bluuebunny/crossref_metadata_2025_split huggingface dataset:
An interferon lambda 4-associated variant in the hepatitis C virus RNA polymerase affects viral replication in infected cells
Authors: Bamford Connor G G , McLauchlan John 
Published: June 2020

AbstractHostIFNL4haplotype status contributes to the development of chronic hepatitis C virus infection in individuals who are acutely infected with the virus.In silicostudies revealed that specific amino acid variants at multiple sites on the HCV polyprotein correlate with functional single nucleotide polymorphisms (SNPs) in theIFNL4locus. Thus, SNPs at theIFNL4locus may select variants that influence virus replication and thereby outcome of infection. Here, we examine the most significantlyIFNL4-associated amino acid variants that lie in the ‘Lambda (L) 2 loop’ of the HCV NS5B RNA polymerase. L2 loop variants were introduced into both sub-genomic replicon and full-length infectious clones of HCV and viral replication examined in the presence and absence of exogenous IFNλ4. Our data demonstrate that while mutation of NS5B L2 loop affects replication, individualIFNL4-associated variants have modest but consistent effects on replication both in the presence and absence of IFNλ4. Given the strong genetic association between these variants andIFNL4, these data suggest a nuanced effect of each individual position on viral replication, the combined effect of which might mediate resistance to the effects of IFNλ4.
Transcription repressor protein ZBTB25 interacts with HDAC1 in macrophages infected withMycobacterium tuberculosis,and its inhibition leads to autophagy and killing of the intracellular pathogen
Authors: Madhavan Aravind , Arun K B , Pushparajan Akhil Raj , Balaji M , Kumar R Ajay 
Published: June 2020

AbstractDownregulation of host gene expression is one of the key strategies adopted by intracellular pathogens such asMycobacterium tuberculosis(MTB) for their survival and subsequent pathogenesis. In a previous study, we have shown that HDAC1 levels go up in macrophages infected with MTB and it hypoacetylates histone H3 at the promoter ofIL-12Bgene leading to its downregulation. Here we show that after infection with MTB, the levels of the phosphorylated form of HDAC1 increase significantly in macrophages. Employing immunoprecipitation and LC-MS/MS, we found transcriptional repressor protein ZBTB25 associates with HDAC1 silencing complex along with transcriptional corepressor Sin3a. By chromatin immunoprecipitation and PCR analyses, we found that phosphorylated HDAC1, Sin3a, and ZBTB25 are recruited to the promoter ofIL-12Bto downregulate its expression in infected macrophages. Knocking down ofZBTB25enhanced release of IL-12p40 from infected macrophages. Interestingly, the treatment of infected macrophages with CI994 (inhibitor of HDAC1) or dithiopyridine (inhibitor of ZBTB25) promoted the colocalization of LC3 (microtubule-associated protein 1A/1B-light chain 3, a marker for autophagy) and MTB in autophagosomes. Induction of autophagy resulted in the killing of intracellular MTB. Enhanced phosphorylation of JAK2 and STAT4 was observed in macrophages upon CI994 and dithiopyridine treatment, and inhibition of JAK2/STAT4 negated the killing of intracellular mycobacteria suggesting a possible role of these proteins in the autophagy-mediated killing of intracellular MTB.
Functional characterization of SARS-CoV-2 infection suggests a complex inflammatory response and metabolic alterations
Authors: Trilla-Fuertes Lucía , Ramos Ricardo , Blanca-López Natalia , López-Camacho Elena , Martín-Pedraza Laura , Ryan Murua Pablo , Díaz-Almirón Mariana , Llorens Carlos , Gabaldón Toni , Moya Andrés , Fresno Vara Juan Ángel , Gámez-Pozo Angelo 
Published: June 2020

