bluuebunny/crossref_metadata_2025_split huggingface dataset:
Multiplexed detection of SARS-CoV-2 genomic and subgenomic RNA using in situ hybridization
Authors: Acheampong Kofi K. , Schaff Dylan L. , Emert Benjamin L. , Lake Jonathan , Reffsin Sam , Shea Emily K. , Comar Courtney E. , Litzky Leslie A. , Khurram Nigar A. , Linn Rebecca L. , Feldman Michael , Weiss Susan R. , Montone Kathleen T. , Cherry Sara , Shaffer Sydney M. 
Published: August 2021

AbstractThe widespread Coronavirus Disease 2019 (COVID-19) is caused by infection with the novel coronavirus SARS-CoV-2. Currently, we have a limited toolset available for visualizing SARS-CoV-2 in cells and tissues, particularly in tissues from patients who died from COVID-19. Generally, single-molecule RNA FISH techniques have shown mixed results in formalin fixed paraffin embedded tissues such as those preserved from human autopsies. Here, we present a platform for preparing autopsy tissue for visualizing SARS-CoV-2 RNA using RNA FISH with amplification by hybridization chain reaction (HCR). We developed probe sets that target different regions of SARS-CoV-2 (including ORF1a and N) as well as probe sets that specifically target SARS-CoV-2 subgenomic mRNAs. We validated these probe sets in cell culture and tissues (lung, lymph node, and placenta) from infected patients. Using this technology, we observe distinct subcellular localization patterns of the ORF1a and N regions, with the ORF1a concentrated around the nucleus and the N showing a diffuse distribution across the cytoplasm. In human lung tissue, we performed multiplexed RNA FISH HCR for SARS-CoV-2 and cell-type specific marker genes. We found viral RNA in cells containing the alveolar type 2 (AT2) cell marker gene (SFTPC) and the alveolar macrophage marker gene (MARCO), but did not identify viral RNA in cells containing the alveolar type 1 (AT1) cell marker gene (AGER). Moreover, we observed distinct subcellular localization patterns of viral RNA in AT2 cells and alveolar macrophages, consistent with phagocytosis of infected cells. In sum, we demonstrate the use of RNA FISH HCR for visualizing different RNA species from SARS-CoV-2 in cell lines and FFPE autopsy specimens. Furthermore, we multiplex this assay with probes for cellular genes to determine what cell-types are infected within the lung. We anticipate that this platform could be broadly useful for studying SARS-CoV-2 pathology in tissues as well as extended for other applications including investigating the viral life cycle, viral diagnostics, and drug screening.
SARS-CoV-2 Spike Affinity and Dynamics Exclude the Strict Requirement of an Intermediate Host
Authors: Castelli Matteo , Scietti Luigi , Clementi Nicola , Cavallaro Mattia , Faravelli Silvia , Pinnola Alberta , Criscuolo Elena , Diotti Roberta Antonia , Clementi Massimo , Forneris Federico , Mancini Nicasio 
Published: August 2021

AbstractSARS-CoV-2 proximal origin is still unclear, limiting the possibility of foreseeing other spillover events with pandemic potential. Here we propose an evolutionary model based on the thorough dissection of SARS-CoV-2 and RaTG13 – the closest bat relative – spike dynamics, kinetics and binding to ACE2. Our results indicate that both spikes share nearly identical, high affinities for Rhinolophus affinis bat and human ACE2, pointing out to negligible species barriers directly related to receptor binding. Also, SARS-CoV-2 spike shows a higher degree of dynamics and kinetics optimization that favors ACE2 engagement. Therefore, we devise an affinity-independent evolutionary process that likely took place in R. affinis bats and limits the eventual involvement of other animal species in initiating the pandemic to the role of vector.
Role of YAP in early ectodermal specification and a Huntington’s Disease model of human neurulation
Authors: Piccolo Francesco M. , Kastan Nathaniel R. , Haremaki Tomomi , Tian Qingyun , Laundos Tiago L. , De Santis Riccardo , Carroll Thomas S. , Luo Ji-Dung , Gnedeva Ksenia , Etoc Fred , Hudspeth A. J. , Brivanlou Ali H. 
Published: August 2021

