bluuebunny/crossref_metadata_2025_split huggingface dataset:
Association of Family Functioning on Youth Physical Activity and Sedentary Behavior
Authors: Loprinzi Paul D. 
Published: August 2014

Objective:High family functioning is associated with reduced depression symptoms, better academic achievement, less disordered eating, and better metabolic control among youth. However, we know very little about the role of family functioning on physical activity and sedentary behavior among youth, which was the purpose of this study.Methods:Data from the 2003 and 2011–2012 cycles of the National Survey of Children’s Health were used. A total of 61,226 parents/guardians from the 2003 cycle and 40,446 parents/guardians from the 2011–2012 cycle (total n = 101,672) across all 50 states and the District of Columbia were examined. Parents answered questions related to family functioning and their child’s (age 6–17 yrs) physical activity and sedentary behavior.Results:Results were the same across both cycles; after adjustments, youth engaged in less physical activity if the family had worse family functioning (β = −0.06, P < .001). Similarly, youth engaged in more sedentary behavior if the family had worse family functioning (β = 0.05, P < .001).Conclusion:This research suggests that youth are more active and engage in less sedentary behavior if their family has greater family functioning.
Combinatorial Strategies for Improving Multiple-Stress Resistance in Industrially Relevant Escherichia coli Strains
Authors: Lennen Rebecca M. , Herrgård Markus J. 
Published: August 2014

ABSTRACT

High-cell-density fermentation for industrial production of chemicals can impose numerous stresses on cells due to high substrate, product, and by-product concentrations; high osmolarity; reactive oxygen species; and elevated temperatures. There is a need to develop platform strains of industrial microorganisms that are more tolerant toward these typical processing conditions. In this study, the growth of six industrially relevant strains of
Escherichia coli
was characterized under eight stress conditions representative of fed-batch fermentation, and strains W and BL21(DE3) were selected as platforms for transposon (Tn) mutagenesis due to favorable resistance characteristics. Selection experiments, followed by either targeted or genome-wide next-generation-sequencing-based Tn insertion site determination, were performed to identify mutants with improved growth properties under a subset of three stress conditions and two combinations of individual stresses. A subset of the identified loss-of-function mutants were selected for a combinatorial approach, where strains with combinations of two and three gene deletions were systematically constructed and tested for single and multistress resistance. These approaches allowed identification of (i) strain-background-specific stress resistance phenotypes, (ii) novel gene deletion mutants in
E. coli
that confer single and multistress resistance in a strain-background-dependent manner, and (iii) synergistic effects of multiple gene deletions that confer improved resistance over single deletions. The results of this study underscore the suboptimality and strain-specific variability of the genetic network regulating growth under stressful conditions and suggest that further exploration of the combinatorial gene deletion space in multiple strain backgrounds is needed for optimizing strains for microbial bioprocessing applications.
Golgi UDP-GlcNAc:Polypeptide
O
-α-
N
-Acetyl-
d
-Glucosaminyltransferase 2 (TcOGNT2) Regulates Trypomastigote Production and Function in Trypanosoma cruzi
Authors: Koeller Carolina M. , van der Wel Hanke , Feasley Christa L. , Abreu Fernanda , da Rocha Juliana Dutra Barbosa , Montalvão Fabrício , Fampa Patrícia , dos Reis Flávia C. G. , Atella Georgia C. , Souto-Padrón Thaís , West Christopher M. , Heise Norton 
Published: August 2014

