bluuebunny/crossref_metadata_2025_split huggingface dataset:
Local recruitment of DNA repair proteins enhances CRISPR-ssODN-HDR editing
Authors: Jillette Nathaniel , Zhu Jacqueline J. , Cheng Albert W. 
Published: April 2022

AbstractCRISPR-Cas technologies enable precise editing of genomic sequences. One major way to introduce precise editing is through homology directed repair (HDR) of DNA double strand breaks (DSB) templated by exogenously supplied single-stranded oligodeoxyribonucleotides (ssODN). Competing pathways determine the outcome of edits. Non-homologous end-joining pathways produce destructive insertions/deletions (indels) at target sites and are dominant over the precise homology directed repair pathways. In this study, we aim to favor HDR and use two strategies to recruit DNA repair proteins (DRPs) to Cas9 cut site, the Casilio-DRP approach that recruits RNA-binding protein-tethered DRPs to target site via aptamers appended to guide RNA; and the 53BP1-DRP approach that recruits DRPs to DSBs via DSB-sensing activity of 53BP1. We conducted two screens using these approaches and identified DRPs such as FANCF and BRCA1 that when recruited to Cas9 cut site, enhance ssODN-templated HDR and increase the proportion of precise edits. This study provides not only new constructs for enhanced CRISPR-ssODN-HDR but also a collection of DRP fusions for studying DNA repair processes.
Genomic surveillance unfolds the dynamics of SARS-CoV-2 transmission and divergence in Bangladesh over the past two years
Authors: Shishir Tushar Ahmed , Jannat Taslimun , Naser Iftekhar Bin 
Published: April 2022

AbstractThe highly pathogenic virus SARS-CoV-2 has shattered the healthcare system of the world causing the COVID-19 pandemic since first detected in Wuhan, China. Therefore, scrutinizing the genome structure and tracing the transmission of the virus has gained enormous interest in designing appropriate intervention strategies to control the pandemic. In this report, we examined 4622 sequences from Bangladesh and found that they belonged to thirty-five major PANGO lineages, while Delta alone accounted for 39%, and 78% were from just four primary lineages. Our research has also shown Dhaka to be the hub of viral transmission and observed the virus spreading back and forth across the country at different times by building a transmission network. The analysis resulted in 7659 unique mutations, with an average of 24.61 missense mutations per sequence. Moreover, our analysis of genetic diversity and mutation patterns revealed that eight genes were under negative selection pressure to purify deleterious mutations, while three genes were under positive selection pressure.ImportanceWith 29,122 deaths, 1.95 million infections and a shattered healthcare system from SARS-CoV-2 in Bangladesh, the only way to avoid further complications is to break the transmission network of the virus. Therefore, it is vital to shedding light on the transmission, divergence, mutations, and emergence of new variants using genomic data analyses and surveillance. Here, we present the geographic and temporal distribution of different SARS-CoV-2 variants throughout Bangladesh over the past two years, and their current prevalence. Further, we have developed a transmission network of viral spreads, which in turn will help take intervention measures. Then we analyzed all the mutations that occurred and their effect on evolution as well as the currently present mutations that could trigger a new variant of concern. In short, together with an ongoing genomic surveillance program, these data will help to better understand SARS-CoV-2, its evolution, and pandemic characteristics in Bangladesh.
TNFα-induced metabolic reprogramming drives an intrinsic anti-viral state
Authors: Ciesla Jessica , Moreno Isreal , Munger Joshua 
Published: April 2022

AbstractCytokines induce an anti-viral state, yet many of the functional determinants responsible for limiting viral infection are poorly understood. Here, we find that TNFα induces significant metabolic remodeling that is critical for its anti-viral activity. Our data demonstrate that TNFα activates glycolysis through the induction of muscle-specific hexokinase (HK2). Further, we show that glycolysis is broadly important for TNFα-mediated anti-viral defense, as its inhibition attenuates TNFα’s ability to limit the replication of evolutionarily divergent viruses. Stable-isotope tracing revealed that TNFα-mediated glycolytic activation promotes the biosynthesis of UDP-sugars (essential precursors of protein glycosylation) and that inhibition of glycolysis prevents the accumulation of several glycosylated anti-viral proteins. Consistent with the importance of glucose-driven glycosylation, glycosyl-transferase inhibition also attenuated TNFα’s ability to promote the anti-viral cell state. Collectively, our data indicate that cytokine-mediated metabolic remodeling is an essential component of the anti-viral response.
Human genomic DNA is widely interspersed with i-motif structures
Authors: Peña Martinez Cristian David , Zeraati Mahdi , Rouet Romain , Mazigi Ohan , Gloss Brian , Chan Chia-Ling , Bryan Tracy M. , Smith Nicole M. , Dinger Marcel E. , Kummerfeld Sarah , Christ Daniel 
Published: April 2022

