and HCC patients with alcohol-associated liver disease evidenced an increasing trend in hepatocyte-EV levels correlating with disease severity, suggesting potential for early diagnosis and prognosis. In conclusion, we present here the Mag-Click-Capture-Release Technology, a customizable and efficient approach for selective isolation of organ-, cell-specific, and disease-relevant EVs from biological samples that can be subsequently released for downstream molecular EV analysis and EV-related functional assays.
Single-cell transcriptomics reveal diverging pathobiology and opportunities for precision targeting in scleroderma-associated versus idiopathic pulmonary arterial hypertension
Authors: Tuhy Tijana , Coursen Julie C. , Graves Tammy , Patatanian Michael , Cherry Christopher , Niedermeyer Shannon E. , Khan Sarah L. , Rosen Darin T. , Croglio Michael P. , Elnashar Mohab , Kolb Todd M. , Mathai Stephen C. , Damico Rachel L. , Hassoun Paul M. , Shimoda Larissa A. , Suresh Karthik , Aldred Micheala A. , Simpson Catherine E. 
Published: October 2024

AbstractIntroductionPulmonary arterial hypertension (PAH) involves progressive cellular and molecular change within the pulmonary vasculature, leading to increased vascular resistance. Current therapies targeting nitric oxide (NO), endothelin, and prostacyclin pathways yield variable treatment responses. Patients with systemic sclerosis-associated PAH (SSc-PAH) often experience worse outcomes than those with idiopathic PAH (IPAH).MethodsLung tissue samples from four SSc-PAH, four IPAH, and four failed donor specimens were obtained from the Pulmonary Hypertension Breakthrough Initiative (PHBI) lung tissue bank. Single-cell RNA sequencing (scRNAseq) was performed using the 10X Genomics Chromium Flex platform. Data normalization, clustering, and differential expression analysis were conducted using Seurat. Additional analyses included gene set enrichment analysis (GSEA), transcription factor activity analysis, and ligand-receptor signaling. Pharmacotranscriptomic screening was performed using the Connectivity Map.ResultsSSc-PAH samples showed a higher proportion of fibroblasts and dendritic cells/macrophages compared to IPAH and donor samples. GSEA revealed enriched pathways related to epithelial-to-mesenchymal transition (EMT), apoptosis, and vascular remodeling in SSc-PAH samples. There was pronounced differential gene expression across diverse pulmonary vascular cell types and in various epithelial cell types in both IPAH and SSc-PAH, with epithelial to endothelial cell signaling observed. Macrophage to endothelial cell signaling was particularly pronounced in SSc-PAH. Pharmacotranscriptomic screening identified TIE2, GSK-3, and PKC inhibitors, among other compounds, as potential drug candidates for reversing SSc-PAH gene expression signatures.DiscussionOverlapping and distinct gene expression patterns exist in SSc-PAH versus IPAH, with significant molecular differences suggesting unique pathogenic mechanisms in SSc-PAH. These findings highlight the potential for precision-targeted therapies to improve SSc-PAH patient outcomes. Future studies should validate these targets clinically and explore their therapeutic efficacy.
Temporal dynamics of inflammatory and transcriptome changes in the myocardium in a murine model of cardiac arrest
Authors: Panigrahi Soumya , Jiang Alice , Enriquez Angela , Tummala Sanjana , Rempinski Donald , Remy Kenneth E. , Rutledge Cody A. 
Published: October 2024

AbstractBackgroundIt is increasingly recognized that inflammatory changes play a significant role in myocardial injury following cardiac arrest. However, the pathophysiological mechanisms responsible for functional changes in the myocardium following the event remain incompletely understood. Here, we characterize the transcriptomic and immunological changes in a mouse model of cardiac arrest to explore the roles of epinephrine and ischemia/reperfusion on myocardial inflammation.Methods and ResultsMale and female mice (C57BL/6J) were divided into three groups: (1) Naïve (anesthesia only), (2) Sham (anesthesia + saline injection + epinephrine), and (3) Arrest (8-minute cardiac arrest followed by resuscitation (anesthesia + potassium chloride + epinephrine). We monitored the animals over 0.5, 1, 3, and 7 days with serial echocardiography. Hearts were harvested for transcriptomic analysis and inflammatory changes as assessed by flow cytometry and immunohistochemistry. Plasma samples were collected for cytokine signaling analysis by multiplex ELISA. Our data indicate that the cardiac arrest group have increased infiltrating neutrophils in the myocardium and elevated plasma levels of pro-inflammatory cytokines, peaking at 0.5 days after the initial insult and subsequently resolving. In contrast, Sham mice displayed less pronounced inflammatory changes, which peaked at 1 day after the procedure. Notably, these inflammatory alterations coincided with cardiac stunning, as demonstrated by echocardiography.ConclusionsIn our murine model of cardiac arrest, epinephrine exposure and ischemia/reperfusion induce significant inflammatory changes in the myocardium with increased infiltrating neutrophils and elevated circulating cytokines which subsequently resolve in a time-dependent manner. These changes correlate with improving cardiac function by echocardiography.
Flagellar motility and the mucus environment influence aggregation mediated antibiotic tolerance ofPseudomonas aeruginosain chronic lung infection
Authors: Higgs Matthew G. , Greenwald Matthew A. , Roca Cristian , Macdonald Jade K. , Sidders Ashelyn E. , Conlon Brian P. , Wolfgang Matthew C. 
Published: October 2024