ABSTRACT

All life cycle stages of the protozoan parasite
Trypanosoma cruzi
are enveloped by mucin-like glycoproteins which, despite major changes in their polypeptide cores, are extensively and similarly
O
-glycosylated.
O
-Glycan biosynthesis is initiated by the addition of αGlcNAc to Thr in a reaction catalyzed by Golgi UDP-GlcNAc:polypeptide
O
-α-
N
-acetyl-
d
-glucosaminyltransferases (ppαGlcNAcTs), which are encoded by
TcOGNT1
and
TcOGNT2
. We now directly show that TcOGNT2 is associated with the Golgi apparatus of the epimastigote stage and is markedly downregulated in both differentiated metacyclic trypomastigotes (MCTs) and cell culture-derived trypomastigotes (TCTs). The significance of downregulation was examined by forced continued expression of TcOGNT2, which resulted in a substantial increase of TcOGNT2 protein levels but only modestly increased ppαGlcNAcT activity in extracts and altered cell surface glycosylation in TCTs. Constitutive TcOGNT2 overexpression had no discernible effect on proliferating epimastigotes but negatively affected production of both types of trypomastigotes. MCTs differentiated from epimastigotes at a low frequency, though they were apparently normal based on morphological and biochemical criteria. However, these MCTs exhibited an impaired ability to produce amastigotes and TCTs in cell culture monolayers, most likely due to a reduced infection frequency. Remarkably, inhibition of MCT production did not depend on TcOGNT2 catalytic activity, whereas TCT production was inhibited only by active TcOGNT2. These findings indicate that TcOGNT2 downregulation is important for proper differentiation of MCTs and functioning of TCTs and that TcOGNT2 regulates these functions by using both catalytic and noncatalytic mechanisms.
Comparative Evaluation of Two Commercial Multiplex Panels for Detection of Gastrointestinal Pathogens by Use of Clinical Stool Specimens
Authors: Khare Reeti , Espy Mark J. , Cebelinski Elizabeth , Boxrud David , Sloan Lynne M. , Cunningham Scott A. , Pritt Bobbi S. , Patel Robin , Binnicker Matthew J. 
Published: August 2014

ABSTRACT

The detection of pathogens associated with gastrointestinal disease may be important in certain patient populations, such as immunocompromised hosts, the critically ill, or individuals with prolonged disease that is refractory to treatment. In this study, we evaluated two commercially available multiplex panels (the FilmArray gastrointestinal [GI] panel [BioFire Diagnostics, Salt Lake City, UT] and the Luminex xTag gastrointestinal pathogen panel [GPP] [Luminex Corporation, Toronto, Canada]) using Cary-Blair stool samples (
n
= 500) submitted to our laboratory for routine GI testing (e.g., culture, antigen testing, microscopy, and individual real-time PCR). At the time of this study, the prototype (non-FDA-cleared) FilmArray GI panel targeted 23 pathogens (14 bacterial, 5 viral, and 4 parasitic), and testing of 200 μl of Cary-Blair stool was recommended. In contrast, the Luminex GPP assay was FDA cleared for the detection of 11 pathogens (7 bacterial, 2 viral, and 2 parasitic), but had the capacity to identify 4 additional pathogens using a research-use-only protocol. Importantly, the Luminex assay was FDA cleared for 100 μl raw stool; however, 100 μl Cary-Blair stool was tested by the Luminex assay in this study. Among 230 prospectively collected samples, routine testing was positive for one or more GI pathogens in 19 (8.3%) samples, compared to 76 (33.0%) by the FilmArray and 69 (30.3%) by the Luminex assay.
Clostridium difficile
(12.6 to 13.9% prevalence) and norovirus genogroup I (GI)/GII (5.7 to 13.9% prevalence) were two of the pathogens most commonly detected by both assays among prospective samples. Sapovirus was also commonly detected (5.7% positive rate) by the FilmArray assay. Among 270 additional previously characterized samples, both multiplex panels demonstrated high sensitivity (>90%) for the majority of targets, with the exception of several pathogens, notably
Aeromonas
sp. (23.8%) by FilmArray and
Yersinia enterocolitica
(48.1%) by the Luminex assay. Interestingly, the FilmArray and Luminex panels identified mixed infections in 21.1% and 13.0% of positive prospective samples, respectively, compared to only 8.3% by routine methods.
The M33 G Protein-Coupled Receptor Encoded by Murine Cytomegalovirus Is Dispensable for Hematogenous Dissemination but Is Required for Growth within the Salivary Gland
Authors: Bittencourt Fabiola M. , Wu Shu-En , Bridges James P. , Miller William E. 
Published: August 2014

