bluuebunny/crossref_metadata_2025_split huggingface dataset:
Assessing genome-wide dynamic changes in enhancer activity during early mESC differentiation by FAIRE-STARR-seq
Authors: Glaser Laura V. , Steiger Mara , Fuchs Alisa , van Bömmel Alena , Einfeldt Edda , Chung Ho-Ryun , Vingron Martin , Meijsing Sebastiaan H. 
Published: June 2021

AbstractEmbryonic stem cells (ESCs) can differentiate into any given cell type and therefore represent a versatile model to study the link between gene regulation and differentiation. To quantitatively assess the dynamics of enhancer activity during the early stages of murine ESC differentiation, we analyzed accessible genomic regions using STARR-seq, a massively parallel reporter assay. This resulted in a genome-wide quantitative map of active mESC enhancers, in pluripotency and during the early stages of differentiation. We find that only a minority of accessible regions is active and that such regions are enriched near promoters, characterized by specific chromatin marks, enriched for distinct sequence motifs, and modeling shows that active regions can be predicted from sequence alone. Regions that change their activity upon retinoic acid-induced differentiation are more prevalent at distal intergenic regions when compared to constitutively active enhancers. Further, analysis of differentially active enhancers verified the contribution of individual TF motifs toward activity and inducibility as well as their role in regulating endogenous genes. Notably, the activity of retinoic acid receptor alpha (RARα) occupied regions can either increase or decrease upon the addition of its ligand, retinoic acid, with the direction of the change correlating with spacing and orientation of the RARα consensus motif and the co-occurrence of additional sequence motifs. Together, our genome-wide enhancer activity map elucidates features associated with enhancer activity levels, identifies regulatory regions disregarded by computational prediction tools, and provides a resource for future studies into regulatory elements in mESCs.
Arthritis Flares Mediated by Tissue Resident Memory T Cells in the Joint
Authors: Chang Margaret H , Levescot Anaïs , Nelson-Maney Nathan , Blaustein Rachel B , Winden Kellen D , Morris Allyn , Wactor Alexandra , Balu Spoorthi , Grieshaber-Bouyer Ricardo , Wei Kevin , Henderson Lauren A , Iwakura Yoichiro , Clark Rachael A , Rao Deepak A , Fuhlbrigge Robert C , Nigrovic Peter A 
Published: June 2021

AbstractAlthough rheumatoid arthritis is a systemic disease, flares typically occur in a subset of joints that is distinctive for each patient. Pursuing this intriguing pattern, we show that arthritis recurrence is mediated by long-lived synovial resident memory T cells (TRM). In three murine models, CD8+ cells bearing TRM markers remain in previously inflamed joints during remission. These cells are bona fide TRM, exhibiting failure to migrate from joint to joint, preferential uptake of fatty acids, and long-term residency. Disease flares result from TRM activation by antigen, leading to CCL5-mediated recruitment of circulating effector cells. Correspondingly, TRM depletion ameliorates recurrence in a site-specific manner. Human rheumatoid arthritis joint tissues contain a comparable CD8+-predominant TRM population, most evident in late-stage non-inflamed synovium, exhibiting limited T cell receptor diversity and a pro-inflammatory transcriptomic signature. Together, these findings establish synovial TRM cells as a targetable mediator of disease chronicity in autoimmune arthritis.
Prenatal cadmium exposure alters proliferation in mouse CD4+ T cells via LncRNA Snhg7
Authors: McCall Jamie L. , Varney Melinda E. , Dziadowicz Sebastian A. , Hall Casey , Blethen Kathryn , Hu Gangqing , Barnett John B. , Martinez Ivan 
Published: June 2021

