bluuebunny/crossref_metadata_2025_split huggingface dataset:
c-Myc Represses Transcription of Epstein-Barr Virus Latent Membrane Protein 1 Early after Primary B Cell Infection
Authors: Price Alexander M. , Messinger Joshua E. , Luftig Micah A. 
Published: November 2017

ABSTRACT
Recent evidence has shown that the Epstein-Barr virus (EBV) oncogene LMP1 is not expressed at high levels early after EBV infection of primary B cells, despite its being essential for the long-term outgrowth of immortalized lymphoblastoid cell lines (LCLs). In this study, we found that expression of LMP1 increased 50-fold between 7 days postinfection and the LCL state. Metabolic labeling of nascent transcribed mRNA indicated that this was primarily a transcription-mediated event. EBNA2, the key viral transcription factor regulating LMP1, and CTCF, an important chromatin insulator, were recruited to the LMP1 locus similarly early and late after infection. However, the activating histone H3K9Ac mark was enriched at the LMP1 promoter in LCLs relative to that in infected B cells early after infection. We found that high c-Myc activity in EBV-infected lymphoma cells as well as overexpression of c-Myc in an LCL model system repressed LMP1 transcription. Finally, we found that chemical inhibition of c-Myc both in LCLs and early after primary B cell infection increased LMP1 expression. These data support a model in which high levels of endogenous c-Myc activity induced early after primary B cell infection directly repress LMP1 transcription.

IMPORTANCE
EBV is a highly successful pathogen that latently infects more than 90% of adults worldwide and is also causally associated with a number of B cell malignancies. During the latent life cycle, EBV expresses a set of viral oncoproteins and noncoding RNAs with the potential to promote cancer. Critical among these is the viral latent membrane protein LMP1. Prior work suggests that LMP1 is essential for EBV to immortalize B cells, but our recent work indicates that LMP1 is not produced at high levels during the first few weeks after infection. Here we show that transcription of the LMP1 gene can be negatively regulated by a host transcription factor, c-Myc. Ultimately, understanding the regulation of EBV oncogenes will allow us to better treat cancers that rely on these viral products for survival.
Peripheral Blood Biomarkers of Disease Outcome in a Monkey Model of Rift Valley Fever Encephalitis
Authors: Wonderlich Elizabeth R. , Caroline Amy L. , McMillen Cynthia M. , Walters Aaron W. , Reed Douglas S. , Barratt-Boyes Simon M. , Hartman Amy L. 
Published: November 2017

ABSTRACT

Rift Valley Fever (RVF) is an emerging arboviral disease of livestock and humans. Although the disease is caused by a mosquito-borne virus, humans are infected through contact with, or inhalation of, virus-laden particles from contaminated animal carcasses. Some individuals infected with RVF virus (RVFV) develop meningoencephalitis, resulting in morbidity and mortality. Little is known about the pathogenic mechanisms that lead to neurologic sequelae, and thus, animal models that represent human disease are needed. African green monkeys (AGM) exposed to aerosols containing RVFV develop a reproducibly lethal neurological disease that resembles human illness. To understand the disease process and identify biomarkers of lethality, two groups of 5 AGM were infected by inhalation with either a lethal or a sublethal dose of RVFV. Divergence between lethal and sublethal infections occurred as early as 2 days postinfection (dpi), at which point CD8
+
T cells from lethally infected AGM expressed activated caspase-3 and simultaneously failed to increase levels of major histocompatibility complex (MHC) class II molecules, in contrast to surviving animals. At 4 dpi, lethally infected animals failed to demonstrate proliferation of total CD4
+
and CD8
+
T cells, in contrast to survivors. These marked changes in peripheral blood cells occur much earlier than more-established indicators of severe RVF disease, such as granulocytosis and fever. In addition, an early proinflammatory (gamma interferon [IFN-γ], interleukin 6 [IL-6], IL-8, monocyte chemoattractant protein 1 [MCP-1]) and antiviral (IFN-α) response was seen in survivors, while very late cytokine expression was found in animals with lethal infections. By characterizing immunological markers of lethal disease, this study furthers our understanding of RVF pathogenesis and will allow the testing of therapeutics and vaccines in the AGM model.