AbstractCovid-19, caused by the SARS-CoV-2 virus, has reached the category of a worldwide pandemic. Even though intensive efforts, no effective treatments or a vaccine are available. Molecular characterization of the transcriptional response in Covid-19 patients could be helpful to identify therapeutic targets. In this study, RNAseq data from peripheral blood mononuclear cell samples from Covid-19 patients and healthy controls was analyzed from a functional point of view using probabilistic graphical models. Two networks were built: one based on genes differentially expressed between healthy and infected individuals and another one based on the 2,000 most variable genes in terms of expression in order to make a functional characterization. In the network based on differentially expressed genes, two inflammatory response nodes with different tendencies were identified, one related to cytokines and chemokines, and another one related to bacterial infections. In addition, differences in metabolism, which were studied in depth using Flux Balance Analysis, were identified. SARS-CoV2-infection caused alterations in glutamate, methionine and cysteine, and tetrahydrobiopterin metabolism. In the network based on 2,000 most variable genes, also two inflammatory nodes with different tendencies between healthy individuals and patients were identified. Similar to the other network, one was related to cytokines and chemokines. However, the other one, lower in Covid-19 patients, was related to allergic processes and self-regulation of the immune response. Also, we identified a decrease in T cell node activity and an increase in cell division node activity. In the current absence of treatments for these patients, functional characterization of the transcriptional response to SARS-CoV-2 infection could be helpful to define targetable processes. Therefore, these results may be relevant to propose new treatments.
Modeling Heterogeneity of Triple-Negative Breast Cancer Uncovers a Novel Combinatorial Treatment Overcoming Primary Drug Resistance
Authors: Lamballe Fabienne , Ahmad Fahmida , Vinik Yaron , Castellanet Olivier , Daian Fabrice , Müller Anna-Katharina , Köhler Ulrike A. , Bailly Anne-Laure , Josselin Emmanuelle , Castellano Rémy , Cayrou Christelle , Charafe-Jauffret Emmanuelle , Mills Gordon B. , Géli Vincent , Borg Jean-Paul , Lev Sima , Maina Flavio 
Published: June 2020

AbstractTriple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype characterized by a remarkable molecular heterogeneity. Currently, there are no effective druggable targets and advanced preclinical models of the human disease. Here, we generated a unique mouse model (MMTV-R26Metmice) of mammary tumors driven by a subtle increase in the expression of the wild-type MET receptor.MMTV-R26Metmice develop spontaneous, exclusive TNBC tumors, recapitulating primary resistance to treatment of patients. Proteomic profiling ofMMTV-R26Mettumors and machine learning approach showed that the model faithfully recapitulates inter-tumoral heterogeneity of human TNBC. Further signaling network analysis highlighted potential druggable targets, of which co-targeting of WEE1 and BCL-XL synergistically killed TNBC cells and efficiently induced tumor regression. Mechanistically, BCL-XL inhibition exacerbates the dependency of TNBC cells on WEE1 function, leading to Histone H3 and phosphoS33RPA32 upregulation, RRM2 downregulation, cell cycle perturbation, mitotic catastrophe and apoptosis. Our study introduces a unique, powerful mouse model for studying TNBC formation and evolution, its heterogeneity, and for identifying efficient therapeutic targets.
Can colony size of honeybees (Apis mellifera) be used as predictor for colony losses due toVarroa destructorduring winter?
Authors: van Dooremalen Coby , van Langevelde Frank 
Published: June 2020

AbstractFor more than three decades, honeybee colonies (Apis mellifera) experience high losses during winter, and these losses are still continuing. It is crucial that beekeepers monitor their colonies closely and anticipate losses early enough to apply mitigating actions. We tested whether colony size can be used as early predictor for potential colony losses, in particular due to the parasitic miteVarroa destructor. V. destructoris one of the most important causes for these losses. Such early predictor for potentialV. destructorinduced losses is especially relevant as measuringV. destructorload in colonies is difficult and cumbersome. During three years, we monitored colonies with high and lowV. destructorload from July until March of the next year. We found that differences in colony size were only visible after November, even though we lost almost all colonies every winter in the group with highV. destructorload. In the Northern hemisphere, November is considered to be too late for beekeepers to strengthen colonies in preparation for winter. We therefore argue that early-warning signs for potential colony losses due toV. destructorare urgently needed to allow beekeepers preventing winter losses. We discuss the role of precision apiculture to monitor the health and productivity of honeybee colonies.
The small RNA ErsA plays a role in the regulatory network ofPseudomonas aeruginosapathogenicity in airways infection
Authors: Ferrara Silvia , Rossi Alice , Ranucci Serena , De Fino Ida , Bragonzi Alessandra , Cigana Cristina , Bertoni Giovanni 
Published: June 2020

