bluuebunny/crossref_metadata_2025_split huggingface dataset:
Single Nucleus RNA Sequencing of Human Pancreatic Islets In Vitro and In Vivo Identifies New Gene Sets and Three β-Cell Subpopulations with Different Transcriptional Profile
Authors: Kang Randy B. , Li Yansui , Rosselot Carolina , Zhang Tuo , Siddiq Mustafa , Rajbhandari Prashant , Stewart Andrew F. , Scott Donald K. , Garcia-Ocana Adolfo , Lu Geming 
Published: May 2022

AbstractSingle-cell RNA sequencing (scRNA-seq) has provided valuable insights into human islet cell types and their corresponding stable gene expression profiles. However, this approach requires cell dissociation that complicates its utility in vivo and provides limited information on the active transcriptional status of islet cells. On the other hand, single-nucleus RNA sequencing (snRNA-seq) does not require cell dissociation and affords enhanced information from intronic sequences that can be leveraged to identify actively transcribing genes in islet cell populations. Here, we first sought to compare scRNA-seq and snRNA-seq analysis of human islets in vitro using exon reads or combined exon and intron reads, respectively. Datasets reveal similar human islet cell clusters using both approaches. In the snRNA-seq data, however, the top differentially expressed genes in human islet endocrine cells are not the canonical genes but a new set of non-canonical gene markers including ZNF385D, TRPM3, LRFN2, PLUT (β cells), PTPRT, FAP, PDK4, LOXL4 (α cells), LRFN5, ADARB2, ERBB4, KCNT2 (δ cells) and CACNA2D3, THSD7A, CNTNAP5, RBFOX3 (γ cells). Notably, these markers also accurately define endocrine cell populations in human islet grafts in vivo. Further, by integrating the information from nuclear and cytoplasmic transcriptomes, we identify three β-cell sub-clusters: an active INS mRNA transcribing cluster (β1), an intermediate INS mRNA-transcribing cluster (β2), and a mature INS mRNA rich cluster (β3). These display distinct gene expression patterns representing different biological dynamic states both in vitro and in vivo. Interestingly, the INS mRNA rich cluster (β3) becomes the predominant sub-cluster in vivo. In summary, snRNA-seq analysis of human islet cells is a previously unrecognized tool that can be accurately employed for improved identification of human islet cell types and their transcriptional status in vivo.
Cooperative assembly confers regulatory specificity and long-term genetic circuit stability
Authors: Bragdon Meghan D. J. , Patel Nikit , Chuang James , Levien Ethan , Bashor Caleb J. , Khalil Ahmad S. 
Published: May 2022

ABSTRACTIn eukaryotes, links in gene regulatory networks are often maintained through cooperative self-assembly between transcriptional regulators (TRs) and DNA cis-regulatory motifs, a strategy widely thought to enable highly specific regulatory connections to be formed between otherwise weakly-interacting, low-specificity molecular components. Here, we directly test whether this regulatory strategy can be used to engineer regulatory specificity in synthetic gene circuits constructed in yeast. We show that circuits composed of artificial zinc-finger TRs can be effectively insulated from aberrant misregulation of the host cell genome by using cooperative multivalent TR assemblies to program circuit connections. As we demonstrate in experiments and mathematical models, assembly-mediated regulatory connections enable mitigation of circuit-driven fitness defects, resulting in genetic and functional stability of circuits in long-term continuous culture. Our naturally-inspired approach offers a simple, generalizable means for building evolutionarily robust gene circuits that can be scaled to a wide range of host organisms and applications.
PP6 regulation of Aurora A-TPX2 limits NDC80 phosphorylation and mitotic spindle size
Authors: Sobajima Tomoaki , Kowalczyk Katarzyna M. , Skylakakis Stefanos , Hayward Daniel , Fulcher Luke J. , Neary Colette , Batley Caleb , Kurlekar Samvid , Roberts Emile , Gruneberg Ulrike , Barr Francis A. 
Published: May 2022