IMPORTANCE
Rift Valley Fever (RVF) is an important emerging viral disease for which we lack both an effective human vaccine and treatment. Encephalitis and neurological disease resulting from RVF lead to death or significant long-term disability for infected people. African green monkeys (AGM) develop lethal neurological disease when infected with RVF virus by inhalation. Here we report the similarities in disease course between infected AGM and humans. For the first time, we examine the peripheral immune response during the course of infection in AGM and show that there are very early differences in the immune response between animals that survive infection and those that succumb. We conclude that AGM are a novel and suitable monkey model for studying the neuropathogenesis of RVF and for testing vaccines and therapeutics against this emerging viral pathogen.
HupB Is a Bacterial Nucleoid-Associated Protein with an Indispensable Eukaryotic-Like Tail
Authors: Hołówka Joanna , Trojanowski Damian , Ginda Katarzyna , Wojtaś Bartosz , Gielniewski Bartłomiej , Jakimowicz Dagmara , Zakrzewska-Czerwińska Jolanta 
Published: November 2017

ABSTRACT

In bacteria, chromosomal DNA must be efficiently compacted to fit inside the small cell compartment while remaining available for the proteins involved in replication, segregation, and transcription. Among the nucleoid-associated proteins (NAPs) responsible for maintaining this highly organized and yet dynamic chromosome structure, the HU protein is one of the most conserved and highly abundant. HupB, a homologue of HU, was recently identified in mycobacteria. This intriguing mycobacterial NAP is composed of two domains: an N-terminal domain that resembles bacterial HU, and a long and distinctive C-terminal domain that contains several PAKK/KAAK motifs, which are characteristic of the H1/H5 family of eukaryotic histones. In this study, we analyzed the
in vivo
binding of HupB on the chromosome scale. By using PALM (photoactivated localization microscopy) and ChIP-Seq (chromatin immunoprecipitation followed by deep sequencing), we observed that the C-terminal domain is indispensable for the association of HupB with the nucleoid. Strikingly, the
in vivo
binding of HupB displayed a bias from the origin (
oriC
) to the terminus (
ter
) of the mycobacterial chromosome (numbers of binding sites decreased toward
ter
). We hypothesized that this binding mode reflects a role for HupB in organizing newly replicated
oriC
regions. Thus, HupB may be involved in coordinating replication with chromosome segregation.

IMPORTANCE
We currently know little about the organization of the mycobacterial chromosome and its dynamics during the cell cycle. Among the mycobacterial nucleoid-associated proteins (NAPs) responsible for chromosome organization and dynamics, HupB is one of the most intriguing. It contains a long and distinctive C-terminal domain that harbors several PAKK/KAAK motifs, which are characteristic of the eukaryotic histone H1/H5 proteins. The HupB protein is also known to be crucial for the survival of tubercle bacilli during infection. Here, we provide
in vivo
experimental evidence showing that the C-terminal domain of HupB is crucial for its DNA binding. Our results suggest that HupB may be involved in organizing newly replicated regions and could help coordinate chromosome replication with segregation. Given that tuberculosis (TB) remains a serious worldwide health problem (10.4 million new TB cases were diagnosed in 2015, according to WHO) and new multidrug-resistant
Mycobacterium tuberculosis
strains are continually emerging, further studies of the biological function of HupB are needed to determine if this protein could be a prospect for novel antimicrobial drug development.
The NOX Family of Proteins Is Also Present in Bacteria
Authors: Hajjar Christine , Cherrier Mickaël V. , Dias Mirandela Gaëtan , Petit-Hartlein Isabelle , Stasia Marie José , Fontecilla-Camps Juan C. , Fieschi Franck , Dupuy Jérôme 
Published: November 2017

ABSTRACT

Transmembrane NADPH oxidase (NOX) enzymes have been so far only characterized in eukaryotes. In most of these organisms, they reduce molecular oxygen to superoxide and, depending on the presence of additional domains, are called NOX or dual oxidases (DUOX). Reactive oxygen species (ROS), including superoxide, have been traditionally considered accidental toxic by-products of aerobic metabolism. However, during the last decade it has become evident that both O
2
•−
and H
2
O
2
are key players in complex signaling networks and defense. A well-studied example is the production of O
2
•−
during the bactericidal respiratory burst of phagocytes; this production is catalyzed by NOX2. Here, we devised and applied a novel algorithm to search for additional
NOX
genes in genomic databases. This procedure allowed us to discover approximately 23% new sequences from bacteria (in relation to the number of NOX-related sequences identified by the authors) that we have added to the existing eukaryotic NOX family and have used to build an expanded phylogenetic tree. We cloned and overexpressed the identified
nox
gene from
Streptococcus pneumoniae
and confirmed that it codes for an NADPH oxidase. The membrane of the
S. pneumoniae
NOX protein (SpNOX) shares many properties with its eukaryotic counterparts, such as affinity for NADPH and flavin adenine dinucleotide, superoxide dismutase and diphenylene iodonium inhibition, cyanide resistance, oxygen consumption, and superoxide production. Traditionally, NOX enzymes in eukaryotes are related to functions linked to multicellularity. Thus, the discovery of a large family of NOX-related enzymes in the bacterial world brings up fascinating questions regarding their role in this new biological context.