AbstractDNA i-motif structures are formed in the nucleus of human cells and are believed to provide critical genomic regulation. While the existence of i-motif structures in human cells has been demonstrated by immunofluorescent staining and by characterisation of select model genes, the abundance and distribution of such structures in the human genome has remained unclear. Here we utilize high affinity i-motif immunoprecipitation followed by sequencing to map i-motifs in human genomic DNA. Validated by biolayer interferometry and circular dichroism spectroscopy, our approach identified over 650,000 i-motif structures in human genomic DNA. The i-motif structures are widely distributed throughout the human genome and are common among highly expressed genes and in genes upregulated in G0/G1 cell cycle phase. Our findings provide experimental evidence for the widespread formation of i-motif structures in human genomic DNA.
Architecture of the biofilm-associated archaic CupE pilus fromPseudomonas aeruginosa
Authors: Böhning Jan , Dobbelstein Adrian , Sulkowski Nina , Eilers Kira , von Kügelgen Andriko , Tarafder Abul K. , Alva Vikram , Filloux Alain , Bharat Tanmay A. M. 
Published: April 2022

AbstractChaperone-Usher Pathway (CUP) pili are major adhesins in Gram-negative bacteria, mediating bacterial adherence to biotic and abiotic surfaces. While classical CUP pili have been extensively characterized, little is known about so-called archaic CUP pili, which are phylogenetically widespread and promote biofilm formation by several human pathogens. In this study, we present the electron cryomicroscopy structure of the archaic CupE pilus from the opportunistic human pathogenPseudomonas aeruginosa. We show that CupE pili consist of CupE1 subunits arranged in a zigzag architecture, with an N-terminal donor β-strand extending from each subunit into the next, where it is anchored by hydrophobic interactions, resulting in an overall flexible pilus arrangement. Imaging CupE pili on the surface ofP. aeruginosacells using electron cryotomography shows that CupE pili adopt variable curvatures in response to their environment, which may facilitate their role in promoting cohesion between bacterial cells. Finally, bioinformatic analysis shows the widespread abundance ofcupEgenes in isolates ofP. aeruginosaand the co-occurrence ofcupEwith othercupclusters, suggesting interdependence ofcuppili in regulating bacterial adherence within biofilms. Taken together, our study provides insights into the architecture of archaic CUP pili and their role in promoting cellular adhesion and biofilm formation inP. aeruginosa.
Sustained plumage divergence despite weak genomic differentiation and broad sympatry in sister species of Australian woodswallows (Artamus spp.)
Authors: Peñalba Joshua V. , Peters Jeffrey L. , Joseph Leo 
Published: April 2022

AbstractPlumage divergence can function as a strong premating barrier when species come into secondary contact. When it fails to do so, the results are often genome homogenization and phenotypic hybrids at the zone of contact. This is not the case in the largely sympatric masked woodswallow and white-browed woodswallow species (Passeriformes: Artamidae: Artamus spp) complex in Australia where phenotypic integrity is sustained despite no discernible mitochondrial structure in earlier work. This lack of structure may suggest recent divergence, ongoing gene flow or both, and phenotypic hybrids are reported albeit rarely. Here, we further assessed the population structure and differentiation across the species’ nuclear genomes using ddRAD-seq. As found in the mitochondrial genome, no structure or divergence within or between the two species was detected in the nuclear genome. This coarse sampling of the genome nonetheless revealed peaks of differentiation around the genes SOX5 and Axin1. Both are involved in the Wnt//μ-catenin signaling pathway, which regulates feather development. Reconstruction of demographic history and estimation of parameters supports a scenario of secondary contact. Our study informs how divergent plumage morphs may arise and be sustained despite whole-genome homogenization and reveals new candidate genes potentially involved in plumage divergence.
Orexin cells efficiently decode blood glucose dynamics to drive adaptive behavior
Authors: Viskaitis Paulius , Tesmer Alexander L. , Karnani Mahesh M. , Arnold Myrtha , Donegan Dane , Bracey Ed F. , Grujic Nikola , Peleg-Raibstein Daria , Burdakov Denis 
Published: April 2022