AbstractAmplification of the mitotic kinase Aurora A or loss of its regulator protein phosphatase 6 (PP6) have emerged as drivers of genome instability. Cells lacking PPP6C, the catalytic subunit of PP6, have amplified Aurora A activity and as we show here, enlarged mitotic spindles which fail to hold chromosomes tightly together in anaphase, causing defective nuclear structure. Using functional genomics to shed light on the processes underpinning these changes, we discover synthetic lethality between PPP6C and the kinetochore protein NDC80. We find that NDC80 is phosphorylated on multiple N-terminal sites during spindle formation by Aurora A-TPX2, exclusively at checkpoint-silenced, microtubule-attached kinetochores. NDC80 phosphorylation persists until spindle disassembly in telophase, is increased in PPP6C-knockout cells and, and is Aurora B-independent. An Aurora-phosphorylation-deficient NDC80-9A mutant reduces spindle size and suppresses defective nuclear structure in PPP6C-knockout cells. By regulating NDC80 phosphorylation by Aurora A-TPX2, PP6 plays an important role in mitotic spindle formation and size control, and thus the fidelity of cell division.
Phenotypic variation and quantitative trait loci for resistance to southern anthracnose and clover rot in red clover
Authors: Frey Lea A. , Vleugels Tim , Ruttink Tom , Schubiger Franz X. , Pegard Marie , Skøt Leif , Grieder Christoph , Studer Bruno , Roldán-Ruiz Isabel , Kölliker Roland 
Published: May 2022

AbstractRed clover (Trifolium pratense L.) is an important forage legume of temperate regions, particularly valued for its high yield potential and its high forage quality. Despite substantial breeding progress during the last decades, continuous improvement of cultivars is crucial to ensure yield stability in view of newly emerging diseases or changing climatic conditions. The high amount of genetic diversity present in red clover ecotypes, landraces and cultivars provides an invaluable, but often unexploited resource for the improvement of key traits such as yield, quality, and resistance to biotic and abiotic stresses.A collection of 397 red clover accessions was genotyped using a pooled genotyping-by-sequencing approach with 200 plants per accession. Resistance to the two most pertinent diseases in red clover production, southern anthracnose caused by Colletotrichum trifolii, and clover rot caused by Sclerotinia trifoliorum, was assessed using spray inoculation. The mean survival rate for southern anthracnose was 22.9% and the mean resistance index for clover rot was 34.0%. Genome-wide association analysis revealed several loci significantly associated with resistance to southern anthracnose and clover rot. Most of these loci are in coding regions. One quantitative trait locus (QTL) on chromosome 1 explained 16.8% of the variation in resistance to southern anthracnose. For clover rot resistance we found eight QTL, explaining together 80.2% of the total phenotypic variation. The SNPs associated with these QTL provide, once validated, a promising resource for marker-assisted selection in existing breeding programs, facilitating the development of novel cultivars with increased resistance against two devastating fungal diseases of red clover.Key messageHigh variability for and candidate loci associated with resistance to southern anthracnose and clover rot in a worldwide collection of red clover provide a first basis for genomics-assisted breeding.
Loss of heterozygosity spectrum varies with ploidy levels in natural yeast populations
Authors: Dutta Abhishek , Dutreux Fabien , Schacherer Joseph 
Published: May 2022

AbstractThe appearance of genomic variations such as loss of heterozygosity (LOH) has a significant impact on phenotypic diversity observed in a population. Recent large-scale yeast population genomic surveys have shown a high frequency of these events in natural isolates and more particularly in polyploids. However, the frequency, extent, and spectrum of LOH in polyploid organisms have never been explored and is poorly characterized to date. Here, we accumulated 5,163 LOH events over 1,875 generations in 76 mutation accumulation (MA) lines comprising nine natural heterozygous diploid, triploid, and tetraploid natural S. cerevisiae isolates from different ecological and geographical origins. We found that the rate and spectrum of LOH are variable across ploidy levels. Of the total accumulated LOH events, 8.5%, 21%, and 70.5% of them were found in diploid, triploid, and tetraploid MA lines, respectively. Our results clearly shows that the frequency of generated LOH events increases with ploidy level. In fact, the cumulative LOH rates were estimated to be 9.3 × 10−3, 2.2 × 10−2, and 8.4 × 10−2 events per division for diploids, triploids, and tetraploids, respectively. In addition, a clear bias towards the accumulation of interstitial and short LOH tracts is observed in triploids and tetraploids compared to diploids. The variation of the frequency and spectrum of LOH events across ploidy level could be related to the genomic instability, characterizing higher ploidy isolates.Highlights-Yeast isolates with a high ploidy level (e.g., 3n and 4n) accumulate a larger number of LOH events compared to diploids.-The type of events and the LOH spectrum are both variable according to the ploidy levels.-A clear bias towards the accumulation of short LOH tracts is observed in polyploids compared to diploids.-The variation in LOH spectrum is most likely related to genomic instability present in higher ploidy strains.
Glucocorticoid receptor activation during human microglial differentiation leads to genomic instability and senescence
Authors: Wei Jingzhang , Arber Charles , Wray Selina , Hardy John , Piers Thomas M , Pocock Jennifer M 
Published: May 2022

