bluuebunny/crossref_metadata_2025_split huggingface dataset:
Serum-free medium increases the replication rate of the avian coronavirus infectious bronchitis virus in chicken embryo kidney cells
Authors: Quinteros José A. , Browning Glenn F. , Noormohammadi Amir H. , Stevenson Mark A. , Coppo Mauricio J. C. , Loncoman Carlos A. , Ficorilli Nino , Lee Sang-Won , Diaz-Méndez Andrés 
Published: May 2021

AbstractInfectious bronchitis virus (IBV), an avian coronavirus, can be isolated and cultured in tracheal organ cultures (TOCs), embryonated eggs and cell cultures. TOCs and embryonated eggs are commonly used for viral isolation but use of these is laborious and expensive. Cell cultures have been used only with IBV strains that have previously been adapted to grow under laboratory conditions, and not for primary isolation. Previous studies using the coronavirus porcine epidemic diarrhoea virus (PEDV) have suggested that foetal bovine serum (FBS), a common component of cell culture media, can inhibit the adsorption of coronaviruses onto the host cell membrane receptors. In the present study, the replication of IBV in primary chicken embryo kidney (CEK) cell cultures and the Leghorn hepatocellular carcinoma (LMH) cell line was examined using two different cell culture media, one containing FBS and the other containing yeast extract (YE). A reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assay was used to quantify viral RNA copies in cell lysates. The highest concentrations of viral genomes were observed when the cell culture medium did not contain FBS. Examination of the infectivity of virus grown in CEK cell cultures was examined by titration in embryonated chicken eggs, demonstrating that the cell lysate from CEK cell cultures in medium without FBS contained a higher median embryo infectious dose (EID50) than that from CEK cell cultures in medium containing FBS. These results suggest that improved replication of IBV in cell cultures can be achieved by the omission of FBS from the cell culture medium. This may enhance the potential for production of vaccines in cell culture and facilitate the isolation of emergent IBV strains in cell cultures.
Sex- and Subtype-Specific Adaptations in Excitatory Signaling Onto Deep-Layer Prelimbic Cortical Pyramidal Neurons After Chronic Alcohol Exposure
Authors: Hughes Benjamin A. , O’Buckley Todd K. , Boero Giorgia , Herman Melissa A. , Morrow A. Leslie 
Published: May 2021

ABSTRACTLong-term alcohol use results in behavioral deficits including impaired working memory, elevated anxiety, and blunted inhibitory control that are associated with prefrontal cortical (PFC) dysfunction. Preclinical observations demonstrate multiple impairments in GABAergic neurotransmission onto deep-layer principal cells (PCs) in prelimbic cortex that suggest dependence-related cortical dysfunction is the product of elevated excitability in these cells. Despite accumulating evidence showing alcohol-induced changes in interneuron signaling onto PCs differ between sexes, there is limited data explicitly evaluating sex-specific ethanol effects on excitatory signaling onto deep-layer PCs that may further contribute to deficits in PFC-dependent behaviors. To address this, we conducted electrophysiological and behavioral tests in both male and female Sprague-Dawley rats to evaluate the effects of chronic ethanol exposure. Among our observations, we report a marked enhancement in glutamatergic signaling onto deep-layer PCs in male, but not female, rats after alcohol exposure. This phenomenon was furthermore specific to a sub-class of PC, sub-cortically projecting Type-A cells, and coincided with enhanced anxiety-like behavior, but no observable deficit in working memory. In contrast, female rats displayed an alcohol-induced facilitation in working memory performance with no change in expression of anxiety-like behavior. Together, these results suggest fundamental differences in alcohol effects on cell activity, cortical sub-circuits, and PFC-dependent behaviors across male and female rats.
Cell-free glycoengineering of the recombinant SARS-CoV-2 spike glycoprotein
Authors: Ruhnau Johannes , Grote Valerian , Juarez-Osorio Mariana , Bruder Dunja , Rapp Erdmann , Rexer Thomas F. T. , Reichl Udo 
Published: May 2021

