bluuebunny/crossref_metadata_2025_split huggingface dataset:
Systemic sterile induced-co-expression of IL-12 and IL-18 drives IFN-γ-dependent activation of microglia and recruitment of MHC-II-expressing inflammatory monocytes into the brain
Authors: Gaviglio Emilia A. , Peralta Ramos Javier M. , Arroyo Daniela S. , Bussi Claudio , Iribarren Pablo , Rodriguez-Galan Maria C. 
Published: July 2020

ABSTRACTThe development of neuroinflammation, as well as the progression of several neurodegenerative diseases, has been associated with the activation and mobilization of the peripheral immune system due to systemic inflammation. However, the mechanism by which this occurs remains unclear. Herein, we addressed the effect of systemic, endotoxin-free induced-co-expression of IL-12 and IL-18 in the establishment of a novel cytokine-mediated model of neuroinflammation. Following peripheral hydrodynamic shear of IL-12 plus IL-18 cDNAs in C57BL/6 mice, we induced systemic and persistent level of IL-12, which in turn promoted the elevation of circulating pro-inflammatory cytokines TNF-α and IFN-γ, accompanied with splenomegaly. Moreover, even though we identified an increased gene expression of both TNF-α and IFN-γ in the brain, only TNF-α was shown to be dispensable, revealing an IFN-γ-dependent activation of microglia and the recruitment of leukocytes, particularly of highly activated inflammatory monocytes. Taken together, our results argue for a systemic cytokine-mediated establishment and development of neuroinflammation, having identified IFN-γ as a potential target for immunotherapy.
Epigenetic age-predictions in mice using pyrosequencing, droplet digital PCR or barcoded bisulfite amplicon sequencing
Authors: Han Yang , Nikolić Miloš , Gobs Michael , Franzen Julia , de Haan Gerald , Geiger Hartmut , Wagner Wolfgang 
Published: July 2020

AbstractAge-associated DNA methylation reflects aspects of biological aging - therefore epigenetic clocks for mice can help to elucidate the impact of treatments or genetic background on the aging process in this model organism. Initially, age-predictors for mice were trained on genome-wide DNA methylation profiles, whereas we have recently described a targeted assay based on pyrosequencing of DNA methylation at only three CG dinucleotides (CpGs). Here, we have re-evaluated pyrosequencing approaches in comparison to droplet digital PCR (ddPCR) and barcoded bisulfite amplicon sequencing (BBA-seq). At individual CpGs the correlation of DNA methylation with chronological age was slightly higher for pyrosequencing and ddPCR as compared to BBA-seq. On the other hand, BBA-seq revealed that neighboring CpGs tend to be stochastically modified in murine age-associated regions. Furthermore, the binary sequel of methylated and non-methylated CpGs in individual reads can be used for single-read predictions, which may reflect heterogeneity in epigenetic aging. In comparison to C57BL/6 mice the epigenetic age-predictions using BBA-seq were also accelerated in the shorter-lived DBA/2 mice, and in C57BL/6 mice with a lifespan quantitative trait locus of DBA/2 mice. Taken together, we describe further optimized and alternative targeted methods to determine epigenetic clocks in mice.
Overexpression of key sterol pathway enzymes in two model marine diatoms alters sterol profiles inPhaeodactylum tricornutum
Authors: Jaramillo-Madrid Ana Cristina , Abbriano Raffaela , Ashworth Justin , Fabris Michele , Ralph Peter J. 
Published: July 2020

