bluuebunny/crossref_metadata_2025_split huggingface dataset:
Restricted proliferation during neurogenesis contributes to regionalization of the amphioxus nervous system
Authors: Gattoni Giacomo , Andrews Toby GR , Benito-Gutiérrez Elia 
Published: December 2021

AbstractThe central nervous system of the cephalochordate amphioxus consists of a dorsal neural tube with an anterior brain. Two decades of gene expression analyses in developing amphioxus embryos have shown that despite the lack of overt segmentation the amphioxus neural tube is highly regionalized at the molecular level. However, little is known about the mechanisms that generate such precise regionalization. Proliferation is a key driver of pattern formation and cell type diversification, but in amphioxus it has never been studied in detail nor in the specific context of neurogenesis. Here, we describe the dynamics of cell division during the formation of the central nervous system in amphioxus embryos and its contributions to the regionalization of the neural axis. We show that after gastrulation, proliferation pauses to become spatially restricted to the anterior and posterior ends of the neural tube at neurula stages. Only at the onset of larval life, proliferation resumes in the central part of the nervous system. By marking specific populations and inhibiting cell division during neurulation, we demonstrate that proliferation in the anterior cerebral vesicle is required to establish the full cell type repertoire of the frontal eye complex and the putative hypothalamic region of the amphioxus brain, while posterior proliferating progenitors, which were found here to derive from the dorsal lip of the blastopore, contribute to elongate the caudal floor plate. Between these proliferative domains, we find trunk nervous system differentiation is independent from cell division, which decreases during neurulation and resumes at the early larval stage. Taken together, our results highlight multiple roles for proliferation in shaping the amphioxus nervous system.
Distinct patterns of genetic variation at low-recombining genomic regions represent haplotype structure
Authors: Ishigohoka Jun , Bascón-Cardozo Karen , Bours Andrea , Fuß Janina , Rhie Arang , Mountcastle Jacquelyn , Haase Bettina , Chow William , Collins Joanna , Howe Kerstin , Uliano-Silva Marcela , Fedrigo Olivier , Jarvis Erich D. , Pérez-Tris Javier , Illera Juan Carlos , Liedvogel Miriam 
Published: December 2021

AbstractGenetic variation of the entire genome represents population structure, yet individual loci can show distinct patterns. Such deviations identified through genome scans have often been attributed to effects of selection instead of randomness. This interpretation assumes that long enough genomic intervals average out randomness in underlying genealogies, which represent local genetic ancestries. However, an alternative explanation to distinct patterns has not been fully addressed: too few genealogies to average out the effect of randomness. Specifically, distinct patterns of genetic variation may be due to reduced local recombination rate, which reduces the number of genealogies in a genomic window. Here, we associate distinct patterns of local genetic variation with reduced recombination rates in a songbird, the Eurasian blackcap (Sylvia atricapilla), using genome sequences and recombination maps. We find that distinct patterns of local genetic variation reflect haplotype structure at low-recombining regions either shared in most populations or found only in a few populations. At the former species-wide low-recombining regions, genetic variation depicts conspicuous haplotypes segregating in multiple populations. At the latter population-specific low-recombining regions, genetic variation represents variance among cryptic haplotypes within the low-recombining populations. With simulations, we confirm that these distinct patterns of haplotype structure evolve due to reduced recombination rate, on which the effects of selection can be overlaid. Our results highlight that distinct patterns of genetic variation can emerge through evolution of reduced local recombination rate. Recombination landscape as an evolvable trait therefore plays an important role determining the heterogeneous distribution of genetic variation along the genome.
A Single-copy Knock In Translating Ribosome ImmunoPrecipitation (SKI TRIP) tool kit for tissue specific profiling of actively translated mRNAs in C. elegans
Authors: Wester Laura E. , Lanjuin Anne , Bruckisch Emanuel H. W. , Perez Matos Maria C. , Heintz Caroline , Denzel Martin S. , Mair William B. 
Published: December 2021