ABSTRACTObjectivePrenatal cadmium (Cd) exposure leads to immunotoxic phenotypes in the offspring affecting coding and non-coding genes. Recent studies have shown that long non-coding RNAs (lncRNAs) are integral to T cell regulation. Here, we investigated the role of long non-coding RNA small nucleolar RNA hostgene 7 (lncSnhg7) in T cell proliferation.MethodsRNA sequencing was used to analyze the expression of lncRNAs in splenic CD4+ T cells with and without CD3/CD28 stimulation. Next, T cells isolated from offspring exposed to control or Cd water throughout mating and gestation were analyzed with and without stimulation with anti-CD3/CD28 beads. Quantitative qPCR and western blotting were used to detect RNA and protein levels of specific genes. Overexpression of a miR-34a mimic was achieved using nucleofection. Apoptosis was measured using flow cytometry and luminescence assays. Flow cytometry was also used to measure T cell proliferation in culture.ResultsWe identified 23 lncRNAs that were differentially expressed in stimulated versus unstimulated T cells, including lncSnhg7. LncSnhg7 and a downstream protein, GALNT7, are upregulated in T cells from offspring exposed to Cd during gestation. Overexpression of miR-34a, a regulator of lncSnhg7 and GALNT7, suppresses GALNT7 protein levels in primary T cells, but not in a mouse T lymphocyte cell line. The T cells isolated from Cd-exposed offspring exhibit increased proliferation after activation in vitro, but Treg suppression and CD4+ T cell apoptosis are not affected by prenatal Cd exposure.ConclusionPrenatal Cd exposure alters the expression of lncRNAs during T cell activation. The induction of lncSnhg7 is enhanced in splenic T cells from Cd offspring resulting in the upregulation of GALNT7 protein and increased proliferation following activation. miR-34a overexpression decreased GALNT7 expression suggesting that the lncSnhg7/miR-34a/GALNT7 is an important pathway in primary CD4+ T cells. These data highlight the need to understand the consequences of environmental exposures on lncRNA functions in non-cancerous cells as well as the effects in utero.
Physical basis for distinct basal and mechanically-gated activity of the human K+ channel TRAAK
Authors: Rietmeijer Robert A. , Sorum Ben , Li Baobin , Brohawn Stephen G. 
Published: June 2021

SummaryTRAAK is a mechanosensitive two-pore domain K+ (K2P) channel localized to nodes of Ranvier in myelinated neurons. TRAAK deletion in mice results in mechanical and thermal allodynia and gain-of-function mutations cause the human neurodevelopmental disorder FHEIG. TRAAK displays basal and stimulus-gated activities typical of K2Ps, but the mechanistic and structural differences between these modes are unknown. Here, we demonstrate that basal and mechanically-gated openings are distinguished by their conductance, kinetics, and structure. Basal openings are low conductance, short duration, and occur through a channel with an interior cavity exposed to the surrounding membrane. Mechanically-gated openings are high conductance, long duration, and occur through a channel that is sealed to the surrounding membrane. Our results explain how dual modes of activity are produced by a single ion channel and provide a basis for the development of state-selective pharmacology with the potential to treat disease.
High-throughput, label-free and slide-free histological imaging by computational microscopy and unsupervised learning
Authors: Zhang Yan , Kang Lei , Li Xiufeng , Wong Ivy H. M. , Wong Terence T. W. 
Published: June 2021

AbstractRapid and high-resolution histological imaging with minimal tissue preparation has long been a challenging and yet captivating medical pursue. Here, we propose a promising and transformative histological imaging method, termed computational high-throughput autofluorescence microscopy by pattern illumination (CHAMP). With the assistance of computational microscopy, CHAMP enables high-throughput and label-free imaging of thick and unprocessed tissues with large surface irregularity at an acquisition speed of 10 mm2/10 seconds with 1.1-µm lateral resolution. Moreover, the CHAMP image can be transformed into a virtually stained histological image (Deep-CHAMP) through unsupervised learning within 15 seconds, where significant cellular features are quantitatively extracted with high accuracy. The versatility of CHAMP is experimentally demonstrated using mouse brain/kidney tissues prepared with various clinical protocols, which enables a rapid and accurate intraoperative/postoperative pathological examination without tissue processing or staining, demonstrating its great potential as an assistive imaging platform for surgeons and pathologists to provide optimal adjuvant treatment.
Peroxidasin protein expression and enzymatic activity in metastatic melanoma cell lines are associated with invasive potential
Authors: Paumann-Page Martina , Kienzl Nikolaus F. , Motwani Jyoti , Bathish Boushra , Paton Louise N. , Magon Nick J. , Sevcnikar Benjamin , Furtmüller Paul G. , Traxlmayr Michael W. , Obinger Christian , Eccles Mike R. , Winterbourn Christine C. 
Published: June 2021