AbstractBacterial small RNAs play a remarkable role in the regulation of functions involved in host-pathogen interaction. ErsA is a small RNA ofPseudomonas aeruginosathat contributes to the regulation of bacterial virulence traits such as biofilm formation and motility. Shown to take part in a regulatory circuit under the control of the envelope stress response sigma factor σ22, ErsA targets post-transcriptionally the key virulence-associated genealgC. Moreover, ErsA contributes to biofilm development and motility through the post-transcriptional modulation of the transcription factor AmrZ. Intending to evaluate the regulatory relevance of ErsA in the pathogenesis of respiratory infections, we analyzed the impact of ErsA-mediated regulation on the virulence potential ofP. aeruginosaand the stimulation of the inflammatory response during the infection of bronchial epithelial cells and a murine model. Furthermore, we assessed ErsA expression in a collection ofP. aeruginosaclinical pulmonary isolates and investigated the link of ErsA with acquired antibiotic resistance by generating anersAgene deletion mutant in a multidrug-resistantP. aeruginosastrain which has long been adapted in the airways of a cystic fibrosis (CF) patient. Our results show that the ErsA-mediated regulation is relevant for theP. aeruginosapathogenicity during acute infection and contributes to the stimulation of the host inflammatory response. Besides, ErsA could be subjected to selective pressure forP. aeruginosapatho-adaptation and acquirement of resistance to antibiotics commonly used in clinical practice during chronic CF infections. Our findings establish the role of ErsA as an important regulatory element in the host-pathogen interaction.Author summaryPseudomonas aeruginosais one of the most critical multi-drug resistant opportunistic pathogen in humans, able to cause both lethal acute and chronic lung infections. Thorough knowledge of the regulatory mechanisms involved in the establishment and persistence of the airways infections byP. aeruginosaremains elusive. Emerging candidates as molecular regulators of pathogenesis inP. aeruginosaare small RNAs, which act post-transcriptionally as signal transducers of host cues. Known for being involved in the regulation of biofilm formation and responsive to envelope stress response, we show that the small RNA ErsA can play regulatory roles in acute infection, stimulation of host inflammatory response, mechanisms of acquirement of antibiotic resistance and adaptation during the chronic lung infections of cystic fibrosis patients. Elucidating the complexity of the networks regulating host-pathogen interaction is crucial to identify novel targets for future therapeutic applications.
Phosphoproteomics after nitrate treatments reveal an important role for PIN2 phosphorylation in control of root system architecture
Authors: Vega Andrea , Fredes Isabel , O’Brien José , Shen Zhouxin , Ötvös Krisztina , Benkova Eva , Briggs Steven P. , Gutiérrez Rodrigo A. 
Published: June 2020

AbstractNitrate is an important signaling molecule that commands genome-wide gene expression changes that impact metabolism, physiology, plant growth and development. Although gene expression responses to nitrate at the mRNA level have been characterized in great detail, the impact of nitrate signaling at the proteome level has been much less explored. Most signaling pathways involve post-translational modifications of key protein factors and chiefly among these modifications is protein phosphorylation. In an effort to identify new components involved in nitrate responses in plants, we performed analyses of theArabidopsis thalianaroot phosphoproteome in response to nitrate treatments via liquid chromatography coupled to tandem mass spectrometry. We identified 268 phosphoproteins that show significant changes at 5 min or 20 min after nitrate treatments. The large majority of these proteins (96%) are coded by genes that are not modulated at the expression level in response to nitrate treatments in publicly available transcriptome data. Proteins identified by 5 min include potential signaling-components such as kinases or transcription factors. In contrast, by 20 min, proteins identified were associated with protein binding, transporter activity or hormone metabolism functions. Interestingly, the phosphorylation profile ofNITRATE TRANSPORTER 1.1(NRT1.1)mutant plants in response to nitrate at 5 min was significantly different (95%) as compared to wild-type plants. This result is consistent with the role of NRT1.1 as a key component of a nitrate signaling pathway that involves phosphoproteomic changes. Our integrative bioinformatics analysis highlights auxin transport as an important mechanism modulated by nitrate signaling at the post-translational level. We experimentally validated the role of PIN2 phosphorylation in both primary and lateral root growth responses to nitrate. Our data provide new insights into the phosphoproteome and identifies novel protein components that are regulated post-translationally, such as PIN2, in nitrate responses inArabidopsis thalianaroots.
Generation of SARS-CoV-2 S1 spike glycoprotein putative antigenic epitopes in vitro by intracellular aminopeptidases
Authors: Stamatakis George , Samiotaki Martina , Mpakali Anastasia , Panayotou George , Stratikos Efstratios 
Published: June 2020