AbstractSterols are a class of triterpenoid molecules with diverse functional roles in eukaryotic cells, including intracellular signaling and regulation of cell membrane fluidity. Diatoms are a dominant eukaryotic phytoplankton group that produce a wide diversity of sterol compounds. The enzymes 3-hydroxy-3-methyl glutaryl CoA reductase (HMGR) and squalene epoxidase (SQE) have been reported to be rate-limiting steps in sterol biosynthesis in other model eukaryotes; however, the extent to which these enzymes regulate triterpenoid production in diatoms is not known. To probe the role of these two metabolic nodes in the regulation of sterol metabolic flux in diatoms, we independently over-expressed two versions of the nativeHMGRand a conventional, heterologous SQE gene in the diatomsThalassiosira pseudonanaandPhaeodactylum tricornutum. Overexpression of these key enzymes resulted in significant differential accumulation of downstream sterol pathway intermediates inP. tricornutum. HMGR-mVenus overexpression resulted in the accumulation of squalene, cycloartenol, and obtusifoliol, while cycloartenol and obtusifoliol accumulated in response to heterologous NoSQE-mVenus overexpression. In addition, accumulation of the end-point sterol 24-methylenecholesta-5,24(24’)-dien-3β-ol was observed in allP. tricornutumoverexpression lines, and campesterol increased 3-fold inP. tricornutumlines expressing NoSQE-mVenus. Minor differences in end-point sterol composition were also found inT. pseudonana, but no accumulation of sterol pathway intermediates was observed. Despite the successful manipulation of pathway intermediates and individual sterols inP. tricornutum, total sterol levels did not change significantly in transformed lines, suggesting the existence of tight pathway regulation to maintain total sterol content.
Base-edited CAR T Cells for combinational therapy against T cell malignancies
Authors: Georgiadis Christos , Rasaiyaah Jane , Gkazi Soragia Athina , Preece Roland , Etuk Aniekan , Christi Abraham , Qasim Waseem 
Published: July 2020

AbstractTargeting T cell malignancies using chimeric antigen receptor (CAR) T cells is hindered by ‘T v T’ fratricide against shared antigens such as CD3 and CD7. Genome-editing can overcome such hurdles through targeted disruption of problematic shared antigens. Base editing offers the possibility of seamless disruption of gene expression through the creation of stop codons or elimination of splice donor or acceptor sites. We describe the generation of fratricide resistant, T cells by orderly removal of shared antigens such as TCR/CD3 and CD7 ahead of lentiviral mediated expression of CARs specific for CD3 or CD7. Molecular interrogation of base edited cells confirmed virtual elimination of chromosomal translocation events detected in conventional Cas9 treated cells. Interestingly, co-culture of 3CAR and 7CAR cells resulted in ‘self-enrichment’ yielding populations that were 99.6% TCR-/CD3/-CD7-. 3CAR or 7CAR cells were able to exert specific cytotoxicity against their relevant target antigen in leukaemia lines with defined CD3 and/or CD7 expression as well as primary T-ALL cells. Co-cultured 3CAR/7CAR cells exhibited the highest level of cytotoxicity against T-ALL targets expressing both targetin vitroand anin vivohuman:murine chimeric model. While APOBEC editors can reportedly exhibit guide-independent deamination of both DNA and RNA, we found no evidence of promiscuous base conversion activity affecting CAR antigen specific binding regions which may otherwise redirect T cell specificity. Combinational infusion of fratricide resistant anti-T CAR T cells may enable enhanced molecular remission ahead of allogeneic haematopoietic stem cell transplantation for T cell malignancies.
The ectomycorrhizal community of urban linden trees in Gdańsk, Poland
Authors: Olchowik Jacek , Suchocka Marzena , Jankowski Paweł , Malewski Tadeusz , Hilszczańska Dorota 
Published: July 2020

AbstractThe linden tree (Tiliaspp.) is a popular tree for landscaping and urban environments in central and northwest European countries, and it is one of the most popular in cities in Poland. Ectomycorrhizal fungi form a symbiosis with many urban tree species and protect the host plant from heavy metals and against salinity. The aim of this study was to characterize the ECM fungal community of urban linden trees along the tree damage gradient. The study was performed on two homogeneous sites located in the centre of the city of Gdańsk, in northern Poland. The vitality assessment of urban linden trees was made according to Roloff’s classification. Tree damage classes were related to soil characteristics using principal component analysis. The five ectomycorrhizal fungal species were shared among all four tree damage classes, andCenococcum geophilumwas found to be the most abundant and frequent ectomycorrhizal fungal species in each class. Park soil had significantly lower pH and Na, Cl and Pb content than street soils. Our knowledge of ectomycorrhizal communities in urban areas is still limited, and these findings provide new insights into ectomycorrhizal distribution patterns in urban areas.
Repurposing Novobiocin for activity against latency associatedMycobacterium tuberculosisdrug target nicotinate-nucleotide adenylyltransferase (Rv2421c)
Authors: Cloete Ruben , Shahbaaz Mohd , Grobbelaar Melanie , Sampson Samantha L. , Christoffels Alan 
Published: July 2020