ABSTRACTPseudomonas aeruginosafrequently causes chronic lung infection in individuals with muco-obstructive airway diseases (MADs). ChronicP. aeruginosainfections are difficult to treat, primarily owing to antibiotic treatment failure, which is often observed in the absence of antimicrobial resistance. In MADs,P. aeruginosaforms biofilm-like aggregates within the luminal mucus. While the contribution of mucin hyperconcentration towards antibiotic tolerance has been described, the mechanism for mucin driven antibiotic tolerance and the influence of aggregates have not been fully elucidated. In this study, we investigated the contribution of flagellar motility towards aggregate formation as it relates to the diseased mucus environment. We found that loss of flagellar motility resulted in increasedP. aeruginosaaggregation and tolerance to multiple classes of antibiotics. Further, we observed differential roles in antimicrobial tolerance of themotABandmotCDstators, which power the flagellum. Additionally, we found that control offliCexpression was important for aggregate formation and antibiotic tolerance as a strain constitutively expressingfliCwas unable to form aggregates and was highly susceptible to treatment. Lastly, we demonstrate that neutrophil elastase, an abundant immune mediator and biomarker of chronic lung infection, promotes aggregation and antibiotic tolerance by impairing flagellar motility. Collectively, these results highlight the key role of flagellar motility in aggregate formation and antibiotic tolerance and deepens our understanding of how the MADs lung environment promotes antibiotic tolerance ofP. aeruginosa.IMPORTANCEAntibiotic recalcitrance of chronicPseudomonas aeruginosainfections in muco-obstructive airway diseases is a primary driver of mortality. Mechanisms that drive antibiotic tolerance are poorly understood. We investigated motility phenotypes related toP. aeruginosaadaptation and antibiotic tolerance in the diseased mucus environment. Loss of flagellar motility drives antibiotic tolerance by promoting aggregate formation. Regulation of flagellar motility appears to be a key step in aggregate formation as the inability to turn off flagellin expression resulted in poor aggregate formation and increased antibiotic susceptibility. These results deepen our understanding of the formation of antibiotic tolerant aggregates within the MADs airway and opens novel avenues and targets for treatment of chronicP. aeruginosainfections.
Differential Responses of Lung and Intestinal Microbiota to SARS-CoV-2 Infection: A Comparative Study of the Wuhan and Omicron Strains in K18-hACE2 tg Mice
Authors: Kim Chae Won , Ku Keun Bon , Hwang Insu , Jung Hi Eun , Kim Kyun-Do , Lee Heung Kyu 
Published: October 2024

AbstractThe COVID-19 pandemic, caused by SARS-CoV-2, has led to the emergence of viral variants with distinct characteristics. We investigated the differential effects of the original Wuhan strain and the emergent Omicron variant of SARS-CoV-2 using a K18-hACE2 transgenic mouse model. We compared the mortality rates, viral loads, and histopathological changes in lung and tracheal tissues, as well as alterations in the lung and intestinal microbiota following infection. We observed significant differences in disease severity, with the Wuhan strain causing higher mortality and more severe lung damage than the Omicron variant. Furthermore, microbiome analyses revealed distinct shifts in microbiota associated with infection by each variant, suggesting that microbiome-related mechanisms might influence disease outcomes. This comprehensive comparison enhances our understanding of COVID-19 pathogenesis and highlights the importance of microbiome dynamics in viral infections, providing insights for future therapeutic and preventive strategies.ImportanceUnderstanding the differential impacts of SARS-CoV-2 variants is crucial for effective public health response and treatment development. This study provides insights into the pathogenesis of the original Wuhan strain and the Omicron variant of SARS-CoV-2, revealing significant differences in host mortality, viral load, and lung pathology. The use of the K18-hACE2 transgenic mouse model enables detailed examination of these differences in a controlled setting. Furthermore, this study highlights the importance of the microbiome in modulating disease severity and host responses to viral infections. By uncovering distinct microbial shifts associated with infection by different SARS-CoV-2 variants, this study suggests potential microbiome-related mechanisms that might be targeted to mitigate disease outcomes.
The PUF RNA-binding protein, FBF-2, maintains stem cells without binding to RNA
Authors: Carrick Brian H. , Crittenden Sarah L. , Linsley MaryGrace , Dos Santos Stephany J. Costa , Wickens Marvin , Kimble Judith 
Published: October 2024