ABSTRACTPeroxidasin, a heme peroxidase, has been shown to play a role in cancer progression. mRNA expression has been reported to be upregulated in metastatic melanoma cell lines and connected to the invasive phenotype, but little is known about how peroxidasin acts in cancer cells. We have analyzed peroxidasin protein expression and activity in eight metastatic melanoma cell lines using an ELISA developed with an in-house peroxidasin binding protein. RNAseq data analysis confirmed high peroxidasin mRNA expression in the five cell lines classified as invasive and low expression in the three non-invasive cell lines. Protein levels of peroxidasin were higher in the cell lines with an invasive phenotype. Active peroxidasin was secreted to the cell culture medium, where it accumulated over time, and peroxidasin protein levels in the medium were also much higher in invasive than non-invasive cell lines. The only well-established physiological role of peroxidasin is in the formation of a sulfilimine bond, which cross-links collagen IV in basement membranes via catalyzed oxidation of bromide to hypobromous acid. We found that peroxidasin secreted from melanoma cells formed sulfilimine bonds in uncross-linked collagen IV, confirming peroxidasin activity and hypobromous acid formation. Moreover, 3-bromotyrosine, a stable product of hypobromous acid reacting with tyrosine residues, was detected in invasive melanoma cells, substantiating that their expression of peroxidasin generates hypobromous acid, and showing that it does not exclusively react with collagen IV, but also with other biomolecules.
Spatial cell type mapping of the oligodendrocyte lineage in the mouse juvenile and adult CNS with in situ sequencing
Authors: Hilscher Markus M. , Langseth Christoffer Mattsson , Kukanja Petra , Yokota Chika , Nilsson Mats , Castelo-Branco Gonçalo 
Published: June 2021

ABSTRACTOligodendrocytes show transcriptional heterogeneity but the regional and functional implications of this heterogeneity are less clear. Here, we applyin situsequencing (ISS) to simultaneously probe the expression of 124 marker genes of distinct oligodendrocyte populations, providing comprehensive maps of corpus callosum, cingulate, motor and somatosensory cortex in the brain, as well as gray (GM) and white matter (WM) regions in the spinal cord, at juvenile and adult stages. We systematically compare abundances of these populations and investigate the neighboring preference of distinct oligodendrocyte populations. As previously described, we observed that oligodendrocyte lineage progression is more advanced in the juvenile spinal cord compared to the brain. Additionally, myelination is ongoing in the adult corpus callosum while it is mostly completed in the cortex. Interestingly, we found a medial-to-lateral gradient of oligodendrocyte lineage progression in the juvenile cortex, which could be linked to arealization, as well as a deep-to-superficial gradient with mature oligodendrocytes preferentially accumulating in the deeper layers of the cortex. We observed differences in abundances and population dynamics over time between GM and WM regions in the brain and spinal cord, indicating regional differences within GM and WM. We also found that oligodendroglia populations’ neighboring preferences are altered from the juvenile to the adult CNS. Thus, our ISS dataset reveals spatial heterogeneity of the oligodendrocyte lineage progression in the brain and spinal cord, which could be relevant to further investigate functional heterogeneity of oligodendroglia.
Cytotoxic and micronuclei inducing effects of petroleum ether fraction of leaf aqueous extract of Clerodendrum viscosum Vent. in Allium cepa root tip cells
Authors: Roy Sujit , Kundu Lalit Mohan , Roy Gobinda Chandra , Barman Manabendu , Ray Sanjib 
Published: June 2021

SummaryClerodendrum viscosum is a traditionally used medicinal plant. The present study aimed to analyze a detailed cytotoxic effect of the nonpolar petroleum ether fraction (AQPEF) of leaf aqueous extract of C. viscosum Vent. (LAECV) in Allium cepa root tip cells. The LAECV was fractionated with petroleum ether and tested for A. cepa toxicity at early hours (2 and 4 h treatment) at concentrations 0, 50, 100, 150 and 200 µg mL-1. The highest aberrant cell percentage (10.45±0.46%) was scored from 150 µg mL-1 followed by100 µg mL-1 (8.75±0.26%) concentration at 4 h treated samples. The AQPEF treatment induced a significant (p<0.0001) increase in micronuclei frequency; 4.31±0.33, 5.08±0.13, 5.05±0.22 and 3.05±0.37% respectively at concentrations 50, 100, 150, and 200 µg mL-1. The highest polyploid frequency (20.14±0.68 %) induced with 100 µg mL-1 of AQPEF at 16 h recovery. 150 µg mL-1 is the most effective concentration of AQPEF to decipher its activity similar to colchicine (150 µg mL-1). In summary, the present study indicates petroleum ether is suitable for extraction of the active phytochemicals of LAECV having cytotoxic effects on A. cepa root tip cells. The AQPEF has colchicine like micronuclei, polyploidy, and mitotic abnormality inducing potentials in A. cepa root apical meristem cells.
Effect of differences in mechanical stress in vivo on the onset and progression of knee osteoarthritis
Authors: Arakawa Kohei , Takahata Kei , Oka Yuichiro , Ozone Kaichi , Nakagaki Sumika , Enomoto Saaya , Murata Kenji , Kanemura Naohiko , Kokubun Takanori 
Published: June 2021