AbstractNicotinamide-nucleotide adenylyl transferase (Rv2421c) was selected as a potential drug target, because it has been shown,in vitro, to be essential forMycobacterium tuberculosisgrowth. It is conserved between mycobacterium species, is up-regulated during dormancy, has a known 3D crystal structure and has no known human homologs. A model of Rv2421c in complex with nicotinic acid adenine dinucleotide and magnesium ion was constructed and subject to virtual ligand screening against the Prestwick Chemical Library and the ZINC database, which yielded 155 potential hit molecules. 3D-QSAR studies of the 155 drug molecules indicated five compounds with similar inhibitory efficiencies compared to known inhibitors of Rv2421c. Molecular docking validation and molecular dynamics simulation analysis of the top five compounds indicated that the identified inhibitor molecules bind to Rv2421c with comparable efficiency as the substrate DND. Subsequentin vitrotesting of the five compounds identified Novobiocin sodium salt with activity againstMycobacterium tuberculosisat 50 μM, 25μM and weakly at 10μM concentrations. Although, Novobiocin salt targetsMycobacterium tuberculosisDNA gyrase B our studies suggest that it has the potential to be repurposed to inhibit Rv2421c. Subsequentin silicostructural analysis of known Novobiocin sodium salt derivatives against Rv2421c suggest promising alternatives for the treatment ofMycobacterium tuberculosis.Author SummaryRv2421c has been shown to be essential forMycobacterium tuberculosisgrowth, shares no homology to known proteins in the human host, is conserved between various Mycobacterium species, is up-regulated during the non-replicative metabolic growth phase, making it an attractive drug target. It has a known 3D structure which has been exploited to screen for putative compounds within the Prestwick chemical library and ZINC database, resulting in the successful identification of 155 candidate compounds. Thereafter 3D-QSAR, molecular docking and molecular dynamics simulation studies were used to prioritize five potential compounds. Of the five compounds testedin vitro, only one, a Novobiocin disodium salt, showed activity againstMycobacterium tuberculosisat 50, 25 and weakly at 10 μM concentrations. Novobiocin is known to targetMycobacterium tuberculosisDNA gyrase B, but emerging resistance stimulated us to seek derivatives to target Rv2421c as alternatives for the treatment ofMycobacterium tuberculosis. Docking studies supported the higher binding affinities of Novobiocin derivatives to Rv2421c compared to DNA gyrase B. Future studies will involve testing these Novobiocin derivatives for activity againstMycobacterium tuberculosis.
Acrylamide Inhibits Vaccinia Virus Through Vimentin-independent Anti-Viral Granule Formation
Authors: Wood Jennifer J , White Ian J , Mercer Jason 
Published: July 2020

AbstractThe replication and assembly of vaccinia virus (VACV), the prototypic poxvirus, occurs exclusively in the cytoplasm of host cells. While the role of cellular cytoskeletal components in these processes remains poorly understood, vimentin - a type III intermediate filament - has been shown to associate with viral replication sites and to be incorporated into mature VACV virions. Here we employed chemical and genetic approaches to further investigate the role of vimentin during the VACV lifecycle. The collapse of vimentin filaments, using acrylamide, was found to inhibit VACV infection at the level of genome replication, intermediate- and late- gene expression. However, we found that CRISPR-mediated knockout of vimentin did not impact VACV replication. Combining these tools, we demonstrate that acrylamide treatment results in the formation of antiviral granules (AVGs) known to mediate translational inhibition of many viruses. We conclude that vimentin is dispensable for poxvirus replication and assembly and that acrylamide, as a potent inducer of AVGs during VACV infection, serves to bolster cell’s antiviral response to poxvirus infection.Summary StatementAcrylamide inhibits poxvirus replication by inducing anti-viral granules and blocking translation. This inhibition is independent of the effect of acrylamide on vimentin filaments which were found to be dispensable for viral replication and assembly.
EdiTyper: a high-throughput tool for analysis of targeted sequencing data from genome editing experiments
Authors: Yahi Alexandre , Hoffman Paul , Brandt Margot , Mohammadi Pejman , Tatonetti Nicholas P. , Lappalainen Tuuli 
Published: July 2020