ABSTRACT

Human cytomegalovirus (HCMV) is a pathogen found worldwide and is a serious threat to immunocompromised individuals and developing fetuses. Due to the species specificity of cytomegaloviruses, murine cytomegalovirus (MCMV) has been used as a model for
in vivo
studies of HCMV pathogenesis. The MCMV genome, like the genomes of other beta- and gammaherpesviruses, encodes G protein-coupled receptors (GPCRs) that modulate host signaling pathways presumably to facilitate viral replication and dissemination. Among these viral receptors, the M33 GPCR carried by MCMV is an activator of CREB, NF-κB, and phospholipase C-β signaling pathways and has been implicated in aspects of pathogenesis
in vivo
, including persistence in the salivary glands of BALB/c mice. In this study, we used immunocompetent
n
on
o
bese
d
iabetic (NOD) and immunocompromised
N
OD-
s
cid-
g
amma (NSG) mice to further investigate the salivary gland defect exhibited by M33 deficiency. Interestingly, we demonstrate that virus with an M33 deletion (ΔM33) can replicate in the salivary gland of immunocompromised animals, albeit with a 400-fold growth defect compared with the growth of wild-type virus. Moreover, we determined that M33 does not have a role in cell-associated hematogenous dissemination but is required for viral amplification once the virus reaches the salivary gland. We conclude that the reduced replicative capacity of the ΔM33 virus is due to a specific defect occurring within the localized environment of the salivary gland. Importantly, since the salivary gland represents a site essential for persistence and horizontal transmission, an understanding of the mechanisms of viral replication within this site could lead to the generation of novel therapeutics useful for the prevention of HCMV spread.

IMPORTANCE
Human cytomegalovirus infects the majority of the American people and can reside silently in infected individuals for the duration of their lives. Under a number of circumstances, the virus can reactivate, leading to a variety of diseases in both adults and developing babies, and therefore, identifying the function of viral proteins is essential to understand how the virus spreads and causes disease. We aim to utilize animal models to study the function of an important class of viral proteins termed G protein-coupled receptors with the ultimate goal of developing inhibitors to these proteins that could one day be used to prevent viral spread.
Mutagenesis of the Catalytic and Cleavage Site Residues of the Hypovirus Papain-Like Proteases p29 and p48 Reveals Alternative Processing and Contributions to Optimal Viral RNA Accumulation
Authors: Jensen Kenneth S. , Nuss Donald L. 
Published: August 2014

ABSTRACT

The positive-stranded RNA genome of the prototypic virulence-attenuating hypovirus CHV-1/EP713 contains two open reading frames (ORF), each encoding an autocatalytic papain-like leader protease. Protease p29, derived from the N-terminal portion of ORF A, functions as a suppressor of RNA silencing, while protease p48, derived from the N-terminal portion of ORF B, is required for viral RNA replication. The catalytic and cleavage site residues required for autoproteolytic processing have been functionally mapped
in vitro
for both proteases but not confirmed in the infected fungal host. We report here the mutagenesis of the CHV-1/EP713 infectious cDNA clone to define the requirements for p29 and p48 cleavage and the role of autoproteolysis in the context of hypovirus replication. Mutation of the catalytic cysteine and histidine residues for either p29 or p48 was tolerated but reduced viral RNA accumulation to ca. 20 to 50% of the wild-type level. Mutation of the p29 catalytic residues caused an accumulation of unprocessed ORF A product p69. Surprisingly, the release of p48 from the ORF B-encoded polyprotein was not prevented by mutation of the p48 catalytic and cleavage site residues and was independent of p29. The results show that, while dispensable for hypovirus replication, the autocatalytic processing of the leader proteases p29 and p48 contributes to optimal virus RNA accumulation. The role of the predicted catalytic residues in autoproteolytic processing of p29 was confirmed in the infected host, while p48 was found to also undergo alternative processing independent of the encoded papain-like protease activities.

IMPORTANCE
Hypoviruses are positive-strand RNA mycoviruses that attenuate virulence of their pathogenic fungal hosts. The prototypic hypovirus CHV-1/EP713, which infects the chestnut bight fungus
Cryphonetria parasitica
, encodes two papain-like autocatalytic leader proteases, p29 and p48, that also have important functions in suppressing the RNA silencing antiviral defense response and in viral RNA replication, respectively. The mutational analyses of the CHV-1/EP713 infectious cDNA clone, reported here, define the requirements for p29 and p48 cleavage and the functional importance of autoproteolysis in the context of hypovirus replication and exposed an alternative p48 processing pathway independent of the encoded papain-like protease activities. These findings provide additional insights into hypovirus gene expression, replication, and evolution and inform ongoing efforts to engineer hypoviruses for interrogating and modulating fungal virulence.
Influenza A Virus Acquires Enhanced Pathogenicity and Transmissibility after Serial Passages in Swine
Authors: Wei Kai , Sun Honglei , Sun Zhenhong , Sun Yipeng , Kong Weili , Pu Juan , Ma Guangpeng , Yin Yanbo , Yang Hanchun , Guo Xin , Chang Kin-Chow , Liu Jinhua 
Published: August 2014