AbstractPresentation of antigenic peptides by MHCI is central to cellular immune responses against viral pathogens. While adaptive immune responses versus SARS-CoV-2 can be of critical importance to both recovery and vaccine efficacy, how protein antigens from this pathogen are processed to generate antigenic peptides is largely unknown. Here, we analyzed the proteolytic processing of overlapping precursor peptides spanning the entire sequence of the S1 spike glycoprotein of SARS-CoV-2, by three key enzymes that generate antigenic peptides, aminopeptidases ERAP1, ERAP2 and IRAP. All enzymes generated shorter peptides with sequences suitable for binding onto HLA alleles, but with distinct specificity fingerprints. ERAP1 was the most efficient in generating peptides 8-11 residues long, the optimal length for HLA binding, while IRAP was the least efficient. The combination of ERAP1 with ERAP2 greatly limited the variability of peptide sequences produced. Less than 7% of computationally predicted epitopes were found to be produced experimentally, suggesting that aminopeptidase processing may constitute a significant filter to epitope presentation. These experimentally generated putative epitopes could be prioritized for SARS-CoV-2 immunogenicity studies and vaccine design. We furthermore propose that this in vitro trimming approach could constitute a general filtering method to enhance the prediction robustness for viral antigenic epitopes.
Ferric Heme as a CO/NO Sensor in the Nuclear Receptor Reverbβ by Coupling Gas binding to Electron Transfer
Authors: Sarkar Anindita , Carter Eric L. , Harland Jill B. , Speelman Amy L. , Lehnert Nicolai , Ragsdale Stephen W. 
Published: June 2020

AbstractRev-Erbβ is a nuclear receptor that couples circadian rhythm, metabolism, and inflammation.1-7Heme binding to the protein modulates its function as a repressor, its stability, its ability to bind other proteins, and its activity in gas sensing.8-11Rev-Erbβ binds Fe3+-heme tighter than Fe2+-heme, suggesting its activities may be regulated by the heme redox state.9Yet, this critical role of heme redox chemistry in defining the protein’s resting state and function is unknown. We demonstrate by electrochemical and whole-cell electron paramagnetic resonance experiments that Rev-Erbβ exists in the Fe3+form within the cell essentially allowing the protein to be heme-replete even at low concentrations of labile heme in the nucleus. However, being in the Fe3+redox state contradicts Rev-Erb’s known function as a gas sensor, which dogma asserts must be a Fe2+protein This paper explains why the resting Fe3+-state is congruent both with heme-binding and cellular gas sensing. We show that the binding of CO/NO elicits a striking increase in the redox potential of the Fe3+/Fe2+couple, characteristic of an EC mechanism in which the unfavorableElectrochemical reduction of heme is coupled to the highly favorableChemical reaction of gas binding, making the reduction spontaneous. Thus, Fe3+-Rev-Erbβ remains heme-loaded, crucial for its repressor activity, and only undergoes reduction when diatomic gases are present. This work has broad implications for hemoproteins where ligand-triggered redox changes cause conformational changes influencing protein’s function or inter-protein interactions, like NCoR1 for Rev-Erbβ. This study opens up the possibility of CO/NO-mediated regulation of the circadian rhythm through redox changes in Rev-Erbβ.
Movement signaling in ventral pallidum and dopaminergic midbrain is gated by behavioral state in singing birds
Authors: Chen Ruidong , Gadagkar Vikram , Roeser Andrea C. , Puzerey Pavel A. , Goldberg Jesse H. 
Published: June 2020

AbstractMovement-related neuronal discharge in ventral tegmental area (VTA) and ventral pallidum (VP) is inconsistently observed across studies. One possibility is that some neurons are movement-related and others are not. Another possibility is that the precise behavioral conditions matter - that a single neuron can be movement related under certain behavioral states but not others. We recorded single VTA and VP neurons in birds transitioning between singing and non-singing states, while monitoring body movement with microdrive-mounted accelerometers. Many VP and VTA neurons exhibited body movement-locked activity exclusively when the bird was not singing. During singing, VP and VTA neurons could switch off their tuning to body movement and become instead precisely time-locked to specific song syllables. These changes in neuronal tuning occurred rapidly at state boundaries. Our findings show that movement-related activity in limbic circuits can be gated by behavioral context.Significance statementNeural signals in the limbic system have long been known to represent body movements as well as reward. Here we show that single neurons dramatically change their tuning to movements when a bird starts to sing.
COPII Sec23 proteins form isoform-specific ER exit sites with differential effects on polarized growth
Authors: Chang Mingqin , Wu Shu-Zon , Ryken Samantha E. , O’Sullivan Jacquelyn E. , Bezanilla Magdalena 
Published: June 2020