AbstractTranslating Ribosome Affinity Purification (TRAP) methods have emerged as a powerful approach to profile actively translated transcripts in specific cell and tissue types. Epitope tagged ribosomal subunits are expressed in defined cell populations and used to pull down ribosomes and their associated mRNAs, providing a snapshot of cell type-specific translation occurring in that space and time. Current TRAP toolkits available to the C. elegans community have been built using multi-copy arrays, randomly integrated in the genome. Here we introduce a Single-copy Knock In Translating Ribosome ImmunoPrecipitation (SKI TRIP) tool kit, a collection of C. elegans strains engineered by CRISPR in which tissue specific expression of FLAG tagged ribosomal subunit protein RPL-22 is driven by cassettes present in single copy from defined sites in the genome. In depth characterization of the SKI TRIP strains and methodology shows that 3xFLAG tagged RPL-22 expressed from its endogenous locus or within defined cell types incorporates into actively translating ribosomes and can be used to efficiently and cleanly pull-down cell type specific transcripts without impacting overall mRNA translation or fitness of the animal. We propose SKI TRIP use for the study of processes that are acutely sensitive to changes in translation, such as aging.
ERK2 MAP kinase regulates SUFU binding by multisite phosphorylation of GLI1
Authors: Bardwell A. Jane , Wu Beibei , Sarin Kavita Y. , Waterman Marian L. , Atwood Scott X. , Bardwell Lee 
Published: December 2021

AbstractThere is considerable evidence that crosstalk between the Hedgehog and MAPK signaling pathways occurs in several types of cancer, and contributes to the emergence of clinical resistance to Hedgehog pathway inhibitors. Here we show that MAP kinase-mediated phosphorylation weakens the binding of the GLI1 transcription factor to its negative regulator SUFU. ERK2 phosphorylates GLI1 on three evolutionarily-conserved target sites (S102, S116 and S130) located near the high-affinity binding site for SUFU; these phosphorylations cooperate to weaken the affinity of GLI1-SUFU binding by over 25 fold. Phosphorylation of any one, or even any two, of the three sites does not result in the level of SUFU release seen when all three sites are phosphorylated. Tumor-derived mutations in R100 and S105, residues bordering S102, also diminish SUFU binding, collectively defining a novel evolutionarily-conserved SUFU-affinity-modulating region. In cultured mammalian cells, GLI1 variants containing phosphomimetic substitutions of S102, S116 and S130 displayed an increased ability to drive transcription. We conclude that of multisite phosphorylation of GLI1 by ERK2 or other MAP kinases weakens GLI1-SUFU binding, thereby facilitating GLI1 activation and contributing to both physiological and pathological crosstalk.
Single-cell RNA sequencing reveals a role for reactive oxygen species and peroxiredoxins in fatty-acid-induced rat ß-cell proliferation
Authors: Vivoli Alexis , Ghislain Julien , Filali-Mouhim Ali , Angeles Zuraya Elisa , Castell Anne-Laure , Sladek Robert , Poitout Vincent 
Published: December 2021

SUMMARYThe functional mass of insulin-secreting pancreatic β cells expands to maintain glucose homeostasis in the face of nutrient excess, in part via replication of existing β cells. To decipher the underlying molecular mechanisms, we assessed β-cell proliferation in isolated rat islets exposed to glucose and oleate or palmitate for 48 h and analyzed the transcriptional response by single-cell RNA sequencing. Unsupervised clustering of pooled β cells identified subpopulations, including proliferating β cells. β-cell proliferation increased in response to oleate but not palmitate. Both fatty acids enhanced the expression of genes involved energy metabolism and mitochondrial activity. Comparison of proliferating vs. non-proliferating β cells and pseudotime ordering suggested the involvement of reactive oxygen species (ROS) and peroxiredoxin signaling. Accordingly, the antioxidant N-acetyl cysteine and the peroxiredoxin inhibitor Conoidin A both blocked oleate-induced β-cell proliferation. Our data reveal a key role for ROS signaling in β-cell proliferation in response to nutrients.
Structure of anchorless RML prion reveals motif variation between strains
Authors: Hoyt Forrest , Standke Heidi G. , Artikis Efrosini , Schwartz Cindi L. , Hansen Bryan , Li Kunpeng , Hughson Andrew G. , Manca Matteo , Thomas Olivia R. , Raymond Gregory J. , Baron Gerald S. , Caughey Byron , Kraus Allison 
Published: December 2021