AbstractGenome editing experiments are generating an increasing amount of targeted sequencing data with specific mutational patterns indicating the success of the experiments and genotypes of clonal cell lines. We present EdiTyper, a high-throughput command line tool specifically designed for analysis of sequencing data from polyclonal and monoclonal cell populations from CRISPR gene editing. It requires simple inputs of sequencing data and reference sequences, and provides comprehensive outputs including summary statistics, plots, and SAM/BAM alignments. Analysis of simulated data showed that EdiTyper is highly accurate for detection of both single nucleotide mutations and indels, robust to sequencing errors, as well as fast and scalable to large experimental batches. EdiTyper is available in github (https://github.com/LappalainenLab/edityper) under the MIT license.
Nanopore sequencing reveals full-length Tropomyosin 1 isoforms and their regulation by RNA binding proteins during rat heart development
Authors: Cao Jun , Routh Andrew L. , Kuyumcu-Martinez Muge N. 
Published: July 2020

ABSTRACTAlternative splicing (AS) contributes to the diversity of the proteome by producing multiple isoforms from a single gene. Although short-read RNA sequencing methods have been the gold standard for determining AS patterns of genes, they have a difficulty in defining full length mRNA isoforms assembled using different exon combinations. Tropomyosin 1 (TPM1) is an actin binding protein required for cytoskeletal functions in non-muscle cells and for contraction in muscle cells.Tpm1undergoes AS regulation to generate muscle versus non-muscle TPM1 protein isoforms with distinct physiological functions. It is unclear which full lengthTpm1isoforms are produced via AS and how they are regulated during heart development. To address these, we utilized nanopore long-read cDNA sequencing without gene-specific PCR amplification. In rat hearts, we identified full lengthTpm1isoforms composed of distinct exons with specific exon linkages. We showed thatTpm1undergoes AS transitions during embryonic heart development such that muscle-specific exons are connected together generating predominantly muscle specificTpm1isoforms in adult hearts. We found that the RNA binding protein RBFOX2 controls AS of ratTpm1exon 6a, which is important for cooperative actin binding. Furthermore, RBFOX2 regulatesTpm1AS of exon 6a antagonistically to the RNA binding protein PTBP1. In sum, we defined full lengthTpm1isoforms with different exon combinations that are tightly regulated during cardiac development and provided insights into regulation ofTpm1AS by RNA binding proteins. Our results demonstrate that nanopore sequencing is an excellent tool to determine fulllength AS variants of muscle enriched genes.
Quantitative sub-cellular acyl-CoA analysis reveals distinct nuclear regulation
Authors: Trefely Sophie , Huber Katharina , Liu Joyce , Noji Michael , Stransky Stephanie , Singh Jay , Doan Mary T. , Lovell Claudia D. , von Krusenstiern Eliana , Jiang Helen , Bostwick Anna , Pepper Hannah L. , Izzo Luke , Zhao Steven , Xu Jimmy P. , Bedi Kenneth C. , Eduardo Rame J. , Bogner-Strauss Juliane G. , Mesaros Clementina , Sidoli Simone , Wellen Kathryn E. , Snyder Nathaniel W. 
Published: July 2020

SummaryQuantitative sub-cellular metabolomic measurements can yield crucial insights into the roles of metabolites in cellular processes, but are subject to multiple confounding factors. We developedStableIsotopeLabeling ofEssential nutrients in cellCulture –Sub-cellularFractionation (SILEC-SF), which uses isotope labeled internal standard controls that are present throughout fractionation and processing to quantify acyl-Coenzyme A thioesters in sub-cellular compartments by liquid chromatography-mass spectrometry. We tested SILEC-SF in a range of sample types and examined the compartmentalized responses to oxygen tension, cellular differentiation, and nutrient availability. Application of SILEC-SF to the challenging analysis of the nuclear compartment revealed a nuclear acyl-CoA profile distinct from that of the cytosol, with notable nuclear enrichment of propionyl-CoA. Using isotope tracing we identified the branched chain amino acid (BCAA) isoleucine as a major metabolic source of nuclear propionyl-CoA and histone propionylation, thus revealing a new mechanism of crosstalk between metabolism and the epigenome.
Adaptation of spontaneous activity in the developing visual cortex
Authors: Wosniack Marina E. , Kirchner Jan H. , Chao Ling-Ya , Zabouri Nawal , Lohmann Christian , Gjorgjieva Julijana 
Published: July 2020