AbstractThe Hippo pathway, a highly conserved signaling cascade that functions as an integrator of molecular signals and biophysical states, ultimately impinges upon the transcription coactivator Yes-associated protein 1 (YAP). Hippo-YAP signaling has been shown to play key roles both at the early embryonic stages of implantation and gastrulation, and later during neurogenesis. To explore YAP’s potential role in neurulation, we used self-organizing neuruloids grown from human embryonic stem cells on micropatterned substrates. We identified YAP activation as a key lineage determinant, first between neuronal ectoderm and non-neuronal ectoderm, and later between epidermis and neural crest, indicating that YAP activity can enhance the effect of BMP4 stimulation and therefore affect ectodermal specification at this developmental stage. Because aberrant Hippo-YAP signaling has been implicated in the pathology of Huntington’s Disease (HD), we used isogenic mutant neuruloids to explore the relationship between signaling and the disease. We found that HD neuruloids demonstrate ectopic activation of gene targets of YAP and that pharmacological reduction of YAP’s transcriptional activity can partially rescue the HD phenotype.
Genome scale CRISPR Cas9a knockout screen reveals genes that controls glioblastoma susceptibility to the alkylating agent temozolomide
Authors: Awah Chidiebere U , Winter Jan , Ogunwobi Olorunseun O. 
Published: August 2021

AbstractGlioblastoma is the most fatal of all primary human brain tumors with 14 months survival, at best. The mainstay therapy for this tumor involves temozolomide, surgery, radiotherapy and tumor treating electric field. Cancer resistance to commonly available chemotherapeutics remains a major challenge in glioblastoma patients receiving treatment and unfavorably impact their overall survival and outcome. However, the lack of progress in this area could be attributed to lack of tools to probe unbiasedly at the genome wide level the coding and non-coding elements contribution on a large scale for factors that control resistance to chemotherapeutics. Understanding the mechanisms of resistance to chemotherapeutics will enable precision medicine in the treatment of cancer patients.CRISPR Cas9a has emerged as a functional genomics tool to study at genome level the factors that control cancer resistance to drugs. Recently, we used genome wide CRISPR-Cas9a screen to identify genes responsible for glioblastoma susceptibility to etoposide. We extended our inquiry to understand genes that control glioblastoma response to temozolomide by using genome scale CRISPR. This study shows that the unbiased genome-wide loss of function approach can be applied to discover genes that influence tumor resistance to chemotherapeutics and contribute to chemoresistance in glioblastoma.
Functionally distinct roles for eEF2K in the control of ribosome availability and p-body abundance in sensory neurons
Authors: Smith Patrick R. , Loerch Sarah , Kunder Nikesh , Stanowick Alexander D. , Lou Tzu-Fang , Campbell Zachary T. 
Published: August 2021

AbstractProcessing bodies (p-bodies) are a prototypical phase-separated RNA-containing granule. Their abundance is highly dynamic and has been linked to translation. Yet, the molecular mechanisms responsible for coordinate control of the two processes are unclear. Here, we uncover key roles for eEF2 kinase (eEF2K) in the control of ribosome availability and p-body abundance. eEF2K acts on a sole known substrate, eEF2, to inhibit translation. We find that the eEF2K agonist nelfinavir abolishes p-bodies specifically in sensory neurons and impairs translation. To probe the latter, we used cryo-electron microscopy. Nelfinavir stabilizes vacant 80S ribosomes. They contain SERBP1 in place of mRNA and eEF2 in the acceptor site. Phosphorylated eEF2 associates with inactive ribosomes that resist splittingin vitro. Collectively, the data suggest that eEF2 phosphorylation defines a population of inactive ribosomes resistant to recycling and protected from degradation. Thus, eEF2K activity is central to both p-body abundance and ribosome availability in sensory neurons.
ELICE16INDURES®: a plant immune-priming activator targeting jasmonate metabolism via TIFY and RHOMBOID proteins inHordeum vulgare
Authors: Hegedűs Géza , Kiniczky Márta , Nagy Ágnes , Pekker Péter , Lang Balázs , Gracza Lajos , Pallos József Péter , Thomas-Nyári Zsófia , Decsi Kincső , Kutasy Barbara , Székvári Kinga , Juhász Ákos , Virág Eszter 
Published: August 2021

