bluuebunny/crossref_metadata_2025_split huggingface dataset:
A-type lamins are critical for the recruitment of RPA and RAD51 to stalled replication forks to maintain fork stability
Authors: Graziano Simona , Coll-Bonfill Nuria , Teodoro-Castro Barbara , Kuppa Sahiti , Jackson Jessica , Shashkova Elena , Mahajan Urvashi , Vindigni Alessandro , Antony Edwin , Gonzalo Susana 
Published: June 2021

ABSTRACTLamins provide a nuclear scaffold for compartmentalization of genome function that is important for genome integrity. The mechanisms whereby lamins regulate genome stability remain poorly understood. Here, we demonstrate a crucial role for A-type lamins preserving the integrity of the replication fork (RF) during replication stress (RS). We find that lamins bind to nascent DNA strands, especially during RS, and ensure the recruitment of fork protective factors RPA and RAD51. These ssDNA-binding proteins, considered the first and second responders to RS respectively, play crucial roles in the stabilization, remodeling and repair of the stalled fork to ensure proper restart and genome stability. Reduced recruitment of RPA and RAD51 upon lamins depletion elicits replication fork instability (RFI) depicted by MRE11 nuclease-mediated degradation of nascent DNA, RS-induced accumulation of DNA damage, and increased sensitivity to replication inhibitors. Importantly, in contrast to cells deficient in various homology recombination repair proteins, the RFI phenotype of lamins-depleted cells is not linked to RF reversal. This suggests that the point of entry of nucleases is not the reversed fork, but regions of ssDNA generated during RS that are not protected by RPA and RAD51. Consistently, RFI in lamins-depleted cells is rescued by forced elevation of the heterotrimeric RPA complex or RAD51. These data unveil a clear involvement of structural nuclear proteins in the protection of ssDNA from the action of nucleases during RS by warranting proper recruitment of ssDNA binding proteins RPA and RAD51 to stalled RFs. In support of this model, we show physical interaction between RPA and lamins. Our study also suggests that RS is a major source of genomic instability in laminopathies and in lamins-depleted tumors.
Cis-Regulatory Hubs: a new 3D Model of Complex Disease Genetics with an Application to Schizophrenia
Authors: Mangnier Loïc , Joly-Beauparlant Charles , Droit Arnaud , Bilodeau Steve , Bureau Alexandre 
Published: June 2021

AbstractThe 3-dimensional (3D) conformation of the chromatin creates complex networks of noncoding regulatory regions (distal elements) and promoters impacting gene regulation. Despite the importance of the role of noncoding regions in complex diseases, little is known about their interplay within regulatory hubs and implication in multigenic diseases like schizophrenia. Here we show that cis-regulatory hubs (CRHs) in neurons highlight functional interactions between distal elements and promoters, providing a model to explain epigenetic mechanisms involved in complex diseases. CRHs represent a new 3D model, where distal elements interact to create a complex network of active genes. In a disease context, CRHs highlighted strong enrichments in schizophrenia-associated genes, schizophrenia-associated SNPs and schizophrenia heritability compared to equivalent structures. Finally, CRHs exhibit larger proportions of genes differentially expressed in schizophrenia compared to promoter-distal element pairs or TADs. CRHs thus capture causal regulatory processes improving the understanding of complex disease etiology such as schizophrenia. These multiple lines of genetic and statistical evidence support CRHs as 3D models to study dysregulation of gene expression in complex diseases more generally.Summary BlurbGenes and their regulatory elements are organized within 3D networks in neurons which model functional structures and explain schizophrenia genetic etiology.
Vigilance and behavioral state-dependent modulation of cortical neuronal activity throughout the sleep/wake cycle
Authors: Brécier Aurélie , Borel Mélodie , Urbain Nadia , Gentet Luc J. 
Published: June 2021