AbstractSpontaneous activity drives the establishment of appropriate connectivity in different circuits during brain development. In the mouse primary visual cortex, two distinct patterns of spontaneous activity occur before vision onset: local low-synchronicity events originating in the retina, and global high-synchronicity events originating in the cortex. We sought to determine the contribution of these activity patterns to jointly organize network connectivity through different activity-dependent plasticity rules. We found that local events shape cortical input selectivity and topography, while global events have a homeostatic role regulating connection strength. To generate robust selectivity, we predicted that global events should adapt their amplitude to the history of preceding cortical activation, and confirmed by analyzingin vivospontaneous cortical activity. This adaptation led to the sparsification of spontaneous activity on a slower timescale during development, demonstrating the remarkable capacity of the developing sensory cortex to acquire sensitivity to visual inputs after eye-opening.
Genetically-encoded fluorescent biosensor for rapid detection of protein expression
Authors: Eason Matthew G. , Pandelieva Antonia T. , Mayer Marc M. , Khan Safwat T. , Garcia Hernan G. , Chica Roberto A. 
Published: July 2020

AbstractFluorescent proteins are widely used as fusion tags to detect protein expressionin vivo. To become fluorescent, these proteins must undergo chromophore maturation, a slow process with a half-time of 5 to >30 min that causes delays in real-time detection of protein expression. Here, we engineer a genetically-encoded fluorescent biosensor to enable detection of protein expression within seconds in live cells. This sensor for transiently-expressed proteins (STEP) is based on a fully matured but dim green fluorescent protein in which pre-existing fluorescence increases 11-foldin vivofollowing the specific and rapid binding of a protein tag (Kd120 nM,kon1.7 × 105M−1s−1). In liveE. colicells, our STEP biosensor enables detection of protein expression twice as fast as the use of standard fluorescent protein fusions. Our biosensor opens the door to the real-time study of short-timescale processes in research model animals with high spatiotemporal resolution.
The mode of expression divergence inDrosophilafat body is infection-specific
Authors: Ramirez-Corona Bryan A. , Fruth Stephanie , Ofoegbu Oluchi , Wunderlich Zeba 
Published: July 2020

AbstractTranscription is controlled by the interactions ofcis-acting DNA elements with diffusibletrans-acting factors. Changes incisortransfactors can drive expression divergence within and between species, and the relative prevalence of each can reveal the evolutionary history and pressures that drive expression variation. Previous work delineating the mode of expression divergence in animals has largely used whole body expression measurements in a single condition. Sincecis-acting elements often drive expression in a subset of cell types or conditions, these measurements may not capture the complete contribution ofcis-acting changes. Here, we quantify the mode of expression divergence in theDrosophilafat body, the primary immune organ, in several conditions. We performed allele-specific expression analysis using two geographically distinct lines ofD. melanogasterand their F1 hybrids. We measured expression in the absence of infection and in separate infections with Gram-negativeS. marcescensor Gram-positiveE. faecalisbacteria, which trigger the two primary signaling pathways in theDrosophilainnate immune response. The mode of expression divergence strongly depends on the condition, withtrans-acting effects dominating in response to Gram-positive infection andcis-acting effects dominating in Gram-negative and pre-infection conditions. Expression divergence in several receptor proteins may underlie the infection-specifictranseffects. Before infection, when the fat body has a metabolic role, there are many compensatory effects, changes incisandtransthat counteract each other to maintain expression levels. This work demonstrates that within a single tissue, the mode of expression divergence varies between conditions and suggests that these differences reflect the diverse evolutionary histories of host-pathogen interactions.