AbstractPriming activity of plant-based allelochemicals is advanced research nowadays meaning a high potential in sustainable agriculture. The ELICE16INDURES®(RIMPH LTD, Hungary) plant conditioner of CO2botanical extracts is rich in plant-active ingredients such as phenolic compounds, alkaloids, and flavonoids formulated in small multilamellar liposomes. This product was investigatedin autumn barley (Hordeum vulgare). Field experiments of ELICE16INDURES showed augmented NDVI values interconnected with higher photosynthetic activity and yield increase. Background of the better vitality of plants was investigated by whole genomic gene expression profiling and showed an enhanced response to wounding, jasmonic acid, oxidative detoxification, and chloroplast activity. Among top50 differentially expressed genes the TIFY domain protein TIFY11B and RHOMBOID-like protein 2 related to JA signaling were up-regulated in field-collected samples. Phytotron experiments of barley were set up to validate and evaluate the transcriptomic effect of ELICE16INDURES. Well-studied priming active agents such as salicylic acid and beta-aminobutyric acid were compared with ELICE16INDURES and confirmed as priming inducer material with positive regulation of TIFY11B, TIFY3B, TIFY9, TIF10A, and RHOMBOID like protein 2 by using NGS GEx and RT-qPCR methods.One-sentence summaryELICE16INDURES®is a plant conditioner agent with a high amount of allelochemicals encapsulated into small multilamellar liposomes and found as an immune priming activator tested inH. vulgarefield and phytotron cultures.
Androgen synthesis inhibition but not gonadectomy reduces persistence during strategy set-shifting and reversal learning in male rats
Authors: Tomm Ryan J. , Seib Désirée R. , Kachkovski George V. , Schweitzer Helen R. , Tobiansky Daniel J. , Floresco Stan B. , Soma Kiran K. 
Published: August 2021

ABSTRACTAndrogens regulate behavioural flexibility, which is essential to adapt to a changing environment and depends on the medial prefrontal cortex (mPFC). Testosterone (T) administration decreases behavioural flexibility. It is well known that T is produced in the gonads, but T is also produced in the mesocorticolimbic system, which modulates behavioural flexibility. It is unclear how T produced in the brain versus the gonads influences behavioural flexibility. Here, we assess the effects of the androgen synthesis inhibitor abiraterone acetate (ABI) and long-term gonadectomy (GDX) on behavioural flexibility in two paradigms. In Experiment 1, ABI independent of GDX reduced the number of trials to criterion and perseverative errors in a strategy set-shifting task. Similarly, in Experiment 2, ABI but not GDX reduced perseverative errors in a reversal learning task. In subjects from Experiment 1, we also examined tyrosine hydroxylase immunoreactivity (TH-ir), and ABI but not GDX increased TH-ir in the mPFC. Our findings suggest that neurally-produced androgens modulate behavioural flexibility via modification of dopamine signalling in the mesocorticolimbic system. These results suggest novel roles for neurosteroids and possible side effects of ABI treatment for prostate cancer.
MRG proteins are shared by multiple protein complexes with distinct functions
Authors: Devoucoux Maëva , Roques Céline , Lachance Catherine , Lashgari Anahita , Joly-Beauparlant Charles , Jacquet Karine , Alerasool Nader , Prudente Alexandre , Taipale Mikko , Droit Arnaud , Lambert Jean-Philippe , Hussein Samer M.I. , Côté Jacques 
Published: August 2021