ABSTRACTObjectiveThe effect of the type of mechanical stress on OA onset has not been clarified. The aim of this study was to establish a new model that reproduces the type and increase and decrease of mechanical stress in vivo and to clarify the differences in the mechanism of knee OA onset and progression among the models.DesignTo reproduce the difference in mechanical stress, we used the anterior cruciate ligament transection (ACL-T) model and the destabilization of the medial meniscus (DMM) model. In addition, we created a controlled abnormal tibial translation (CATT) model and a controlled abnormal tibial rotation (CATR) model that suppressed the joint instability of the ACL-T and DMM model, respectively. These four models reproduced the increase and decrease in shear force due to joint instability and compressive stress due to meniscal dysfunction. We performed joint instability analysis with soft X-ray, micro computed tomography analysis, histological analysis, and immunohistological analysis in 4 and 6 weeks.ResultsJoint instability decreased in the CATT and CATR groups. The meniscus deviated in the DMM and CATR groups. Chondrocyte hypertrophy increased in the ACL-T and DMM groups with joint instability. In the subchondral bone, bone resorption was promoted in the ACL-T and CATT groups, and bone formation was promoted in the DMM and CATR groups.ConclusionsIncreased shear force causes articular cartilage degeneration and osteoclast activation in the subchondral bone. In contrast, increased compressive stress promotes bone formation in the subchondral bone earlier than articular cartilage degeneration occurs.
HyDrop: droplet-based scATAC-seq and scRNA-seq using dissolvable hydrogel beads
Authors: De Rop Florian V. , Ismail Joy N. , González-Blas Carmen Bravo , Hulselmans Gert J. , Flerin Christopher C. , Janssens Jasper , Theunis Koen , Christiaens Valerie M. , Wouters Jasper , Marcassa Gabriele , de Wit Joris , Poovathingal Suresh , Aerts Stein 
Published: June 2021

AbstractSingle-cell RNA-seq and single-cell ATAC-seq technologies are being used extensively to create cell type atlases for a wide range of organisms, tissues, and disease processes. To increase the scale of these atlases, lower the cost, and allow for more specialized multi-ome assays, custom droplet microfluidics may provide complementary solutions to commercial setups. We developed HyDrop, a flexible and generic droplet microfluidic platform encompassing three protocols. The first protocol involves creating dissolvable hydrogel beads with custom oligos that can be released in the droplets. In the second protocol, we demonstrate the use of these beads for HyDrop-ATAC, a low-cost non-commercial scATAC-seq protocol in droplets. After validating HyDrop-ATAC, we applied it to flash-frozen mouse cortex and generated 8,502 high-quality single-cell chromatin accessibility profiles in a single run. In the third protocol, we adapt both the reaction chemistry and the capture sequence of the barcoded hydrogel bead to capture mRNA, and demonstrate a significant improvement in throughput and sensitivity compared to previous open-source droplet-based scRNA-seq assays (Drop-seq and inDrop). Similarly, we applied HyDrop-RNA to flash-frozen mouse cortex and generated 9,508 single-cell transcriptomes closely matching reference single-cell gene expression data. Finally, we leveraged HyDrop-RNA’s high capture rate to analyse a small population of FAC-sorted neurons from the Drosophila brain, confirming the protocol’s applicability to low-input samples and small cells. HyDrop is currently capable of generating single-cell data in high throughput and at a reduced cost compared to commercial methods, and we envision that HyDrop can be further developed to be compatible with novel (multi-) omics protocols.
Long-term culture of fetal monocyte precursorsin vitroallowing the generation ofbona fidealveolar macrophagesin vivo
Authors: Li Fengqi , Okreglicka Katarzyna Maria , Pohlmeier Lea Maria , Schneider Christoph , Kopf Manfred 
Published: June 2021

ABSTRACTTissue-resident macrophage-based immune therapies have been proposed for various diseases. However, generation of sufficient numbers that possess tissue-specific functions remains a major handicap. Here, we show that fetal liver monocytes (FLiMo) cultured with GM-CSF (also known as CSF2) rapidly differentiate into a long-lived, homogeneous alveolar macrophage (AM)-like populationin vitro. CSF2-cultured FLiMo remain the capacity to develop intobona fideAM upon transfer intoCsf2ra-/-neonates and prevent development of alveolar proteinosis and efferocytosis of apoptotic cells for at least 1 yearin vivo. Compared to transplantation of AM-like cells derived from bone marrow macrophages (BMM), CSF2-cFliMo more efficiently engraft empty AM niches in the lung and protect mice from respiratory viral infection. Harnessing the potential of this approach for gene therapy, we restored a disrupted Csf2ra gene in FLiMo and their capacity to develop into AMin vivo. Together, we provide a novel platform for generation of immature AM-like precursors amenable for genetic manipulation, which will be useful to study to dissect AM development and function and pulmonary transplantation therapy.
Heterogeneous mutation rates and spectra in yeast hybrids
Authors: Fijarczyk Anna , Hénault Mathieu , Marsit Souhir , Charron Guillaume , Landry Christian R. 
Published: June 2021