AbstractLike all canonical PUF proteins,C. elegansFBF-2 binds to specific RNAs via tripartite recognition motifs (TRMs). Here we report that an FBF-2 mutant protein that cannot bind to RNA, is nonetheless biologically active and maintains stem cells. This unexpected result challenges the conventional wisdom that RBPs must bind to RNAs to achieve biological activity. Also unexpectedly, FBF-2 interactions with partner proteins can compensate for loss of RNA-binding. FBF-2 only loses biological activity when its RNA-binding and partner interactions are both defective. These findings highlight the complementary contributions of RNA-binding and protein partner interactions to activity of an RNA-binding protein.
Impaired erythroid maturation in murine embryos upon loss of the preeclampsia-associated serine protease prostasin
Authors: Di Carlo Sara , Salas-Bastos Adrian , Castelblanco Mariela Castelblanco , Auberson Muriel , Rumpler Marie , Tournier Malaury , Sommer Lukas , Naveiras Olaia , Hummler Edith 
Published: October 2024

AbstractIn humans, the membrane-bound serine protease prostasin encoded byPrss8is associated with preeclampsia, a gestational hypertension disorder affecting blood supply of the placenta. Mice deficient inPrss8resulted in the death of embryos at embryonic day (E) 14.5 and it was characterized by impaired placental labyrinth maturation and vascularization. A pale phenotype was observed in these embryos, suggesting ineffective erythropoiesis. Thus, in this study we analyzed this phenotype further inPrss8-/-embryos at E11.5 and E12.5. We found a reduced number of fetal erythroblasts in placenta, yolk sac and fetal liver ofPrss8-/-embryos, while the reticulocyte number was increased, suggesting a defective terminal erythroid differentiation. Further, single-cell RNA sequencing (scRNA-seq) analyses of aorta-gonad-mesonephros (AGM) revealed an upregulation of several ribosomal genes associated with Diamond-Blackfan anemia in erythroid cells ofPrss8-/-(KO) embryos. These cells showed a lower capacity to maturate into erythrocytesin vivoand invitro,despite hematopoietic cells (HSCs) being produced normally. We suggested prostasin influenced erythropoiesis in a cell-extrinsic manner, sincePrss8expression was not detected in erythroid cells but highly expressed in ectoderm-like cells within the AGM. Congruently, while yolk sac-derived cells displayed no erythroid maturation defectin vitro, the yolk sac vascular remodeling in KO embryos was impaired as evidenced by reduced secondary branching likely as a consequence of the reduced blood flow. Our findings unveiled a novel role for this serine protease in terminal maturation of erythrocytes in the fetal liver and open new research avenues for understanding the physiological mechanism of prostasin and its pathological implications.Key PointsPrss8deficiency causes transcriptional changes in erythroid progenitor cells in the AGM leading to impaired embryonic erythropoiesisOverexpression of Rpl and Rps genes by erythroid cells lackingPrss8leads to defective erythropoiesis and embryonic lethality
In VivoExpression of an SCA27A-linkedFGF14Mutation Results in Haploinsufficiency and Impaired Firing of Cerebellar Purkinje Neurons
Authors: Ransdell Joseph L. , Brown Samuel P. , Xiao Maolei , Ornitz David M. , Nerbonne Jeanne M. 
Published: October 2024