AbstractBlood glucose variability shapes human brain performance and diverse clinical outcomes. However, it remains poorly understood how blood glucose fluctuations are decoded by genetically-defined neurons to change brain activity and behavior. Recent breakthroughs in genetics and clinical diagnostics identified hypothalamic hypocretin/orexin neurons (HONs) as core determinants of brain activity and adaptive behavior across mammals. Here we show that low-frequency HON population waves are tuned for transmitting information about minute-to-minute temporal features of blood glucose, thus rapidly converting its variability into brain state of behaving mice. Contrary to current theories envisioning glucose-proportional neural responses, the HONs’ response tracked blood glucose gradients, thus generating efficient neural adaptations in anticipation of maximal glucose deviations. Resolving this population response at the single cell level with volumetric multiphoton imaging furthermore revealed glucose-excited and glucose-inhibited HONs, distinctly coupled to body movements in the high-frequency domain. Finally, HON-selective opotogenetics and cell ablation demonstrated that HONs are critical for linking glucose to adaptive behavior. These results provide an insight into how behaviorally influential hypothalamic networks interpret blood glucose variability. This may inform future metrics for efficient prediction of glycemic states in health and disease.
Biochemical characterization of Pectin Methylesterase Inhibitor 3 from Arabidopsis thaliana
Authors: Xu Fan , Gonneau Martine , Faucher Elvina , Habrylo Olivier , Lefebvre Valérie , Domon Jean-Marc , Martin Marjolaine , Sénéchal Fabien , Peaucelle Alexis , Pelloux Jérôme , Höfte Herman 
Published: April 2022

AbstractThe Arabidopsis thaliana PECTIN METHYLESTERASE INHIBITOR 3 (PMEI3) gene is frequently used as a tool to manipulate PME activity in vivo, in studies assessing the role of pectin de-methylesterification in the control of cell expansion. One limitation of these studies is that the exact biochemical activity of this protein has not yet been determined. In this manuscript we produced the protein in Pichia pastoris and characterized its activity in vitro. Like other PMEIs, PMEI3 inhibits PME activity in acidic pH conditions for a variety of cell wall extracts and for purified PME preparations, but doesn’t affect PME activity at neutral pH. This suggests that the previously observed in vivo effects reflect the inhibition of PME activity at low pH. The protein is remarkable heat stable and shows higher activity against PME3 than against PME2, illustrating how different members of the large PMEI family can differ in their specificities towards PME targets. Finally, application of purified PMEI3 on Arabidopsis thaliana seedlings showed a dose-dependent inhibition of homogalacturonan de-methylesterification and root growth. Purified recombinant PMEI3 is therefore a powerful tool to study the connection between pectin methylesterification and cell expansion.
Quantitative Cell Proteomic Atlas: Pathway-scale targeted mass spectrometry for high-resolution functional profiling of cell signaling
Authors: Cifani Paolo , Kentsis Alex 
Published: April 2022

AbstractIn spite of extensive studies of cellular signaling, many fundamental processes such as pathway integration, cross-talk and feedback remain poorly understood. To enable integrated and quantitative measurements of cellular biochemical activities, we have developed the Quantitative Cell Proteomics Atlas (QCPA). QCPA consists of panels of targeted mass spectrometry assays to determine the abundance and stoichiometry of regulatory post-translational modifications of sentinel proteins from most known physiologic and pathogenic signaling pathways in human cells. QCPA currently profiles 1,913 peptides from 469 effectors of cell surface signaling, apoptosis, stress response, gene expression, quiescence, and proliferation. For each protein, QCPA includes triplets of isotopically labeled peptides covering known post-translational regulatory sites to determine their stoichiometries and unmodified protein regions to measure total protein abundance. The QCPA framework incorporates analytes to control for technical variability of sample preparation and mass spectrometric analysis, including TrypQuant, a synthetic substrate for accurate quantification of proteolysis efficiency for proteins containing chemically modified residues. The ability to precisely and accurately quantify most known signaling pathways should enable improved chemoproteomic approaches for the comprehensive analysis of cell signaling and clinical proteomics of diagnostic specimens. QCPA is openly available athttps://qcpa.mskcc.org.
Quantitative analysis of C. elegans transcripts by Nanopore direct-cDNA sequencing reveals terminal hairpins in non trans-spliced mRNAs
Authors: Bernard Florian , Dargère Delphine , Rechavi Oded , Dupuy Denis 
Published: April 2022