Little is known about the structural basis of prion strains. Here we provide a high (3.0 Å) resolution cryo-electron microscopy-based structure of brain-derived fibrils of the mouse anchorless RML scrapie strain which, like the recently determined hamster 263K strain, has a parallel in-register β-sheet-based core. However, detailed comparisons reveal that variations in shared structural motifs provide a basis for prion strain determination.One-sentence summaryCryo-electron microscopy reveals a near-atomic structure of an infectious, brain-derived murine prion fibril and strain differences.
Biophysical and pharmacokinetic characterization of a small-molecule inhibitor of RUNX1/ETO tetramerization with anti-leukemic effects
Authors: Gopalswamy Mohanraj , Kroeger Tobias , Bickel David , Frieg Benedikt , Akter Shahina , Schott-Verdugo Stephan , Viegas Aldino , Pauly Thomas , Mayer Manuela , Przibilla Julia , Reiners Jens , Nagel-Steger Luitgard , Smits Sander H.J. , Groth Georg , Etzkorn Manuel , Gohlke Holger 
Published: December 2021

AbstractAcute myeloid leukemia (AML) is a malignant disease of immature myeloid cells and the most prevalent acute leukemia among adults. The oncogenic homo-tetrameric fusion protein RUNX1/ETO results from the chromosomal translocation t(8;21) and is found in AML patients. The nervy homology region 2 (NHR2) domain of ETO mediates tetramerization; this oligomerization is essential for oncogenic activity. Previously, we identified the first-in-class small-molecule inhibitor of NHR2 tetramer formation, 7.44, which was shown to specifically interfere with NHR2, restore gene expression down-regulated by RUNX1/ETO, inhibit the proliferation of RUNX1/ETO-depending SKNO-1 cells, and reduce the RUNX1/ETO-related tumor growth in a mouse model. However, no biophysical and structural characterization of 7.44 binding to the NHR2 domain has been reported. Likewise, the compound has not been characterized as to physicochemical, pharmacokinetic, and toxicological properties. Here, we characterize the interaction between the NHR2 domain of RUNX1/ETO and 7.44 by biophysical assays and show that 7.44 interferes with NHR2 tetramer stability and leads to an increase in the dimer population of NHR2. The affinity of 7.44 with respect to binding to NHR2 is Klig = 3.95 ± 1.28 μM. By NMR spectroscopy combined with molecular dynamics simulations, we show that 7.44 binds with both heteroaromatic moieties to NHR2 and interacts with or leads to conformational changes in the N-termini of the NHR2 tetramer. Finally, we demonstrate that 7.44 has favorable physicochemical, pharmacokinetic, and toxicological properties. Together with biochemical, cellular, and in vivo assessments, the results reveal 7.44 as a lead for further optimization towards targeted therapy of t(8;21) AML.
Effects of α-crystallin gene knockout on zebrafish lens development
Authors: Posner Mason , Murray Kelly L. , Andrew Brandon , Brdicka Stuart , Roberts Alexis , Franklin Kirstan , Hussen Adil , Kaye Taylor , Kepp Emmaline , McDonald Mathew S. , Snodgrass Tyler , Zientek Keith , David Larry L. 
Published: December 2021

AbstractThe α-crystallin small heat shock proteins contribute to the transparency and refractive properties of the vertebrate eye lens and prevent the protein aggregation that would otherwise produce lens cataracts, the leading cause of human blindness. There are conflicting data in the literature as to what role the α-crystallins may play in early lens development. In this study, we used CRISPR gene editing to produce zebrafish lines with null mutations for each of the three α-crystallin genes (cryaa, cryaba and cryabb). The absence of normal protein was confirmed by mass spectrometry, and lens phenotypes were assessed with differential interference contrast microscopy and histology. Loss of αA-crystallin produced a variety of lens defects with varying severity in larval lenses at 3 and 4 dpf but little substantial change in normal fiber cell denucleation. Loss of either αBa- or full-length αBb-crystallin produced no substantial lens defects. Mutation of each α-crystallin gene did not alter the mRNA levels of the remaining two, suggesting a lack of genetic compensation. These data confirm a developmental role for αA-crystallin in lens development, but the range of phenotype severity suggests that its loss simply increases the chance for defects and that the protein is not essential. Our finding that cryaba and cryabb mutants lack noticeable lens defects is congruent with insubstantial transcript levels in lens epithelial and fiber cells. Future experiments can explore the molecular consequences of cryaa mutation and causes of lens defects in this null mutant, as well as the roles of other genes in lens development and function.
A discovery-based proteomics approach identifies protein disulfide isomerase (PDIA1) as a biomarker of β cell stress in type 1 diabetes
Authors: Syed Farooq , Singhal Divya , Raedschelders Koen , Krishnan Preethi , Bone Robert N. , McLaughlin Madeline R. , Van Eyk Jennifer E. , Mirmira Raghavendra G. , Yang Mei-Ling , Mamula Mark J. , Wu Huanmei , Liu Xiaowen , Evans-Molina Carmella 
Published: December 2021