AbstractMutation rates and spectra vary between species and among populations. Hybridization can contribute to this variation, but its role remains poorly understood. Estimating mutation rates requires controlled conditions where the effect of natural selection can be minimized. One way to achieve this is through mutation accumulation experiments coupled with genome sequencing. Here we investigate 400 mutation accumulation lines initiated from 11 genotypes spanning intra-lineage, inter-lineage and interspecific crosses of the yeasts Saccharomyces paradoxus and S. cerevisiae and propagated for 770 generations. We find significant differences in mutation rates and spectra among crosses, which are not related to the level of divergence of parental strains but are specific to some genotype combinations. Differences in number of generations and departures from neutrality play a minor role, whereas polyploidy and loss of heterozygosity impact mutation rates in some of the hybrid crosses in an opposite way.
Presynaptic Gq-coupled receptors drive biphasic dopamine transporter trafficking that modulates dopamine clearance and motor function
Authors: Kearney Patrick J. , Kahuno Elizabeth , Conklin Tucker L. , Martin Gilles E. , Lubec Gert , Melikian Haley E. 
Published: June 2021

AbstractExtracellular dopamine (DA) levels are constrained by the presynaptic DA transporter (DAT), a major psychostimulant target. Despite its necessity for DA neurotransmission, DAT regulation in situ is poorly understood, and it is unknown whether regulated DAT trafficking impacts dopaminergic signaling and/or behaviors. Leveraging chemogenetics and conditional gene silencing, we found that activating presynaptic Gq-coupled receptors, either hM3Dq or mGluR5, drove rapid biphasic DAT membrane trafficking, with region-specific differences in ventral and dorsal striata. DAT insertion required DRD2 autoreceptors and intact retromer, whereas DAT retrieval required PKC activation and Rit2. Ex vivo voltammetry revealed that DAT trafficking impacts DA clearance. Importantly, dopaminergic mGluR5 silencing elevated surface DAT, which abolished motor learning and was rescued by inhibiting DAT. We found that presynaptic DAT trafficking is complex, multimodal, and region-specific, and identify cell autonomous mechanisms governing presynaptic DAT tone. Importantly, the findings suggest regulated DAT trafficking impacts both DA clearance and motor function.
Parallel valence processing alterations associated with compulsive behavior in SAPAP3 knockout mice and human OCD
Authors: Kajs Bridget L. , van Roessel Peter J. , Davis Gwynne L. , Williams Leanne M. , Rodriguez Carolyn I. , Gunaydin Lisa A. 
Published: June 2021

ABSTRACTAbnormalities in valence processing – the processing of aversive or appetitive stimuli – may be an underrecognized component of obsessive-compulsive disorder (OCD). Independent experimental paradigms have suggested disturbance of emotional valence systems in OCD, yet no standardized assay has been employed to assess both negative and positive valence processing in clinical studies of OCD patients, either at baseline or in response to therapeutic interventions. Additionally, preclinical rodent models are critical for treatment discovery in OCD, yet investigations examining whether rodent models of compulsive behavior similarly show alterations in valence systems have been limited. We sought to establish paradigms for assessing valence processing across both human OCD patients and in a preclinical rodent model: in OCD patients, we used validated behavioral tests to assess explicit and implicit processing of fear-related facial expressions (negative valence) and socially-rewarding happy expressions (positive valence); in the SAPAP3 knockout (KO) mouse model of compulsive behavior, we used auditory fear conditioning and extinction (negative valence) and reward-based operant conditioning (positive valence). We find that OCD patients show enhanced negative and impaired positive valence processing, and that performance on valence processing tasks correlates with clinical measures of OCD severity. We further find that SAPAP3 KO mice show heightened negative and impaired positive valence processing alterations similar to those of OCD patients. Our results show parallel valence processing abnormalities in OCD patients and a preclinical rodent model of compulsive behavior, and suggest valence processing alterations as novel therapeutic targets across a translational research spectrum.