SummaryEarly life stress, prenatal exposure to glucocorticoids (GCs), confers a higher risk of psychiatric and neurodevelopmental disorders in children. Increasingly, the importance of microglia in these disorders has been recognised. Studies on GCs exposure during microglial development have been limited, and there are few, if any, human studies. We established an in vitro model of ELS by continuous pre-expoure of human iPS-microglia to GCs during primitive haematopoiesis (the critical stage of iPS-microglial differentiation) and then examined how this exposure affected the microglial phenotype as they differentiated and matured to microglia. The iPS-microglia predominately expressed glucocorticoid receptors over mineralocorticoid receptors, and the GR-α splice variant. Chronic GCs exposure during primitive haematopoiesis was able to recapitulate in vivo ELS effects. Thus pre-exposure to prolonged GCs resulted in increased type I interferon signalling, the presence of Cyclic GMP-AMP synthase-positive (cGAS) micronuclei, and cellular senescence in the matured iPS-microglia. The findings from this in vitro ELS model have ramifications for the responses of microglia in the pathogenesis of GC-mediated ELS- associated disorders such as schizophrenia, attention-deficit hyperactivity disorder and autistic spectrum disorder.HighlightsHuman iPS-derived-microglia predominantly express glucocorticoid receptor NR3C1 compared with mineralocorticoid receptor NR3C2, and a predominant splice variant of the NR3C1 of GR-α.GC expression shows a differentiation-linked increment from iPSC to iPS-microglia.An early-life stress model was established by exposing iPSC to glucocorticoids during primitive haematopoiesis.RNA-seq analysis revealed that this early glucocorticoid exposure led to enhanced type I interferon inducible gene expression in the subsequent iPS-microglia.Furthermore, micronuclei formation and cellular senescence markers were upregulated in the iPSC-microglia, indicating genomic instability due to early chronic GC exposure.These findings have ramifications for the microglial responses in ELS linked neurodevelopmental disorders such as schizophrenia, attention-deficit hyperactivity disorder and autistic spectrum disorder.Graphical abstract
MPS1 localizes to microtubule-attached kinetochores and actively promotes microtubule release
Authors: Hayward Daniel , Roberts Emile , Gruneberg Ulrike 
Published: May 2022

SummaryIn eukaryotes, the spindle assembly checkpoint protects genome stability in mitosis by preventing anaphase onset until incorrect microtubule-kinetochore attachment geometries have been eliminated and chromosome biorientation has been completed. These error correction and checkpoint processes are linked by two conserved serine/threonine kinases, Aurora B and MPS1 1, 2. In the prevailing model for spindle checkpoint signaling, MPS1 detects microtubule-free kinetochores generated by the Aurora B-dependent error correction pathway, and initiates spindle checkpoint signaling. However, we find that MPS1 initially localizes to microtubule-attached kinetochores in a manner dependent on the relative activities of Aurora B and its counteracting phosphatase PP2A-B56, and then actively promotes microtubule release. Thus, MPS1 is not a passive sensor for the microtubule binding state of kinetochores but actively generates microtubule-free checkpoint signaling kinetochores. MPS1 is thus instrumental for both the initial resolution of incorrect microtubule-kinetochore attachments and the downstream propagation of spindle checkpoint signaling.
Identification and characterisation of potent anti-MRSA phloroglucinol derivatives of Dryopteris crassirhizoma Nakai
Authors: Bhowmick Sumana , Beckmann Manfred , Shen Jianying , Mur Luis A.J. 
Published: May 2022