ABSTRACTGABAergic inhibitory neurons, through their molecular, anatomic and physiological diversity, provide a substrate for the modulation of ongoing cortical circuit activity throughout the sleep-wake cycle. Here, we investigated neuronal activity dynamics of parvalbumin (PV), vasoactive intestinal polypeptide (VIP) and somatostatin (SST) neurons in naturally-sleeping head-restrained mice at the level of layer 2/3 of the primary somatosensory barrel cortex of mice. Through calcium-imaging and targeted single-unit loose-patch or whole-cell recordings, we found that PV action potential (AP) firing activity was largest during both NREM (non-rapid eye movement) and REM sleep stages, that VIP neurons were activated during REM sleep and that the overall activity of SST neurons remained stable throughout the sleep/wake cycle. Analysis of neuronal activity dynamics uncovered rapid decreases in PV cell firing at wake onset followed by a progressive recovery during wake. Simultaneous local field potential (LFP) recordings further revealed that, except for SST neurons, a large proportion of neurons were modulated by ongoing delta and theta waves. During NREM sleep spindles, PV and SST activity increased and decreased, respectively. Finally, we uncovered the presence of whisking behavior in mice during REM sleep and show that the activity of VIP and SST is differentially modulated during awake and sleeping whisking bouts, which may provide a neuronal substrate for internal brain representations occurring during sleep.
Tissue-specific modification of cellular bioelectrical activities using the chemogenetic tool, DREADD, in zebrafish
Authors: Silic Martin R. , Zhang GuangJun 
Published: June 2021

ABSTRACTCellular electronic activity plays an essential role in neuronal communication. Manipulation and visualization of cellular membrane potential remain essential tasks in order to study electrical signaling in living organisms. Light-controlled optogenetic and designed chemical-controlled chemogenetic tools were developed to manipulate cellular electric activities for neuroscience research. One of the most common chemogenetic tools is DREADD (designer receptors exclusively activated by designer drugs). It has been extensively utilized due to its convenience and long-lasting effects in murine and primate models, but not in zebrafish, a leading model organism in various research fields. Here, we first establish multiple tissue-specific transgenic zebrafish lines that express two different DREADDs with a genetically encoded voltage indicator, ASAP2s. We observed voltage changes in zebrafish melanophores, epidermis, and neurons by hM4DGi or rM3DGs receptors measured by ASAP2s fluorescence intensity. Alteration to melanophore bioelectricity by DREADD generated dynamic electric signals and resulted in morphological alterations to pigment cells. We also tested a few agonists and found that the latest generation performs better than clozapine N-oxide (CNO). Collectively, our experiments demonstrate that DREADD can be utilized to manipulate cell-specific membrane potential in the zebrafish model. The availability of this tool in zebrafish will offer a new resource for a variety of bioelectricity research fields such as neuroscience, cardiology, and developmental biology.
Mechanistic target of rapamycin complex 1 (mTORC1) activity occurs predominantly in the periphery of human skeletal muscle fibers, in close proximity to focal adhesion complexes, following anabolic stimuli
Authors: Hodson Nathan , Mazzulla Michael , Kumbhare Dinesh , Moore Daniel R. 
Published: June 2021

AbstractFollowing anabolic stimuli (e.g. mechanical loading and/or amino acid provision) the mechanistic target of rapamycin complex 1 (mTORC1), a master regulator of protein synthesis, translocates toward the cell periphery. However, it is unknown if mTORC1 activity occurs prior to or following this translocation. We therefore aimed to determine the cellular location of mTORC1 activity in human skeletal muscle following anabolic stimuli. Fourteen young, healthy males either ingested a protein-carbohydrate beverage (0.25g/kg protein, 0.75g/kg carbohydrate) alone (n=7, 23±5yrs, 76.8±3.6kg, 13.6±3.8%BF, FED) or following a whole-body resistance exercise bout (n=7, 22±2yrs, 78.1±3.6kg, 12.2±4.9%BF, EXFED). Vastus lateralis muscle biopsies were obtained at rest (PRE) and 120 and 300min following anabolic stimuli. The spatial regulation of mTORC1 activity was assessed through immunofluorescent staining of p-RPS6Ser240/244, an mTORC1-specific phosphorylation event. p-RPS6Ser240/244 measured by immunofluorescent staining or immunoblot was positively correlated (r=0.76, p<0.001). Peripheral staining intensity of p-RPS6Ser240/244 increased above PRE in both FED and EXFED at 120min (~54% and ~138% respectively, p<0.05) but was greater in EXFED at both post-stimuli time points (p<0.05). The peripheral-central ratio of p-RPS6240/244 staining was displayed a similar pattern, suggesting mTORC1 activity occurs predominantly in the periphery of fibers. Moreover, p-RPS6Ser240/244 intensity within paxillin-positive regions, a marker of focal adhesion complexes, was elevated at 120min irrespective of stimulus (p=0.006) before returning to PRE at 300min. These data confirm that mTORC1 activity occurs in the region of human muscle fibers to which mTORC1 translocates following anabolic stimuli and identifies focal adhesion complexes as a potential site of mTORC1 activation in vivo.
Atypical integrative element with strand-biased circularization activity assists interspecies antimicrobial resistance gene transfer from Vibrio alfacsensis
Authors: Nonaka Lisa , Masuda Michiaki , Yano Hirokazu 
Published: June 2021