AbstractNematode mRNA processing involves a trans-splicing step through which a 21nt sequence from a snRNP replaces the original 5’ end of the primary transcript. It has long been held that 70% of C. elegans mRNAs are submitted to trans-splicing. Our recent work suggested that the mechanism is more pervasive but not fully captured by mainstream transcriptome sequencing methods.In this study, we used Oxford Nanopore’s long-read amplification-free sequencing technology to perform a comprehensive analysis of trans-splicing in worms.We demonstrated that spliced leader (SL) sequences presence at the 5’ end of the messengers affected library preparation and generated sequencing artefacts due to their self-complementarity. Consistent with our previous observations, we found evidence of trans-splicing for most genes. However, a subset of genes appears to be only marginally trans-spliced. These messengers all share the capacity to generate a 5’ terminal hairpin structure mimicking the SL structure providing a mechanistic explanation for their non conformity. Altogether, our data provides the most comprehensive quantitative analysis of SL usage to date in C. elegans.
A butterfly pan-genome reveals a large amount of structural variation underlies the evolution of chromatin accessibility
Authors: Ruggieri Angelo A. , Livraghi Luca , Lewis James J. , Evans Elizabeth , Cicconardi Francesco , Hebberecht Laura , Montgomery Stephen H. , Ghezzi Alfredo , Rodriguez-Martinez José Arcadio , Jiggins Chris D. , McMillan W. Owen , Counterman Brian A. , Papa Riccardo , Van Belleghem Steven M. 
Published: April 2022

AbstractDespite insertions and deletions being the most common structural variants (SVs) found across genomes, not much is known about how much these SVs vary within populations and between closely related species, nor their significance in evolution. To address these questions, we characterized the evolution of indel SVs using genome assemblies of three closely related Heliconius butterfly species. Over the relatively short evolutionary timescales investigated, up to 18.0% of the genome was composed of indels between two haplotypes of an individual H. charithonia butterfly and up to 62.7% included lineage-specific SVs between the genomes of the most distant species (11 Mya). Lineage-specific sequences were mostly characterized as transposable elements (TEs) inserted at random throughout the genome and their overall distribution was similarly affected by linked selection as single nucleotide substitutions. Using chromatin accessibility profiles (i.e., ATAC-seq) of head tissue in caterpillars to identify sequences with potential cis-regulatory function, we found that out of the 31,066 identified differences in chromatin accessibility between species, 30.4% were within lineage-specific SVs and 9.4% were characterized as TE insertions. These TE insertions were localized closer to gene transcription start sites than expected at random and were enriched for several transcription factor binding site candidates with known function in neuron development in Drosophila. We also identified 24 TE insertions with head-specific chromatin accessibility. Our results show high rates of structural genome evolution that were previously overlooked in comparative genomic studies and suggest a high potential for structural variation to serve as raw material for adaptive evolution.
Parallel recruitment pathways contribute to synaptonemal complex assembly during mammalian meiosis
Authors: Crichton James H. , Dunce James M. , Baarends Willy M. , Davies Owen R. , Adams Ian R. 
Published: April 2022