AbstractThe baculovirus-insect cell expression system is readily utilized to produce viral glycoproteins for research as well as for subunit vaccines and vaccine candidates, for instance against SARS-CoV-2 infections. However, the glycoforms of recombinant proteins derived from this expression system are inherently different from mammalian cell-derived glycoforms with mainly complex-type N-glycans attached, and the impact of these differences in protein glycosylation on the immunogenicity is severely underinvestigated. This applies also to the SARS-CoV-2 spike glycoprotein, which is the antigen target of all licensed vaccines and vaccine candidates including virus like particles and subunit vaccines that are variants of the spike protein. Here, we expressed the transmembrane-deleted human β-1,2 N-acetlyglucosamintransferases I and II (MGAT1∆TM and MGAT2∆TM) and the β-1,4-galactosyltransferase (GalT∆TM) in E. coli to in-vitro remodel the N-glycans of a recombinant SARS-CoV-2 spike glycoprotein derived from insect cells. In a cell-free sequential one-pot reaction, fucosylated and afucosylated paucimannose-type N-glycans were converted to complex-type galactosylated N-glycans. In the future, this in-vitro glycoengineering approach can be used to efficiently generate a wide range of N-glycans on antigens considered as vaccine candidates for animal trials and preclinical testing to better characterize the impact of N-glycosylation on immunity and to improve the efficacy of protein subunit vaccines.
Disease-linked TDP-43 hyperphosphorylation suppresses TDP-43 condensation and aggregation
Authors: da Silva Lara Gruijs , Simonetti Francesca , Hutten Saskia , Riemenschneider Henrick , Sternburg Erin L. , Pietrek Lisa M. , Gebel Jakob , Dötsch Volker , Edbauer Dieter , Hummer Gerhard , Stelzl Lukas S. , Dormann Dorothee 
Published: May 2021

AbstractPost-translational modifications (PTMs) have emerged as key modulators of protein phase separation and have been linked to protein aggregation in neurodegenerative disorders. The major aggregating protein in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), the RNA-binding protein TDP-43, is hyperphosphorylated in disease on several C-terminal serine residues, which is generally believed to promote TDP-43 aggregation. Here, we show that hyperphosphorylation by Casein kinase 1δ or C-terminal phosphomimetic mutations surprisingly reduce TDP-43 phase separation and aggregation and render TDP-43 condensates more liquid-like and dynamic. Multi-scale simulations reveal reduced homotypic interactions of TDP-43 low complexity domains through enhanced solvation of phosphomimetic residues. Cellular experiments show that phosphomimetic substitutions do not affect nuclear import or RNA regulatory functions of TDP-43, but suppress accumulation of TDP-43 in membrane-less organelles and promote its solubility in neurons. We propose that TDP-43 hyperphosphorylation may be a protective cellular response to counteract TDP-43 aggregation.
Characterization of a triad of genes in cyanophage S-2L sufficient to replace adenine by 2-aminoadenine in bacterial DNA
Authors: Czernecki D. , Bonhomme F. , Kaminski P.A. , Delarue M. 
Published: May 2021

AbstractCyanophage S-2L is known to profoundly alter the biophysical properties of its DNA by replacing all adenines (A) with 2-aminoadenines (Z), which still pair with thymines but with a triple hydrogen bond. It was recently demonstrated that a homologue of adenylosuccinate synthase (PurZ) and a dATP triphosphohydrolase (DatZ) are two important pieces of the metabolism of 2-aminoadenine, participating in the synthesis of ZTGC-DNA. Here, we determine that S-2L PurZ can use either dATP or ATP as a source of energy, thereby also depleting the pool of nucleotides in dATP. Furthermore, we identify a conserved gene (mazZ)located betweenpurZanddatZgenes inSiphoviridaephage genomes, and show that it encodes a (d)GTP-specific diphosphohydrolase, thereby providing the substrate of PurZ in the 2-aminoadenine synthesis pathway. High-resolution crystal structures of S-2L PurZ and MazZ with their respective substrates provide a rationale for their specificities. The Z-cluster made of these three genes –datZ,mazZandpurZ– was expressed inE. coli, resulting in a successful incorporation of 2-aminoadenine in the bacterial chromosomal and plasmidic DNA. This work opens the possibility to study synthetic organisms containing ZTGC-DNA.
Post-stroke administration of the p75 neurotrophin receptor modulator, LM11A-31, attenuates chronic changes in brain metabolism, increases neurotransmitter levels, and improves recovery
Authors: Nguyen Thuy-Vi V. , Crumpacker Rachel H. , Calderon Kylie E. , Garcia Frankie G. , Zbesko Jacob C. , Frye Jennifer B. , Gonzalez Selena , Becktel Danielle A. , Yang Tao , Tavera-Garcia Marco A. , Morrison Helena W. , Schnellmann Rick G. , Longo Frank M. , Doyle Kristian P. 
Published: May 2021