AbstractThe exchange of antimicrobial resistance (AMR) genes between aquaculture and terrestrial microbial populations has emerged as a serious public health concern. However, the nature of the mobile genetic elements in marine bacteria is poorly documented. To gain insight into the genetic mechanisms underlying AMR gene transfer from marine bacteria, we mated a multidrug-resistant Vibrio alfacsensis strain with an Escherichia coli strain, and then determined the complete genome sequences of the donor and the transconjugant strains. Sequence analysis revealed a conjugative multidrug resistance plasmid in the donor strain, which was integrated into the chromosome of the recipient. The plasmid backbone in the transconjugant chromosome was flanked by two copies of a 7.1 kb unclassifiable integrative element harboring a β-lactamase gene. The 7.1 kb element and the previously reported element Tn6283 share four coding sequences, two of which encode the catalytic R-H-R-Y motif of tyrosine recombinases. Polymerase chain reaction and sequencing experiments revealed that these elements generate a circular copy of one specific strand without leaving an empty site on the donor molecule, in contrast to the movement of integron gene cassettes or ICE/IMEs discovered to date. These elements are termed SEs (strand-biased circularizing integrative elements): SE-6945 (the 7.1 kb element) and SE-6283 (Tn6283). The copy number and location of SE-6945 in the chromosome affected the antibiotic resistance levels of the transconjugants. SEs were identified in the genomes of other Vibrio species. Overall, these results suggest that SEs are involved in the spread of AMR genes among marine bacteria.
Genome-Scale Metabolic Model Accurately Predicts Fermentation of Glucose by Chromochloris zofingiensis
Authors: Meagher Michelle , Metcalf Alex , Ramsey S. Alex , Prentice Walter , Boyle Nanette R. 
Published: June 2021

AbstractAlgae have the potential to be sources of renewable fuels and chemicals. One particular strain, Chromochloris zofingiensis, is of interest due to the co-production of triacylglycerols (TAGs) and astaxanthin, a valuable nutraceutical. To aid in future engineering efforts, we have developed the first genome-scale metabolic model on C. zofingiensis, iChr1915. This model includes 1915 genes, 3413 metabolic reactions and 2652 metabolites. We performed detailed biomass composition analysis for three growth conditions: autotrophic, mixotrophic and heterotrophic and used this data to develop biomass formation equations for each growth condition. The completed model was then used to predict flux distributions for each growth condition; interestingly, for heterotrophic growth, the model predicts the excretion of fermentation products due to overflow metabolism. We confirmed this experimentally via metabolomics of spent medium and fermentation product assays. An in silico gene essentiality analysis was performed on this model, as well as a flux variability analysis to test the production capabilities of this organism. In this work, we present the first genome scale metabolic model of C. zofingiensis and demonstrate its use predicting metabolic activity in different growth conditions, setting up a foundation for future metabolic engineering studies in this organism.
Horizontal transposon transfer and its implications for the ancestral ecology of hydrophiine snakes
Authors: Galbraith James D. , Ludington Alastair J. , Sanders Kate L. , Amos Timothy G. , Thomson Vicki A. , Tuipulotu Daniel Enosi , Dunstan Nathan , Edwards Richard J. , Suh Alexander , Adelson David L. 
Published: June 2021

AbstractTransposable elements (TEs), also known as jumping genes, are sequences able to move or copy themselves within a genome. As TEs move throughout genomes they often act as a source of genetic novelty, hence understanding TE evolution within lineages may help in understanding environmental adaptation. Studies into the TE content of lineages of mammals such as bats have uncovered horizontal transposon transfer (HTT) into these lineages, with squamates often also containing the same TEs. Despite the repeated finding of HTT into squamates, little comparative research has examined the evolution of TEs within squamates. Here we examine a diverse family of Australo-Melanesian snakes (Hydrophiinae) to examine if the previously identified, order-wide pattern of variable TE content and activity holds true on a smaller scale. Hydrophiinae diverged from Asian elapids ∼30 Mya and have since rapidly diversified into six amphibious, ∼60 marine and ∼100 terrestrial species which fill a broad range of ecological niches. We find TE diversity and expansion differs between hydrophiines and their Asian relatives and identify multiple HTTs into Hydrophiinae, including three likely transferred into the ancestral hydrophiine from fish. These HTT events provide the first tangible evidence that Hydrophiinae reached Australia from Asia via a marine route.
Whole blood metabolomics of dementia patients reveal classes of disease-linked metabolites
Authors: Teruya Takayuki , Chen Yung-Ju , Kondoh Hiroshi , Fukuji Yasuhide , Yanagida Mitsuhiro 
Published: June 2021