ABSTRACTMRG15/MORF4L1 is a highly conserved protein in eukaryotes that contains a chromodomain recognizing H3K36me3 in chromatin. Intriguingly, it has been reported in the literature to interact with several different factors involved in chromatin modifications, gene regulation, alternative mRNA splicing and DNA repair by homologous recombination. In order to get a complete and reliable picture of associations in physiological conditions, we used genome editing and tandem affinity purification to analyze the stable native interactome of human MRG15, its paralog MRGX/MORF4L2 that lacks the chromodomain, and MRGBP (MRG-binding protein) in isogenic K562 cells. We found stable interchangeable association of MRG15 and MRGX with the NuA4/TIP60 histone acetyltransferase/chromatin remodeler, Sin3B histone deacetylase/demethylase, ASH1L histone methyltransferase and PALB2/BRCA2 DNA repair protein complexes. These associations were further confirmed and analyzed by CRISPR-tagging of endogenous proteins and comparison of expressed isoforms. Importantly, based on structural information, point mutations could be introduced that can specifically disrupt MRG15 association with some complexes but not others. Most interestingly, we also identified a new abundant native complex formed by MRG15/X-MRGBP-BRD8-EP400NL that is functionally similar to the yeast TINTIN (Trimer Independent of NuA4 for Transcription Interactions with Nucleosomes) complex. Our results show that EP400NL, being homologous to the N-terminal region of NuA4/TIP60 subunit EP400, creates TINTIN by competing for BRD8 association. Functional genomics indicate that human TINTIN plays a role in transcription of specific genes. This is most likely linked to the H4ac-binding bromodomain of BRD8 along the H3K36me3-binding chromodomain of MRG15 on the coding region of transcribed genes. Taken together, our data provide a complete detailed picture of human MRG proteins-associated protein complexes which is essential to understand and correlate their diverse biological functions in chromatin-based nuclear processes.HighlightsMRG15 and MRGX are stably associated with several different protein complexes important for genome expression and stability.Several MRG-containing complexes are chromatin modifiers.Specific point mutations in the MRG domain differentially affect associated complexes.A major human complex homologous to the yeast TINTIN complex is identified.The protein EP400NL competes with EP400 to functionally separate TINTIN from the NuA4/TIP60 complex.TINTIN contains a bromodomain and a chromodomain to regulate transcription.
In vitro determination of inhibitory effects by humic substances complexing Zn and Se on SARS-CoV-2 virus replication
Authors: Hajdrik Polett , Pályi Bernadett , Kis Zoltán , Kovács Noémi , Veres Daniel S. , Szigeti Krisztián , Hegedűs Imre , Kovács Tibor , Bergmann Ralf , Máthé Domokos 
Published: August 2021

AbstractHumic substances are well known human nutritional supplement materials and play important performance-enhancing roles as animal feed additives, too. For decades, ingredients of humic substances have also been proven to carry potent antiviral effects against different viruses. Here, the antiviral activity of a humic substance containing ascorbic acid, Se- and Zn2+ ions intended as a nutritional supplement material was investigated against SARS-CoV-2 virus B1.1.7 Variant of Concern (“Alpha Variant”) in a VeroE6 cell line. Results show that this combination has a significant in vitro antiviral effect at a very low concentration range of its intended active ingredients. Even picomolar concentration ranges of humic substances, vitamin C and Zn/Se ions in the given composition were enough to achieve fifty percent viral replication inhibition in the applied SARS-CoV-2 virus inhibition test.
Protection against SARS-CoV-2 Beta Variant in mRNA-1273 Boosted Nonhuman Primates
Authors: Corbett Kizzmekia S. , Gagne Matthew , Wagner Danielle A. , Connell Sarah O’ , Narpala Sandeep R. , Flebbe Dillon R. , Andrew Shayne F. , Davis Rachel L. , Flynn Barbara , Johnston Timothy S. , Stringham Christopher , Lai Lilin , Valentin Daniel , Van Ry Alex , Flinchbaugh Zackery , Werner Anne P. , Moliva Juan I. , Sriparna Manjari , O’Dell Sijy , Schmidt Stephen D. , Tucker Courtney , Choi Angela , Koch Matthew , Bock Kevin W. , Minai Mahnaz , Nagata Bianca M. , Alvarado Gabriela S. , Henry Amy R. , Laboune Farida , Schramm Chaim A. , Zhang Yi , Wang Lingshu , Choe Misook , Boyoglu-Barnum Seyhan , Shi Wei , Lamb Evan , Nurmukhambetova Saule T. , Provost Samantha J. , Donaldson Mitzi M. , Marquez Josue , Todd John-Paul M. , Cook Anthony , Dodson Alan , Pekosz Andrew , Boritz Eli , Ploquin Aurélie , Doria-Rose Nicole , Pessaint Laurent , Andersen Hanne , Foulds Kathryn E. , Misasi John , Wu Kai , Carfi Andrea , Nason Martha C. , Mascola John , Moore Ian N. , Edwards Darin K. , Lewis Mark G. , Suthar Mehul S. , Roederer Mario , McDermott Adrian , Douek Daniel C. , Sullivan Nancy J. , Graham Barney S. , Seder Robert A. 
Published: August 2021