ABSTRACTThe aim of this study was to test whether post-stroke oral administration of a small molecule p75 neurotrophin receptor (p75NTR) modulator (LM11A-31) can augment neuronal survival and improve recovery in a mouse model of stroke. Mice were administered LM11A-31 for up to 12 weeks, beginning 1 week after stroke. Metabolomic analysis revealed that after 2 weeks of daily treatment, mice that received LM11A-31 were distinct from vehicle treated mice by principal component analysis and had higher levels of serotonin, acetylcholine, and dopamine in their ipsilateral hemisphere. LM11A-31 treatment also improved redox homeostasis by restoring reduced glutathione. It also offset a stroke induced reduction in glycolysis by increasing acetyl-CoA. There was no effect on cytokine levels in the infarct. At 13 weeks following stroke, adaptive immune cell infiltration in the infarct was unchanged in LM11A-31 treated mice, indicating that LM11A-31 does not alter the chronic inflammatory response to stroke at the site of the infarct. However, LM11A-31 treated mice had less brain atrophy, neurodegeneration, tau pathology, and microglial activation in other regions of the ipsilateral hemisphere. These findings correlated with improved recovery of motor function on a ladder test, improved sensorimotor and cognitive abilities on a nest construction test, and less impulsivity in an open field test. These data support small molecule modulation of the p75 neurotrophin receptor for preserving neuronal health and function during stroke recovery.SIGNIFICANCE STATEMENTThe findings from this study introduce the p75 neurotrophin receptor as a novel small molecule target for promotion of stroke recovery. Given that LM11A-31 is in clinical trials as a potential therapy for Alzheimer’s disease, it could be considered as a candidate for assessment in stroke or vascular dementia studies.
Shelterin components modulate nucleic acids condensation and phase separation in the context of telomeric DNA
Authors: Soranno Andrea , Incicco J. Jeremías , De Bona Paolo , Tomko Eric J. , Galburt Eric A. , Holehouse Alex S. , Galletto Roberto 
Published: May 2021

AbstractTelomeres are nucleoprotein complexes that protect the ends of chromosomes and are essential for chromosome stability in Eukaryotes. In cells, individual telomeres form distinct globules of finite size that appear to be smaller than expected for bare DNA. Moreover, upon changes in their protein composition, telomeres can cluster to form telomere-induced-foci (TIFs) or co-localize with promyelocytic leukemia (PML) nuclear bodies. The physical basis for collapse of individual telomeres and coalescence of multiple ones remains unclear, as does the relationship between these two phenomena. By combining single-molecule measurements, optical microscopy, turbidity assays, and simulations, we show that the telomere scaffolding protein TRF2 can condense individual DNA chains and drives coalescence of multiple DNA molecules, leading to phase separation and the formation of liquid-like droplets. Addition of the TRF2 binding protein hRap1 modulates phase boundaries and tunes the specificity of solution demixing while simultaneously altering the degree of DNA compaction. Our results suggest that the condensation of single telomeres and formation of biomolecular condensates containing multiple telomeres are two different outcomes driven by the same set of molecular interactions. Moreover, binding partners, such as other telomere components, can alter those interactions to promote single-chain DNA compaction over multiple-chain phase separation.
Early acquisition of threat conditioning in a selectively-bred anxiety-like rat phenotype: regulation by maternal presence and FGF2
Authors: White Amanda M. , Chang Da-Jeong , Hider Joanna H. , Hebda-Bauer Elaine K. , Turner Cortney A. , Sullivan Regina M. , Akil Huda , Debiec Jacek 
Published: May 2021