AbstractTraditional Chinese Medicine (TCM) has been used to treat infectious diseases and could offer potential drug leads. This study evaluates the in vitro antimicrobial activities commercially sourced Dryopteris crassirhizoma Nakai whose authenticity was confirmed by DNA barcoding based on the ribulose bisphosphate carboxylase (rbcL) gene. Powdered rhizomes were sequentially extracted using n-hexane, dichloromethane, ethyl acetate and methanol at ambient temperature. The dried extracts at different concentrations were tested for antimicrobial activities against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus (MRSA), and Mycobacterium smegmatis. D. crassirhizoma extracts exhibited significant antimicrobial activities only against MRSA. Activity-led fractionations of D. crassirhizoma and characterisation by Ultra performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) identified two potent anti-MRSA phloroglucinol derivatives: Norflavaspidic acid AB and flavaspidic acid AB. The impact of norflavaspidic acid AB on MRSA cells was examined using untargeted metabolomic analysis and compared to that of other established antibiotics (all treatments normalized to MIC50 at 6 h). This suggested that norflavaspidic acid AB had a distinctive effect which involved targeting bioenergetic transformation, metabolism, and particularly acetyl CoA, in MRSA cells. No cytotoxicity was observed for norflavaspidic acid AB against murine HepG2 cells. This study requires further experimental validation but can have indicated a naturally available compound that could help counter the threat of clinically relevant strains with antibiotic resistance.
Bayesian species distribution models integrate presence-only and presence-absence data to predict deer distribution and relative abundance
Authors: Morera-Pujol Virginia , Mostert Philip S. , Murphy Kilian , Burkitt Tim , Coad Barry , McMahon Barry J. , Nieuwenhuis Maarten , Morelle Kevin , Ward Alastair , Ciuti Simone 
Published: May 2022

AbstractThe use of georeferenced information on the presence of a species to predict its distribution across a geographic area is one of the most common tools in management and conservation. The collection of high-quality presence-absence data through structured surveys is, however, expensive, and managers usually have more abundant low-quality presence-only data collected by citizen scientists, opportunistic observations, and culling returns for game species. Integrated Species Distribution Models (ISDMs) have been developed to make the most of the data available by combining the higher-quality, but usually less abundant and more spatially restricted presence-absence data, with the lower quality, unstructured, but usually more extensive and abundant presence-only data. Joint-likelihood ISDMs can be run in a Bayesian context using INLA (Integrated Nested Laplace Approximation) methods that allow the addition of a spatially structured random effect to account for data spatial autocorrelation. These models, however, have only been applied to simulated data so far. Here, for the first time, we apply this approach to empirical data, using presence-absence and presence-only data for the three main deer species in Ireland: red, fallow and sika deer. We collated all deer data available for the past 15 years and fitted models predicting distribution and relative abundance at a 25 km2resolution across the island. Models’ predictions were associated to spatial estimate of uncertainty, allowing us to assess the quality of the model and the effect that data scarcity has on the certainty of predictions. Furthermore, we validated the three species-specific models using independent deer hunting returns. Our work clearly demonstrates the applicability of spatially-explicit ISDMs to empirical data in a Bayesian context, providing a blueprint for managers to exploit unused and seemingly unusable data that can, when modelled with the proper tools, serve to inform management and conservation policies.
Deconstructing cold-induced brown adipocyte neogenesis in mice
Authors: Burl Rayanne B. , Rondini Elizabeth A. , Wei Hongguang , Pique-Regi Roger , Granneman James G. 
Published: May 2022

ABSTRACTCold-exposure triggers neogenesis in classic interscapular brown adipose tissue (BAT) that involves activation of b1-adrenergic receptors, proliferation of PDGFRA+ adipose tissue stromal cells (ASCs), and recruitment of immune cells whose phenotypes are presently unknown. Single- cell RNA-sequencing (scRNA-seq) identified three ASC subpopulations that occupied distinct tissue locations. Of these, interstitial ASC1 were found to be direct precursors of new brown adipocytes (BA). Surprisingly, knockout of b1-adrenergic receptors in ASCs did not prevent cold- induced neogenesis, whereas pharmacological activation of the b3-adrenergic receptor on BAs was sufficient, suggesting that signals derived from mature BAs indirectly trigger ASC proliferation and differentiation. In this regard, cold exposure induced the delayed appearance of multiple macrophage and dendritic cell populations whose recruitment strongly correlated with the onset and magnitude of neogenesis across diverse experimental conditions. High resolution immunofluorescence and single molecule fluorescence in situ hybridization demonstrated that cold-induced neogenesis involves dynamic interactions between ASC1 and recruited immune cells that occur on the micrometer scale in distinct tissue regions. Our results indicate that neogenesis is not a reflexive response of progenitors to b-adrenergic signaling, but rather is a complex adaptive response to elevated metabolic demands within brown adipocytes.
Unbiased proteomic and forward genetic screens reveal that mechanosensitive ion channel MSL10 functions at ER-plasma membrane contact sites in Arabidopsis thaliana
Authors: Codjoe Jennette M. , Richardson Ryan A. , Haswell Elizabeth S. 
Published: May 2022