AbstractDementia is caused by factors that damage neurons. We quantified small molecular markers in whole blood of dementia patients, using non-targeted liquid chromatography-mass spectroscopy (LC-MS). Thirty-three metabolites, classified into 5 groups (A-E), differed significantly in dementia patients, compared with healthy elderly subjects. Seven Group A metabolites present in plasma, including quinolinic acid, kynurenine, and indoxyl-sulfate, increased. Possibly they act as neurotoxins in the central nervous system (CNS). The remaining 26 compounds (Groups B-E) decreased, possibly causing a loss of support or protection of the brain in dementia. Six Group B metabolites, normally enriched in red blood cells (RBCs) of healthy subjects, all contain trimethylated ammonium moieties. These metabolites include ergothioneine and structurally related compounds have scarcely been investigated as dementia markers, validating the examination of RBC metabolites. Ergothioneine, a potent anti-oxidant, is significantly decreased in various cognition-related disorders, such as mild cognitive impairment and frailty. Group C compounds, also include some oxidoreductants and are normally abundant in RBCs (NADP+, glutathione, ATP, pantothenate, S-adenosyl-methionine, and gluconate). Their decreased levels in dementia patients may also contribute to depressed brain function. Groups D (12) contains plasma compounds, such as amino acids, glycerophosphocholine, dodecanoyl-carnitine, 2-hydroxybutyrate, which normally protect the brain, but their diminution in dementia may reduce that protection. Seven Group D compounds have been identified previously as dementia markers. Group B-E compounds may be critical to maintain the CNS by acting directly or indirectly. How RBC metabolites act in the CNS and why they diminish so significantly in dementia remain to be determined.Significance StatementDementia is a slowly progressing, chronic, and usually irreversible decline in cognitive function. Mechanistic causes and definitive treatments remain elusive. Using comprehensive metabolomics, we identified 5 groups of metabolites (A-E), 21 of which are novel, possibly useful for diagnosis and therapy of forms of dementia, such as Alzheimer’s disease. Seven Group A compounds may act as neurotoxins, whereas Group B-E compounds may protect the CNS against oxidative stress, maintain energy reserves, supply nutrients and neuroprotective factors. Five metabolites, ergothioneine,S-methyl-ergothioneine, trimethyl-histidine, methionine, and tryptophan identified in this study overlap with those reported for frailty. Interventions for cognitive diseases involving these dementia metabolomic markers may be accomplished either by inhibiting Group A compounds or by supplementing Group B-E compounds in patients.
Escherichia coli recombinant expression of SARS-CoV-2 protein fragments
Authors: McGuire Bailey E. , Mela Julia E. , Thompson Vanessa C. , Cucksey Logan R , Stevens Claire E. , McWhinnie Ralph L. , Winkler Dirk F. H. , Pelech Steven , Nano Francis E. 
Published: June 2021

ABSTRACTWe have developed a method for the inexpensive, high-level expression of antigenic protein fragments of SARS-CoV-2 proteins in Escherichia coli. Our approach used the thermophilic family 9 carbohydrate-binding module (CBM9) as an N-terminal carrier protein and affinity tag. The CBM9 module was joined to SARS-CoV-2 protein fragments via a flexible proline-threonine linker, which proved to be resistant to E. coli proteases. Two CBM9-spike protein fragment fusion proteins and one CBM9-nucleocapsid fragment fusion protein largely resisted protease degradation, while most of the CBM9 fusion proteins were degraded at some site in the SARS-CoV-2 protein fragment. All fusion proteins were expressed in E. coli at about 0.1 g/L, and could be purified with a single affinity binding step using inexpensive cellulose powder. Three purified CBM9-SARS-CoV-2 fusion proteins were tested and found to bind antibody directed to the appropriate SARS-CoV-2 antigenic region. The largest intact CBM9 fusion protein incorporates spike protein amino acids 540-588, which is a conserved region immediately C-terminal to the receptor binding domain that is widely recognized by human convalescent sera and contains a putative protective epitope.
Impact of dose, duration and host immune status on ultrashort telacebec treatment in a mouse model of Buruli ulcer
Authors: Almeida Deepak V. , Converse Paul J. , Komm Oliver , Nuermberger Eric L. 
Published: June 2021