AbstractTemperament is an innate, stable predisposition towards particular emotional and behavioral responses. In humans, certain temperaments are associated with a heightened risk of developing anxiety later in life. Non-human animals, including rodents, also exhibit innate, stable dispositions; these are referred to as behavioral phenotypes. The interaction between behavioral phenotype and early life adverse events is critical for the development of maladaptive anxiety. Rodent studies of typically developing animals have identified a number of mechanisms that protect against aversive experiences in early life. One such mechanism is an early life quiescence of threat learning, which protects against the effects of stress and facilitates safety and attachment learning. However, little is known about the factors that alleviate the effects of early life aversive events on phenotypes vulnerable to pathological anxiety. Here, we examined threat learning and the stress response in selectively-bred infant rats that show an anxiety-like phenotype relative to typically developing animals. We investigated the potential roles of maternal presence and the anxiolytic neurotrophic factor fibroblast growth factor 2 (FGF2) in regulating threat learning and the stress response in infant anxiety-like phenotype animals. We observed that rats selectively-bred for anxiety-like behaviors could acquire conditioned freezing earlier in life than typically developing animals. FGF2 administration on postnatal day 1 (PND 1) and maternal presence during threat conditioning were both capable of suppressing this early emergence of conditioned freezing. However, neither FGF2 nor maternal presence during threat conditioning were associated with reduced corticosterone levels during threat conditioning. Our results suggest that although an anxiety-like phenotype may be associated with early threat learning, environmental factors (such as maternal presence) and pharmacological intervention (such as modulation of the FGF2 system) may be capable of counteracting that early aversive learning. Interventions in vulnerable infants may thus decrease the impact of aversive events.
The sperm protein SPACA4 is required for efficient fertilization in mice
Authors: Herberg Sarah , Fujihara Yoshitaka , Blaha Andreas , Panser Karin , Kobayashi Kiyonori , Larasati Tamara , Novatchkova Maria , Theußl H. Christian , Olszanska Olga , Ikawa Masahito , Pauli Andrea 
Published: May 2021

SUMMARYFertilization is the fundamental process that initiates the development of a new individual in all sexually reproducing species. Despite its importance, our understanding of the molecular players that govern mammalian sperm-egg interaction is incomplete, partly because many of the essential factors found in non-mammalian species do not have obvious mammalian homologs. We have recently identified the Ly6/uPAR protein Bouncer as a new, essential fertilization factor in zebrafish (Herberg et al., 2018). Here, we show that Bouncer’s homolog in mammals, SPACA4, is also required for efficient fertilization in mice. In contrast to fish, where Bouncer is expressed specifically in the egg, SPACA4 is expressed exclusively in the sperm. Male knockout mice are severely sub-fertile, and sperm lacking SPACA4 fail to fertilize wild-type eggsin vitro. Interestingly, removal of the zona pellucida rescues the fertilization defect ofSpaca4-deficient spermin vitro, indicating that SPACA4 is not required for the interaction of sperm and the oolemma but rather of sperm and zona pellucida. Our work identifies SPACA4 as an important sperm protein necessary for zona pellucida penetration during mammalian fertilization.
Oxidized alkyl phospholipids stimulate sodium transport in proximal tubules via PPARγ-dependent pathway
Authors: Mizuno Tomohito , Satoh Nobuhiko , Horita Shoko , Tsukada Hiroyuki , Sato Yusuke , Kume Haruki , Nangaku Masaomi , Nakamura Motonobu 
Published: May 2021