IMPORTANCE
NADPH oxidase (NOX) enzymes have not yet been reported in bacteria. Here, we carried out computational and experimental studies to provide the first characterization of a prokaryotic NOX. Out of 996 prokaryotic proteins showing NOX signatures, we initially selected, cloned, and overexpressed four of them. Subsequently, and based on preliminary testing, we concentrated our efforts on
Streptococcus
SpNOX, which shares many biochemical characteristics with NOX2, the referent model of NOX enzymes. Our work makes possible, for the first time, the study of pure forms of this important family of enzymes, allowing for biophysical and molecular characterization in an unprecedented way. Similar advances regarding other membrane protein families have led to new structures, further mechanistic studies, and the improvement of inhibitors. In addition, biological functions of these newly described bacterial enzymes will be certainly discovered in the near future.
Proofreading-Deficient Coronaviruses Adapt for Increased Fitness over Long-Term Passage without Reversion of Exoribonuclease-Inactivating Mutations
Authors: Graepel Kevin W. , Lu Xiaotao , Case James Brett , Sexton Nicole R. , Smith Everett Clinton , Denison Mark R. 
Published: November 2017

ABSTRACT
The coronavirus (CoV) RNA genome is the largest among the single-stranded positive-sense RNA viruses. CoVs encode a proofreading 3′-to-5′ exoribonuclease within nonstructural protein 14 (nsp14-ExoN) that is responsible for CoV high-fidelity replication. Alanine substitution of ExoN catalytic residues [ExoN(-)] in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and murine hepatitis virus (MHV) disrupts ExoN activity, yielding viable mutant viruses with defective replication, up to 20-fold-decreased fidelity, and increased susceptibility to nucleoside analogues. To test the stability of the ExoN(-) genotype and phenotype, we passaged MHV-ExoN(-) 250 times in cultured cells (P250), in parallel with wild-type MHV (WT-MHV). Compared to MHV-ExoN(-) P3, MHV-ExoN(-) P250 demonstrated enhanced replication and increased competitive fitness without reversion at the ExoN(-) active site. Furthermore, MHV-ExoN(-) P250 was less susceptible than MHV-ExoN(-) P3 to multiple nucleoside analogues, suggesting that MHV-ExoN(-) was under selection for increased replication fidelity. We subsequently identified novel amino acid changes within the RNA-dependent RNA polymerase and nsp14 of MHV-ExoN(-) P250 that partially accounted for the reduced susceptibility to nucleoside analogues. Our results suggest that increased replication fidelity is selected in ExoN(-) CoVs and that there may be a significant barrier to ExoN(-) reversion. These results also support the hypothesis that high-fidelity replication is linked to CoV fitness and indicate that multiple replicase proteins could compensate for ExoN functions during replication.

IMPORTANCE
Uniquely among RNA viruses, CoVs encode a proofreading exoribonuclease (ExoN) in nsp14 that mediates high-fidelity RNA genome replication. Proofreading-deficient CoVs with disrupted ExoN activity [ExoN(-)] either are nonviable or have significant defects in replication, RNA synthesis, fidelity, fitness, and virulence. In this study, we showed that ExoN(-) murine hepatitis virus can adapt during long-term passage for increased replication and fitness without reverting the ExoN-inactivating mutations. Passage-adapted ExoN(-) mutants also demonstrate increasing resistance to nucleoside analogues that is explained only partially by secondary mutations in nsp12 and nsp14. These data suggest that enhanced resistance to nucleoside analogues is mediated by the interplay of multiple replicase proteins and support the proposed link between CoV fidelity and fitness.
Dynamic Proteomics of Herpes Simplex Virus Infection
Authors: Drayman Nir , Karin Omer , Mayo Avi , Danon Tamar , Shapira Lev , Rafael Dor , Zimmer Anat , Bren Anat , Kobiler Oren , Alon Uri 
Published: November 2017