ABSTRACTMechanosensitive (MS) ion channels are an evolutionarily conserved way for cells to sense mechanical forces and transduce them into ionic signals. The channel properties of Arabidopsis thaliana MscS-Like (MSL)10 have been well studied, but how MSL10 signals remains largely unknown. To uncover signaling partners of MSL10, we employed both a proteomic screen and a forward genetic screen; both unexpectedly implicated ER-plasma membrane contact sites (EPCSs) in MSL10 function. The proteomic screen revealed that MSL10 associates with multiple proteins associated with EPCSs. Of these, only VAMP-associated proteins (VAP)27-1 and VAP27-3 interacted directly with MSL10. The forward genetic screen, for suppressors of a gain-of-function MSL10 allele (msl10-3G, MSL10S640L), identified mutations in the synaptotagmin (SYT)5 and SYT7 genes. We also found that EPCSs were expanded in leaves of msl10-3G plants compared to the wild type. Taken together, these results indicate that MSL10 can be found at EPCSs and functions there, providing a new cell-level framework for understanding MSL10 signaling. In addition, placing a mechanosensory protein at EPCS provides new insight into the function and regulation of this type of subcellular compartment.
A critical role for touch neurons in a skin-brain pathway for stress resilience
Authors: Schaffler Melanie D , Johnson Micah , Hing Ben , Kahler Paul , Hultman Ian , Srivastava Sanvesh , Arnold Justin , Blendy Julie N , Hultman Rainbo , Abdus-Saboor Ishmail 
Published: May 2022

AbstractSocial touch can act as a stress buffer, reducing behavioral and physiological responses to stressful scenarios. However, skin-brain touch pathways that promote stress resilience remain unknown. Here, we show that mice with an early life genetic ablation of Mrgprb4-lineage touch neurons display stress vulnerability behaviors in adulthood. Chemogenetic activation of these touch neurons reduced corticosterone levels under mild acute stress conditions. In addition, whole-brain c-Fos activity mapping while chemogenetically turning on these neurons uncovered differential neural activity patterns in brain areas relevant to somatosensation, reward, and affect. To gain mechanistic insight into this skin-brain touch pathway for stress susceptibility, we used multi-circuit neurophysiological recordings across seven brain regions at baseline and after stress in mice that had Mrgprb4-lineage touch neurons ablated in early life. Interestingly, the Mrgprb4-lineage neuron-ablated mice have alterations in local field potential phase directionality and power in the theta frequencies in mesolimbic reward regions, which may underlie our observed stress susceptibility phenotype. Together, these studies revealed that sensory neurons in the skin engage networks across the brain to promote stress resilience.
A Well-Characterized Polycistronic-Like Gene Expression System in Yeast
Authors: Mukherjee Minakshi , Wang Zhen Q. 
Published: May 2022

AbstractEfficient expression of multiple genes is critical to yeast metabolic engineering because of the increasing complexity of engineered pathways. A yeast polycistronic expression system is of particular interest because one promoter can drive the expression of multiple genes. Polycistronic expression thus requires less genetic material and minimizes undesirable recombination due to repeated use of the same promoter. It also decreases the number of DNA parts necessary for cloning a multi-gene construct. 2A viral peptides enable the co-translation of multiple proteins from one mRNA by ribosomal skipping. However, the wide adaptation of this strategy for polycistronic-like gene expression in yeast awaits in-depth characterizations. Additionally, a one-step assembly of such a polycistronic-like system is highly desirable. To this end, we have developed a MoClo compatible 2A peptide-based polycistronic-like system capable of expressing multiple genes from a single promoter in yeast. Characterizing the bi-, tri, and quad-cistronic expression of fluorescent proteins showed high cleavage efficiencies of three 2A peptides. The expression level of each protein decreases as it moves away from the promoter. Additionally, the impact of a C-terminal 2A peptide on protein function is dependent on the protein sequence. Applying the polycistronic-like system for geraniol production resulted in similar or higher titers compared to a conventional monocistronic construct. In summary, this highly-characterized polycistronic-like gene expression system is another tool to facilitate multi-gene expression in yeast.