AbstractThe pleiotropic effects of oxidized phospholipids (oxPLs) have been identified. 1-O-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine (azPC), an oxPL formed from alkyl phosphatidylcholines, is a potent peroxisome proliferator-activated receptor γ (PPARγ) agonist. Although it has been reported that thiazolidinediones can induce volume expansion by enhancing renal sodium and water retention, the role of azPC, an endogenous PPARγ agonist, in renal transport functions is unknown. In the present study, we investigated the effect of azPC on renal proximal tubule (PT) transport using isolated PTs and kidney cortex tissues. We showed that azPC rapidly stimulated Na+/HCO3-cotransporter 1 activity and luminal Na+/H+exchanger (NHE) activities in a dose-dependent manner, at submicromolar concentrations, in isolated PTs from rats and humans. Additionally, the stimulatory effects were completely blocked by a specific PPARγ antagonist, 2-chloro-5-nitro-N-phenylbenzamide (GW9662), and a mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor, PD98059. Treatment with an siRNA against PPARγ significantly suppressed the expression of PPARγ mRNA, and it completely blocked the stimulation of both Na+/HCO3-cotransporter 1 and NHE activities by azPC. Moreover, azPC induced extracellular signal-regulated kinase (ERK) phosphorylation in rat and human kidney cortex tissues, and the induced ERK phosphorylation by azPC was completely suppressed by GW9662 and PD98059. These results suggest that azPC stimulates renal PT sodium-coupled bicarbonate transport via the PPARγ/MEK/ERK pathway. The stimulatory effects of azPC on PT transport may be partially involved in the development of volume expansion.
Simple and efficient heterologous expression of necrosis-inducing effectors using the model plantNicotiana benthamiana
Authors: Dagvadorj Bayantes , Solomon Peter S. 
Published: May 2021

AbstractPlant fungal pathogens cause devastating diseases on cereal plants and threaten global food security. During infection, these pathogens secrete proteinaceous effectors that promote disease. Some of these effectors from necrotrophic plant pathogens induce a cell death response (necrosis), which facilitates pathogen growthin planta. Characterisation of these effectors typically requires heterologous expression and microbial expression systems such as bacteria and yeast are the predominantly used. However, microbial expression systems often require optimization for any given effector and are, in general, not suitable for effectors involving cysteine bridges and posttranslational modifications for activity. Here, we describe a simple and efficient method for expressing such effectors in the model plantNicotiana benthamiana. Briefly, an effector protein is transiently expressed and secreted into the apoplast ofN. benthamianaby Agrobacterium-mediated infiltration. Two-to-three days subsequent to agroinfiltration, the apoplast from the infiltrated leaves is extracted and can be directly used for phenotyping on host plants. The efficacy of this approach was demonstrated by expressing the ToxA, Tox3 and Tox1 necrosis-inducing effectors fromParastagonospora nodorum. All three effectors produced inN. benthamianawere capable of inducing necrosis in wheat lines, and two of three showed visible bands on Coomassie-stained gel. These data suggest thatN. benthamiana-agroinfiltration system is a feasible tool to obtain fungal effectors, especially those that require disulfide bonds and posttranslational modifications. Furthermore, due to the low number of proteins typically observed in the apoplast (compared to intracellular), this simple and high-throughput approach circumvents the requirement to lyse cells and further purify the target proteins that is required in other heterologous systems. Because of its simplicity and potential for high-throughput, this method is highly amenable to the phenotyping of candidate protein effectors on host plants.
Data fusion for abundance estimation: community science augments systematically collected removal-in-time distance sampling data
Authors: Joseph Maxwell B. , Pavlacky David C. , Bartuszevige Anne M. 
Published: May 2021

AbstractEcologists use a variety of systematically and opportunistically sampled count data to estimate bird abundance, and integrating or fusing different datasets has emerged as a critical challenge in recent years. While previous work provides data integration methodology for occupancy (presence/absence) estimation, methods for abundance estimation that account for imperfect detection and disparate survey protocols remains an active area of research. Here we show how to integrate systematically collected removal-in-time distance sampling data from the Integrated Monitoring in Bird Conservation Regions (IMBCR) program with North American Breeding Bird Survey (BBS) point counts and eBird community science observations. Using the Grasshopper Sparrow (Ammodramus savannarum) in the Great Plains of the United States as a focal species, we demonstrate that BBS and eBird data improve predictive performance for IMBCR count data, providing more spatially refined and precise estimates of abundance at regional scales. Data fusion increased predictive performance even despite relatively weak spatial correlations among data sets. The methodology developed here provides a principled way to fuse data when estimating abundance with distance sampling, that accounts for imperfect detection and variable effort.