ABSTRACTBackgroundActivation of stress pathways intrinsic to the β cell are thought to both accelerate β cell death and increase β cell immunogenicity in type 1 diabetes (T1D). However, information on the timing and scope of these responses is lacking.MethodsTo identify temporal and disease-related changes in islet β cell protein expression, data independent acquisition-mass spectrometry was performed on islets collected longitudinally from NOD mice and NOD-SCID mice rendered diabetic through T cell adoptive transfer.FindingsIn islets collected from female NOD mice at 10, 12, and 14 weeks of age, we found a time-restricted upregulation of proteins involved in the maintenance of β cell function and stress mitigation, followed by loss of expression of protective proteins that heralded diabetes onset. Pathway analysis identified EIF2 signaling and the unfolded protein response, mTOR signaling, mitochondrial function, and oxidative phosphorylation as commonly modulated pathways in both diabetic NOD mice and NOD-SCID mice rendered acutely diabetic by adoptive transfer, highlighting this core set of pathways in T1D pathogenesis. In immunofluorescence validation studies, β cell expression of protein disulfide isomerase A1 (PDIA1) and 14-3-3b were found to be increased during disease progression in NOD islets, while PDIA1 plasma levels were increased in pre-diabetic NOD mice and in the serum of children with recent-onset T1D compared to age and sex-matched non-diabetic controls.InterpretationWe identified a common and core set of modulated pathways across distinct mouse models of T1D and identified PDIA1 as a potential human biomarker of β cell stress in T1D.
Applying a Modified Metabarcoding Approach for the Sequencing of Macrofungal Specimens from Fungarium Collections
Authors: Olds C. Gary , Berta-Thompson Jessie W. , Loucks Justin J. , Levy Richard A. , Wilson Andrew W. 
Published: December 2021

AbstractPremise:Fungaria are a largely untapped source for understanding fungal biodiversity. The effort and cost in producing DNA barcode sequence data for large numbers of fungal specimens can be prohibitive. This study applies a modified metabarcoding approach that provides a labor and cost-effective solution for sequencing the fungal DNA barcode from hundreds of specimens at once.Methods:A two-step PCR approach uses nested barcoded primers to nrITS2 sequence data. We applied this to 766 macrofungal specimens that represent a broad taxonomic sampling of the Dikarya, of which 382Lactariusspecimens are used to identify molecular operational taxonomic units (MOTUs) through a phylogenetic approach. Scripts in Python and R were used to organize sequence data and execute packages CutAdapt and DADA2 were used for primer removal and assessing sequence quality. Sequences were compared to NCBI and UNITE databases and Sanger-produced sequences.Results:Specimen taxonomic identities from nrITS2 sequence data are >90% accurate across all specimens sampled. Phylogenetic analysis ofLactariussequences identified 20 MOTUs.Discussion:The results demonstrate the capacity of these methods to produce nrITS2 sequences from large numbers of fungarium specimens. This provides an opportunity to more effectively use fungarium collections in advancing fungal diversity identification and documentation.
Transcriptome-wide analysis of microRNA-mRNA correlations in unperturbed tissue transcriptomes identifies microRNA targeting determinants
Authors: Trinidad Barnech Juan Manuel , Fort Rafael Sebastián , Barnech Guillermo Trinidad , Garat Beatriz , Duhagon María Ana 
Published: December 2021