AbstractCOPII, a coat of proteins that form vesicles on the ER, mediates vesicle traffic from the ER to the Golgi. In contrast to metazoans that have few genes encoding each COPII component, plants have expanded these gene families leading to the hypothesis that plant COPII has functionally diversified. Here, we analyzed the gene families encoding for the Sec23/24 heterodimer in the mossPhyscomitrium (Physcomitrella) patens. InP. patens, Sec23andSec24gene families are each comprised of seven genes. Silencing the Sec23/24 genes revealed isoform specific contributions to polarized growth, with the closely relatedSec23D/EandSec24C/Dessential for protonemal development. Focusing on theSec23gene family, we discovered that loss of Sec23D alters ER morphology, increases ER stress, inhibits trafficking to the Golgi and to the plasma membrane in tip growing protonemata. In contrast, the remaining fiveSec23genes are dispensable for tip growth. WhileSec23A/B/C/F/Gdo not quantitatively affect ER to Golgi trafficking in protonemata, they do contribute to secretion to the plasma membrane. Of the three highly expressed Sec23 isoforms in protonemata, Sec23G forms ER exit sites that are larger than Sec23B and Sec23D and do not overlap with Sec23D. Furthermore, ER exit sites labeled by Sec23B or Sec23G form in the absence of Sec23D. These data suggest thatSec23D/Eform unique ER exit sites contributing to secretion that is essential for tip growing protonemata.
Rolling Circle Amplification is a high fidelity and efficient alternative to plasmid preparation for the rescue of infectious clones
Authors: Marano Jeffrey M. , Chuong Christina , Weger-Lucarelli James 
Published: June 2020

AbstractAlphaviruses (genusAlphavirus; familyTogaviridae) are a medically relevant family of viruses that include chikungunya virus, Eastern equine encephalitis virus, and the emerging Mayaro virus. Infectious cDNA clones of these viruses are necessary molecular tools to understand viral biology and to create effective vaccines. The traditional approach to rescuing virus from an infectious cDNA clone requires propagating large amounts of plasmids in bacteria, which can result in unwanted mutations in the viral genome due to bacterial toxicity or recombination and requires specialized equipment and knowledge to propagate the bacteria. Here, we present an alternative to the bacterial-based plasmid platform that uses rolling circle amplification (RCA), anin vitrotechnology that amplifies plasmid DNA using only basic equipment. We demonstrate that the use of RCA to amplify plasmid DNA is comparable to the use of a midiprepped plasmid in terms of viral yield, albeit with a slight delay in virus recovery kinetics. RCA, however, has lower cost and time requirements and amplifies DNA with high fidelity and with no chance of unwanted mutations due to toxicity. We show that sequential RCA reactions do not introduce mutations into the viral genome and, thus, can replace the need for glycerol stocks or bacteria entirely. These results indicate that RCA is a viable alternative to traditional plasmid-based approaches to viral rescue.ImportanceThe development of infectious cDNA clones is critical to studying viral pathogenesis and for developing vaccines. The current method for propagating clones in bacteria is limited by the toxicity of the viral genome within the bacterial host, resulting in deleterious mutations in the viral genome, which can only be detected through whole-genome sequencing. These mutations can attenuate the virus, leading to lost time and resources and potentially confounding results. We have developed an alternative method of preparing large quantities of DNA that can be directly transfected to recover infectious virus without the need for bacteria by amplifying the infectious cDNA clone plasmid using rolling circle amplification (RCA). Our results indicate that viral rescue from an RCA product produces a viral yield equal to bacterial-derived plasmid DNA, albeit with a slight delay in replication kinetics. The RCA platform, however, is significantly more cost and time-efficient compared to traditional approaches. When the simplicity and costs of RCA are combined, we propose that a shift to an RCA platform will benefit the field of molecular virology and could have significant advantages for recombinant vaccine production.