ABSTRACT
The cellular response to viral infection is usually studied at the level of cell populations. Currently, it remains an open question whether and to what extent cell-to-cell variability impacts the course of infection. Here we address this by dynamic proteomics—imaging and tracking 400 yellow fluorescent protein (YFP)-tagged host proteins in individual cells infected by herpes simplex virus 1. By quantifying time-lapse fluorescence imaging, we analyze how cell-to-cell variability impacts gene expression from the viral genome. We identify two proteins, RFX7 and geminin, whose levels at the time of infection correlate with successful initiation of gene expression. These proteins are cell cycle markers, and we find that the position in the cell cycle at the time of infection (along with the cell motility and local cell density) can reasonably predict in which individual cells gene expression from the viral genome will commence. We find that the onset of cell division dramatically impacts the progress of infection, with 70% of dividing cells showing no additional gene expression after mitosis. Last, we identify four host proteins that are specifically modulated in infected cells, of which only one has been previously recognized. SUMO2 and RPAP3 levels are rapidly reduced, while SLTM and YTHDC1 are redistributed to form nuclear foci. These modulations are dependent on the expression of ICP0, as shown by infection with two mutant viruses that lack ICP0. Taken together, our results provide experimental validation for the long-held notion that the success of infection is dependent on the state of the host cell at the time of infection.

IMPORTANCE
High-throughput assays have revolutionized many fields in biology, both by allowing a more global understanding of biological processes and by deciphering rare events in subpopulations. Here we use such an assay, dynamic proteomics, to study viral infection at the single-cell level. We follow tens of thousands of individual cells infected by herpes simplex virus using fluorescence live imaging. Our results link the state of a cell at the time of virus infection with its probability to successfully initiate gene expression from the viral genome. Further, we identified three cellular proteins that were previously unknown to respond to viral infection. We conclude that dynamic proteomics provides a powerful tool to study single-cell differences during viral infection.
Near-Cognate Codons Contribute Complexity to Translation Regulation
Authors: Glass N. Louise 
Published: November 2017

ABSTRACT

The interplay between translation initiation, modification of translation initiation factors, and selection of start sites on mRNA for protein synthesis can play a regulatory role in the cellular response to stress, development, and cell fate in eukaryotic species by shaping the proteome. As shown by Ivanov et al. (mBio 8:e00844-17, 2017,
https://doi.org/10.1128/mBio.00844-17
), in the filamentous fungus
Neurospora crassa
, both upstream open reading frames (uORFs) and near-cognate start codons negatively or positively regulate the translation of the transcription factor CPC1 and production of CPC1 isoforms, which mediate the cellular response to amino acid starvation. Dissecting the physiological roles that differentiate cellular choice of translation initiation is an important parameter to understanding mechanisms that determine cell fate via gene regulation and protein synthesis.
Diagnostic accuracy of the D-dimer in children
Authors: Kanis Jessica , Hall Cassandra L , Pike Jonathan , Kline Jeffrey A 
Published: November 2017

BackgroundWe sought to measure the diagnostic accuracy of D-dimer in children with suspected pulmonary embolism (PE).MethodsWe queried our electronic medical record for quantitative D-dimer values obtained in all children ages 5–17 over 10 years in our 10-hospital system. Patients who had a D-dimer obtained in the evaluation of PE underwent supervised chart review to extract baseline demographics (age, sex, ethnicity), medical history, laboratory data and imaging results. PE was confirmed by imaging positive for deep vein thrombosis (DVT) or PE and excluded by imaging or no DVT or PE diagnosis within 90 days.ResultsOver a 10-year period, we identified 13 792 orders for D-dimer testing in 2554 unique patients. Chart review indicated that 526 (20.6%) unique patients had D-dimer testing performed in the evaluation of PE (Cohen’s kappa=0.95, 95% CI 0.85 to 1.0). Most D-dimers (465/526, 88%) were ordered in children aged >12 years. Of these 526 children, 34 (6.4%, 95% CI) had a criterion standard positive for new or recurrent PE. The mean D-dimer value was 2104±1394 ng/mL in the 34/34 PE+ children and 586±962 ng/mL in PE− children with a sensitivity of 34/34 (100%, 89% to 100%) and a specificity of 290/492 (58%, 54% to 63%). The area under the receiver operating characteristic curve was 0.90 ((0.9)87–0.94).ConclusionsD-dimer is currently ordered in children for suspected PE in the emergency care setting, mostly in teenagers. The observed lower limit 95% CIs of 89% and 54% for diagnostic sensitivity and the specificity, respectively, suggest if used in patients with low-clinical probability, a normal D-dimer can safely exclude PE in children.
Longitudinal deformation bench testing using a coronary artery model: a new standard?
Authors: Choudhury Tawfiq R , Al-Saigh Salwan , Burley Steve , Li Lin , Shakhshir Nizar , Mirhosseini Nazanin , Wang Tao , Arnous Samer , Khan Muhammad A , Mamas Mamas A , Fraser Douglas G W 
Published: November 2017