AbstractDuring meiosis, the synaptonemal complex (SC) assembles between paired chromosomes, binding them together in close apposition, and facilitating recombination. SC assembly is thought to occur through the hierarchical zipper-like recruitment of axial elements, followed by transverse filaments and then central elements. However, the rapidity of SC formation in mammals has hitherto hindered investigation of its assembly mechanisms and their relationship with recombination. Using super-resolution imaging of separation-of-function mouse mutants, we show that, contrary to the hierarchical assembly model, central element protein SYCE2 is recruited to recombination sites early in SC assembly, and independently of SYCP1-containing transverse filaments. Further, SYCE2-TEX12 binds DNA in vitro, and SYCE2-containing bridges physically link paired chromosomes at recombination sites prior to transverse filament recruitment and chromosome synapsis. These data suggest that mammals integrate parallel recruitment pathways to assemble a mature SC: one recruiting central element proteins to recombination sites, and another recruiting transverse filaments to chromosomes.
Acute administration of a dopamine D2/D3 receptor agonist alters behavioral and neural parameters in adult zebrafish
Authors: Nabinger Débora Dreher , Altenhofen Stefani , Buatois Alexis , Facciol Amanda , Peixoto Julia Vasconcellos , da Silva Julia Maria Kuhl , Rübensam Gabriel , Gerlai Robert , Bonan Carla Denise 
Published: April 2022

AbstractThe dopaminergic neurotransmitter system is involved in numerous brain functions and behavioral processes. Alterations in this neurotransmitter system are associated with the pathogenesis of several human neurological disorders. Pharmacological agents that interact with the dopaminergic system allow the investigation of dopamine-mediated cellular and molecular responses and may elucidate the biological bases of such disorders. The zebrafish, a translationally relevant biomedical research organism, has been successfully employed in prior psychopharmacology studies. Here, we evaluate the effects of quinpirole (a dopamine D2/D3 receptor agonist) in adult zebrafish on behavioral parameters and neurotransmitter levels. Adult zebrafish received intraperitoneal injections of 0.5, 1.0, or 2.0 mg/kg of quinpirole or saline (control group) twice with an inter-injection interval of 48h. All tests were performed 24h after the second injection. After acute quinpirole administration, zebrafish exhibited decreased locomotor activity, increased anxiety-like behaviors and memory impairment compared to control. However, the quinpirole administration did not affect social and aggressive behavior. Quinpirole-treated fish exhibited altered swimming patterns: fish showed stereotypic swimming characterized by repetitive behavior, swimming from corner to corner at the bottom of the tank preceded and followed by episodes of immobility. Moreover, analysis of neurotransmitter levels in the brain demonstrated a significant increase in glutamate and a decrease in serotonin, while no alterations were observed in dopamine. These findings demonstrate that dopaminergic signaling altered by quinpirole administration results in significant changes in behavior and neurotransmitter levels in the central nervous system of zebrafish. Thus, we conclude that the use of quinpirole administration in adult zebrafish may be an appropriate tool for the analysis of mechanisms underlying neurological disorders related to the dopaminergic system.
A silk-based platform to stabilize phenylalanine ammonia-lyase (PAL) for orally administered enzyme replacement therapy
Authors: d’Amone Luciana , Trivedi Vikas D. , Nair Nikhil U. , Omenetto Fiorenzo G. 
Published: April 2022

ABSTRACTPhenylalanine ammonia-lyase (PAL) has gained attention in recent years for the treatment of phenylketonuria (PKU), a genetic disorder that affects ∼1 in 15,000 individuals globally. However, the enzyme is easily degraded by proteases, unstable at room temperature, and is currently administered in PKU patients as daily subcutaneous injections. We report here the stabilization of the PAL from Anabaena variabilis, which is currently used to formulate pegvaliase, through incorporation in a silk fibroin matrix. The combination with silk stabilizes PAL at 37 °C. In addition, in vitro studies showed that inclusion in a silk matrix preserves the biological activity of the enzyme in simulated intestinal fluid, enabling the oral administration of pegvaliase for the treatment of PKU.
Generative Moment Matching Networks for Genotype Simulation
Authors: Perera Maria , Montserrat Daniel Mas , Barrabés Míriam , Geleta Margarita , Giró-i-Nieto Xavier , Ioannidis Alexander G. 
Published: April 2022

AbstractThe generation of synthetic genomic sequences using neural networks has potential to ameliorate privacy and data sharing concerns and to mitigate potential bias within datasets due to under-representation of some population groups. However, there is not a consensus on which architectures, training procedures, and evaluation metrics should be used when simulating single nucleotide polymorphism (SNP) sequences with neural networks. In this paper, we explore the use of Generative Moment Matching Networks (GMMNs) for SNP simulation, we present some architectural and procedural changes to properly train the networks, and we introduce an evaluation scheme to qualitatively and quantitatively assess the quality of the simulated sequences.