AbstractAutosomal dominant mutations inFGF14, which encodes intracellular fibroblast growth factor 14 (iFGF14), underlie spinocerebellar ataxia type 27A (SCA27A), a devastating multisystem disorder resulting in progressive deficits in motor coordination and cognitive function. Mice lacking iFGF14 (Fgf14-/-) exhibit similar phenotypes, which have been linked to iFGF14-mediated modulation of the voltage-gated sodium (Nav) channels that control the high frequency repetitive firing of Purkinje neurons, the main output neurons of the cerebellar cortex. To investigate the pathophysiological mechanisms underlying SCA27A, we developed a targeted knock-in strategy to introduce the first point mutation identified inFGF14into the mouseFgf14locus (Fgf14F145S), we determined the impact ofin vivoexpression of the mutantFgf14F145Sallele on the motor performance of adult animals and on the firing properties of mature Purkinje neurons in acute cerebellar slices. Electrophysiological experiments revealed that repetitive firing rates are attenuated in adultFgf14F145S/+cerebellar Purkinje neurons, attributed to a hyperpolarizing shift in the voltage-dependence of steady-state inactivation of Nav channels. More severe effects on firing properties and Nav channel inactivation were observed in homozygousFgf14F145S/F145SPurkinje neurons. Interestingly, the electrophysiological phenotypes identified in adultFgf14F145S/+andFgf14F145S/F145Scerebellar Purkinje neurons mirror those observed in heterozygousFgf14+/-and homozygousFgf14-/-Purkinje neurons, respectively, suggesting that the mutation results in the loss of the iFGF14 protein. Western blot analysis of lysates from adult heterozygousFgf14F145S/+and homozygousFgf14F145S/F145Sanimals revealed reduced or undetectable, respectively, iFGF14 expression, supporting the hypothesis that the mutant allele results in loss of the iFGF14 protein and that haploinsufficiency underlies SCA27A neurological phenotypes.
Determining zygosity with multiplex Kompetitive Allele-Specific PCR (mxKASP) genotyping
Authors: de Visser Manon C. , Meilink Willem R.M. , Theodoropoulos Anagnostis , Fahrbach Michael , Wielstra Ben 
Published: October 2024

AbstractWe introduce multiplex Kompetitive Allele-Specific PCR (mxKASP): a modification of ‘classical’ KASP genotyping that allows zygosity to be determined in diploid organisms. Rather than targeting a SNP associated with a single marker, mxKASP targets two non-homologous markers. We show proof of concept by applying mxKASP to the balanced lethal system inTriturusnewts, in which individuals are known to possess either: (1) zero copies of the 1A version of chromosome 1 and two copies of the 1B version; (2) one copy of 1A and one copy of 1B; or (3) two copies of 1A and zero copies of 1B. mxKASP is successful in amplifying both a 1A and a 1B marker in a single reaction (if present), allowing the zygosity of individuals to be inferred. We independently confirm our mxKASP results with a multiplex PCR approach. We argue that mxKASP can be applied to rapidly and economically determine zygostity in diploid organisms, for a large number of samples at once.
EEG-VLM Toolbox: Extending voxel-based lesion mapping to multi-dimensional EEG data
Authors: Hardstone Richard , Ostrowski Lauren , Dusang Aliceson N. , López-Larraz Eduardo , Jesser Jessica , Cash Sydney S. , Cramer Steven C. , Hochberg Leigh R. , Ramos-Murguialday Ander , Lin David J. 
Published: October 2024

AbstractFocal brain lesions (such as with stroke) cause functional changes in local and distributed neural systems. While there is a long history of post-stroke neurophysiological assessment using electroencephalography (EEG), the observed neurophysiological changes have rarely been related to specific lesion locations. Therefore, the relationships between anatomical injury and physiological changes after stroke remain unclear. Voxel-based lesion symptom mapping (VLSM) is a tool for statistically relating stroke lesion locations to “symptoms”, but current VLSM methods are restricted to symptoms that can be defined by a single value. Therefore, current VLSM techniques are unable to map the relationships between anatomical injury and multidimensional neurophysiological data such as EEG, which contains rich spatio-temporal information across different channels and frequency bands.Here we present a novel algorithm, EEG Voxel-based Lesion Mapping (EEG-VLM), that produces the set of significant relationships between precise neuroanatomical injury locations and neurophysiology (defined by a cluster of adjacent EEG channels and frequency bands). Further, the algorithm provides statistical analyses to define the overall significance of each neural structure-function relationship by correcting for multiple comparisons using a permutation test. Applying EEG-VLM to a dataset of recordings from chronic stroke patients performing a cued upper extremity movement task, we found that subjects with lesions in frontal subcortical white matter have reduced ipsilesional parietal cue-evoked EEG responses. These results are consistent with damage to a frontal-parietal network that has been associated with impairments in attention. EEG-VLM is a novel and unbiased method for relating neurophysiologic changes after stroke with neuroanatomic lesions. In the context of focal brain lesions associated with neurological impairments, we propose that this method will enable improved mechanistic understanding, facilitate biomarker development, and guide neurorehabilitation strategies.