ObjectivesTo compare susceptibility of five different stent platforms with longitudinal stent deformation (LSD) using a clinically relevant bench testing model simulating both short and long malapposed lengths.BackgroundRecent data suggest that design modifications to the Promus Element stent which led to the Promus Premier stent has reduced susceptibility to LSD. However, susceptibility to LSD at long malapposed lengths has not been tested. Furthermore, the mechanisms behind susceptibility to LSD are as yet unclear.MethodsThe Omega, Integrity, Multilink 8, Biomatrixand Promus Premier stent platforms were tested. The Omega, Integrity and Multilink 8 platforms were used in place of their drug-eluting equivalents. 3.5 mm stents were deployed in a stepped tube with the distal portion fixed and the proximal test section exposed. The force required to compress stents by a fixed distance at different exposed lengths was compared. Symmetrical and point loading were used.ResultsThe Promus Premier was longitudinally as strong as Multilink and Integrity at a short exposed length (4 mm) but weaker, in between Omega and the other platforms, at longer exposed lengths (12 mm). As previously noted, the Omega (Promus Element) platform was significantly weaker than the other stents and Biomatrix was the strongest stent.ConclusionSusceptibility to LSD varies depending on length of malapposed segment when tested using a clinically relevant model as in this study. The mechanisms behind the susceptibility are likely multifactorial, including connector number, strut thickness, connector alignment and ring orientation but remain to be elucidated.
Endocannabinoid-mediated potentiation of nonnociceptive synapses contributes to behavioral sensitization
Authors: Wang Yanqing , Burrell Brian D. 
Published: November 2017

Endocannabinoids, such as 2-arachidonoyl glycerol (2-AG) and anandamide, can elicit long-term depression of both excitatory and inhibitory synapses. This latter effect will result in disinhibition and would therefore be expected to produce an increase in neural circuit output. However, there have been no examples directly linking endocannabinoid-mediated disinhibition to a change in a functional neurobehavioral circuit. The present study uses the well-characterized central nervous system of the medicinal leech, Hirudo verbana, to examine the functional/behavioral relevance of endocannabinoid modulation of an identified afferent synapse. Bath application of 2-AG potentiates synaptic transmission by pressure-sensitive sensory neurons (P cells) as well as the magnitude of the defensive shortening reflex elicited by P-cell stimulation. This potentiation requires activation of TRPV-like channels. Endocannabinoid/TRPV signaling was found to produce sensitization of the shortening reflex elicited by either direct stimulation of nearby nociceptive afferents (N cells) or noxious stimulation applied to skin several segments away. In both cases, heterosynaptic potentiation of P-cell synapses was observed in parallel with an increase in the magnitude of elicited shortening and both synaptic and behavioral effects were blocked by pharmacological inhibition of 2-AG synthesis or TRPV-like channel activation. Serotonin (5-HT) is known to play a critical role in sensitization in Hirudo and other animals, and the 5-HT2receptor antagonist ritanserin also blocked behavioral sensitization and the accompanying synaptic potentiation. These findings suggest a novel, endocannabinoid-mediated contribution to behavioral sensitization that may interact with known 5-HT-dependent modulatory processes.NEW & NOTEWORTHY There is considerable interest in the analgesic potential of cannabinoids. However, there is evidence that the cannabinoid system can have both pro- and antinociceptive effects. This study examines how an endogenous cannabinoid transmitter can potentiate nonnociceptive synapses and enhance their capacity to elicit a nocifensive behavioral response.