ABSTRACTTelacebec (Q203) is a new anti-tuberculosis drug in clinical development with extremely potent activity against Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU). The potency of Q203 has prompted investigation of its potential role in ultra-short, even single-dose, treatment regimens for BU in mouse models. However, the relationships of Q203 dose and duration and host immune status to treatment outcomes remain unclear, as does the risk of emergence of drug resistance with Q203 monotherapy. In the present study, immunocompetent BALB/c and immunocompromised SCID-beige mice were infected in both hind footpads and treated eight weeks later. In both mouse strains, controls received rifampin-clarithromycin; others received Q203 at 0.5 or 2 mg/kg/d for 5 or 10 days. Additionally, BALB/c mice received a single dose of 2.5 or 10 mg/kg or 3.3 mg/kg/d for 3 days. Treatment response was based on changes in footpad swelling and CFU counts at the end of treatment as well as 4 and 13 weeks after stopping treatment. Efficacy depended on total dose more than duration. Total doses of 5-20 mg/kg rendered nearly all BALB/c mice culture-negative 13 weeks post-treatment without selection of Q203-resistant bacteria. Although less potent in SCID-beige mice, Q203 still rendered the majority of footpads culture-negative at total doses of 10-20 mg/kg. However, Q203 resistance was identified in relapse isolates from some SCID-beige mice. Overall, these results support the potential of Q203 monotherapy for single-dose or other ultra-short therapy for BU, although highly immunocompromised hosts may require higher doses or durations and/or combination therapy.
Fusobacterium nucleatum metabolically integrates commensals and pathogens in oral biofilms
Authors: Sakanaka Akito , Kuboniwa Masae , Shimma Shuichi , Alghamdi Samar A. , Mayumi Shota , Lamont Richard J. , Fukusaki Eiichiro , Amano Atsuo 
Published: June 2021

AbstractFusobacterium nucleatum is a common constituent of the oral microbiota in both periodontal health and disease. Previously, we discovered ornithine cross-feeding between F. nucleatum and Streptococcus gordonii, where S. gordonii secretes ornithine via an arginine-ornithine antiporter (ArcD), which in turn supports the growth and biofilm development of F. nucleatum; however, broader metabolic aspects of F. nucleatum within polymicrobial communities and their impact on periodontal pathogenesis have not been addressed. Here, we show that when co-cultured with S. gordonii, F. nucleatum increased amino acid availability to enhance the production of butyrate and putrescine, a polyamine produced by ornithine decarboxylation. Co-culture with Veillonella parvula, another common inhabitant of the oral microbiota, also increased lysine availability, promoting cadaverine production by F. nucleatum. We confirmed that ArcD-dependent ornithine excretion by S. gordonii results in synergistic putrescine production, and mass spectrometry imaging revealed this metabolic capability creates a putrescine-rich microenvironment inside F. nucleatum biofilms. We further demonstrated that polyamines caused significant changes in the biofilm phenotype of a periodontal pathogen, Porphyromonas gingivalis, with putrescine being a potent stimulator of biofilm development and dispersal, and confirmed that F. nucleatum-mediated conversion of ornithine to putrescine enhances biofilm formation by P. gingivalis. Lastly, analysis of plaque samples revealed cooccurrence of P. gingivalis with genetic modules for putrescine production by S. gordonii and F. nucleatum. Overall, our results highlight the ability of F. nucleatum to induce synergistic polyamine production within multi-species consortia, and provide insight into how the trophic web in oral biofilm ecosystems can eventually shape disease-associated communities.Significance StatementPeriodontitis is caused by the pathogenic transition of subgingival microbiota ecosystems, which is accompanied by alterations to microbiome functions including metabolic systems and the establishment of metabolic cross-feeding. While Fusobacterium nucleatum is a major constituent of the periodontal microbiota, its metabolic integration within polymicrobial communities and the impact on periodontal pathogenesis are poorly understood. Here, we report that amino acids supplied by other commensal bacteria induce polyamine production by F. nucleatum, creating polyamine-rich microenvironments. We further show that this trophic web results in enhancement of biofilm formation and dispersal of a periodontal pathogen, Porphyromonas gingivalis. This work provides mechanistic insight into how cooperative metabolism within oral biofilms can tip the balance toward periodontitis.