ABSTRACTNeutralizing antibody responses gradually wane after vaccination with mRNA-1273 against several variants of concern (VOC), and additional boost vaccinations may be required to sustain immunity and protection. Here, we evaluated the immune responses in nonhuman primates that received 100 µg of mRNA-1273 vaccine at 0 and 4 weeks and were boosted at week 29 with mRNA-1273 (homologous) or mRNA-1273.β (heterologous), which encompasses the spike sequence of the B.1.351 (beta or β) variant. Reciprocal ID50pseudovirus neutralizing antibody geometric mean titers (GMT) against live SARS-CoV-2 D614G and the β variant, were 4700 and 765, respectively, at week 6, the peak of primary response, and 644 and 553, respectively, at a 5-month post-vaccination memory time point. Two weeks following homologous or heterologous boost β-specific reciprocal ID50GMT were 5000 and 3000, respectively. At week 38, animals were challenged in the upper and lower airway with the β variant. Two days post-challenge, viral replication was low to undetectable in both BAL and nasal swabs in most of the boosted animals. These data show that boosting with the homologous mRNA-1273 vaccine six months after primary immunization provides up to a 20-fold increase in neutralizing antibody responses across all VOC, which may be required to sustain high-level protection against severe disease, especially for at-risk populations.One-sentence summarymRNA-1273 boosted nonhuman primates have increased immune responses and are protected against SARS-CoV-2 beta infection.
Towards the extended barcode concept: Generating DNA reference data through genome skimming of danish plants
Authors: Chua Physilia Y. S. , Leerhøi Frederik , Langkjær Emilia M. R. , Margaryan Ashot , Noer Christina L. , Richter Stine R. , Restrup Marlene E. , Bruun Hans Henrik , Hartvig Ida , Coissac Eric , Boessenkool Sanne , Alsos Inger G. , Bohmann Kristine 
Published: August 2021

ABSTRACTBackgroundRecently, there has been a push towards the extended barcode concept of utilising chloroplast genomes (cpGenome) and nuclear ribosomal DNA (nrDNA) sequences for molecular identification of plants instead of the standard barcode regions. These extended barcodes has a wide range of applications, including biodiversity monitoring and assessment, primer design, and evolutionary studies. However, these extended barcodes are not well represented in global reference databases. To fill this gap, we generated cpGenomes and nrDNA reference data from genome skims of 184 plant species collected in Denmark. We further explored the application of our generated reference data for molecular identifications of plants in an environmental DNA metagenomics study.ResultsWe assembled partial cpGenomes for 82.1% of sequenced species and full or partial nrDNA sequences for 83.7% of species. We added all assemblies to GenBank, of which chloroplast reference data from 101 species and nuclear reference data from 6 species were not previously represented. On average, we recovered 45 genes per species. The rate of recovery of standard barcodes was higher for nuclear barcodes (>89%) than chloroplast barcodes (< 60%). Extracted DNA yield did not affect assembly outcome, whereas high GC content did so negatively. For thein silicosimulation of metagenomic reads, taxonomic assignments using the reference data generated had better species resolution (94.9%) as compared to GenBank (18.1%) without any identification errors.ConclusionsGenome skimming generates reference data of both standard barcodes and other loci, contributing to the global DNA reference database for plants.
Cost-Benefit Analysis of a Children’s Spectacle Reimbursement Scheme: Evidence for Including Children’s Spectacles in Mongolia’s Social Health Insurance
Authors: Yong Ai Chee , Chuluunkhuu Chimgee , Chan Ving Fai , Stephan Tai , Congdon Nathan , O’Neill Ciaran 
Published: August 2021

AbstractBackground and aimGlobally, 12.8 million children have vision impairment due to uncorrected refractive error (URE). In Mongolia, one in five children needs but do not have access to spectacles. This cost-benefit analysis aims to estimate the net benefits of a children’s spectacles reimbursement scheme in Mongolia.MethodsA willingness-to-pay (WTP) survey using the contingent valuation method was administered to rural and urban Mongolia respondents. The survey assessed WTP in additional annual taxes for any child with refractive error to be provided government-subsidised spectacles. Net benefits were then calculated based on mean WTP (i.e. benefit) and cost of spectacles.ResultsThe survey recruited 50 respondents (mean age 40.2 ± 9.86 years; 78.0% women; 100% response rate) from rural and urban Mongolia. Mean WTP was US$24.00 ± 5.15 (95% CI US$22.55 to 25.35). The average cost of a pair of spectacles in Mongolia is US$15.00. Subtracting the average cost of spectacles from mean WTP yielded a mean positive net benefit of US$9.00.ConclusionA spectacle reimbursement scheme is a potentially cost-effective intervention to address childhood vision impairment due to URE in Mongolia. These preliminary findings support the inclusion of children’s spectacles into existing Social Health Insurance. A much larger random sample could be employed in future research to increase the precision and generalisability of findings.