AbstractMicroRNAs are small RNAs that regulate gene expression through complementary base pairing with their target mRNAs. Given the small size of the pairing region and the large number of mRNAs that each microRNA can control, identifying biologically relevant targets is difficult. Since current knowledge of target recognition and repression has mainly relied on in vitro studies, we sought to determine if the interrogation of gene expression data of unperturbed tissues could yield new insight into these processes. The transcriptome-wide repression of all the microRNA-mRNA canonical interaction sites (seed and 3’-supplementary regions, identified by sole base complementarity) was calculated as a normalized Spearman correlation (Z-score) between the abundance of the transcripts in the PRAD-TCGA tissues (RNA-seq and small RNA-seq data of 546 samples). Using the repression values obtained, we confirmed established properties or microRNA targeting efficacy, such as the preference for gene regions (3’UTR > CDS > 5’UTR), the correspondence between repression and seed length (6mer < 7mer < 8mer), and the contribution to the repression exerted by the 3’-supplementary pairing at nucleotides 13-16 of the microRNA. Our results suggest that the 7mer-A1 seed could be more repressive than the 7mer-m8, while they have similar efficacy when they interact using the 3’-supplementary pairing. The 6mer+suppl sites yielded a normalized Z-score of repression similar to the sole 7mer-A1 seeds, alerting its potential biological relevance. We then used the approach to further characterize the 3’-supplementary pairing using 39 microRNAs that hold repressive 3’-supplementary interactions. The analysis of the bridge between seed and 3’-supplementary pairing sites confirmed the optimum +1 offset previously evidenced, but higher offsets appear to have similar repressive strength. The selected microRNAs show a low GC content at positions 13-16 and base preferences that allow a sequence motif identification. Our study demonstrates that transcriptome-wide analysis of microRNA-mRNA correlations in large, matched RNA-seq and small-RNA-seq data can uncover hints of microRNA targeting determinants operating in the in vivo unperturbed set. Finally, we provide a bioinformatic tool to identify microRNA-mRNA candidate interactions based on sequence complementarity of the seed and 3’-supplementary regions.
Estimating Microbial Population Data from Optical Density
Authors: Mira Portia , Yeh Pamela , Hall Barry G. 
Published: December 2021

AbstractThe spectrophotometer has been used for decades to measure the density of bacterial populations as the turbidity expressed as optical density – OD. However, the OD alone is an unreliable metric and is only proportionately accurate to cell titers to about an OD of 0.1. The relationship between OD and cell titer depends on the configuration of the spectrophotometer, the length of the light path through the culture, the size of the bacterial cells, and the cell culture density. We demonstrate the importance of plate reader calibration to identify the exact relationship between OD and cells/ml. We use four bacterial genera and two sizes of micro-titer plates (96-well and 384-well) and show that the cell/ml per unit OD depends heavily on the bacterial cell size and plate size. We applied our calibration curve to real growth curve data and conclude the cells/ml – rather than OD – is a metric that can be used to directly compare results across experiments, labs, instruments, and species.
sRNAs enriched in outer membrane vesicles of pathogenic Flavobacterium psychrophilum interact with immune genes of rainbow trout
Authors: Chapagain Pratima , Ali Ali , Kidane Destaaalem T. , Farone Mary , Salem Mohamed 
Published: December 2021

AbstractOuter membrane vesicles (OMVs) released by gram-negative bacteria during host-pathogen interactions harbor cargos, such as DNA, RNA, toxins, and virulence factors. We hypothesized that sRNAs carried within OMVs of Flavobacterium psychrophilum interact with host immune genes and affect their expression. OMVs were isolated from F. psychrophilum and visualized using transmission electron microscopy (TEM). RNA-Seq datasets generated from whole-cell F. psychrophilum and their OMVs indicated enrichment of specific sRNAs in the OMVs compared to the parent cell. Fluorescent in situ hybridization (FISH) and confocal microscopy confirmed the expression of a randomly chosen sRNA.Integrated RNA-Seq analyses of host transcriptome and bacterial sRNAs on day 5 post-infection of F. psychrophilum-resistant and -susceptible rainbow trout genetic lines revealed 516 protein-coding, 595 lncRNA, and 116 bacterial sRNA differentially expressed (DE) transcripts. Integrated and network analyses of these DE transcripts revealed immune genes targeted by bacterial sRNAs. On the top of these genes, an isoform encoding anaphase-promoting complex subunit 13 (ANAPC13_1) was highly upregulated and exhibited interaction and reciprocal expression with 21 DE sRNAs enriched in OMVs and/or located in pathogenicity islands (PAIs). In vitro treatment of the rainbow trout epithelial cell line RTgill-W1 with OMVs showed signs of cell autolysis accompanied by dynamic changes in expression of host genes when profiled 24h following treatment. The OMV-enriched sRNAs, soFE013584 and soFE002123, showed high interactions with the protection of telomeres 1 gene (POT1); essential for chromosome stability and cellular viability. Modulation of the host gene expression following OMV-treatment, which favors elements from the phagocytic, endocytic, and antigen presentation pathways in addition to HSP70, HSP90, and cochaperone proteins, provided evidence for a potential role of OMVs in boosting the host immune response. In conclusion, our work identified novel microbial targets and inherent characteristics of OMVs that could open up new avenues of treatment and prevention of fish infections.