ABSTRACT

Genetic and phylogenetic analyses suggest that the pandemic H1N1/2009 virus was derived from well-established swine influenza lineages; however, there is no convincing evidence that the pandemic virus was generated from a direct precursor in pigs. Furthermore, the evolutionary dynamics of influenza virus in pigs have not been well documented. Here, we subjected a recombinant virus (rH1N1) with the same constellation makeup as the pandemic H1N1/2009 virus to nine serial passages in pigs. The severity of infection sequentially increased with each passage. Deep sequencing of viral quasispecies from the ninth passage found five consensus amino acid mutations: PB1 A469T, PA 1129T, NA N329D, NS1 N205K, and NEP T48N. Mutations in the hemagglutinin (HA) protein, however, differed greatly between the upper and lower respiratory tracts. Three representative viral clones with the five consensus mutations were selected for functional evaluation. Relative to the parental virus, the three viral clones showed enhanced replication and polymerase activity
in vitro
and enhanced replication, pathogenicity, and transmissibility in pigs, guinea pigs, and ferrets
in vivo
. Specifically, two mutants of rH1N1 (PB1 A469T and a combination of NS1 N205K and NEP T48N) were identified as determinants of transmissibility in guinea pigs. Crucially, one mutant viral clone with the five consensus mutations, which also carried D187E, K211E, and S289N mutations in its HA, additionally was able to infect ferrets by airborne transmission as effectively as the pandemic virus. Our findings demonstrate that influenza virus can acquire viral characteristics that are similar to those of the pandemic virus after limited serial passages in pigs.

IMPORTANCE
We demonstrate here that an engineered reassortant swine influenza virus, with the same gene constellation pattern as the pandemic H1N1/2009 virus and subjected to only nine serial passages in pigs, acquired greatly enhanced virulence and transmissibility. In particular, one representative pathogenic passaged virus clone, which carried three mutations in the HA gene and five consensus mutations in PB1, PA, NA, NS1, and NEP genes, additionally was able to confer respiratory droplet transmission as effectively as the pandemic H1N1/2009 virus. Our findings suggest that pigs can readily induce adaptive mutational changes to a precursor pandemic-like virus to transform it into a highly virulent and infectious form akin to that of the pandemic H1N1/2009 virus, which underlines the potential direct role of pigs in promoting influenza A virus pathogenicity and transmissibility.
A Chimeric Virus-Mouse Model System for Evaluating the Function and Inhibition of Papain-Like Proteases of Emerging Coronaviruses
Authors: Deng Xufang , Agnihothram Sudhakar , Mielech Anna M. , Nichols Daniel B. , Wilson Michael W. , StJohn Sarah E. , Larsen Scott D. , Mesecar Andrew D. , Lenschow Deborah J. , Baric Ralph S. , Baker Susan C. 
Published: August 2014

ABSTRACT

To combat emerging coronaviruses, developing safe and efficient platforms to evaluate viral protease activities and the efficacy of protease inhibitors is a high priority. Here, we exploit a biosafety level 2 (BSL-2) chimeric Sindbis virus system to evaluate protease activities and the efficacy of inhibitors directed against the papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus (SARS-CoV), a biosafety level 3 (BSL-3) pathogen. We engineered Sindbis virus to coexpress PLpro and a substrate, murine interferon-stimulated gene 15 (ISG15), and found that PLpro mediates removal of ISG15 (deISGylation) from cellular proteins. Mutation of the catalytic cysteine residue of PLpro or addition of a PLpro inhibitor blocked deISGylation in virus-infected cells. Thus, deISGylation is a marker of PLpro activity. Infection of alpha/beta interferon receptor knockout (IFNAR
−/−
) mice with these chimeric viruses revealed that PLpro deISGylation activity removed ISG15-mediated protection during viral infection. Importantly, administration of a PLpro inhibitor protected these mice from lethal infection, demonstrating the efficacy of a coronavirus protease inhibitor in a mouse model. However, this PLpro inhibitor was not sufficient to protect the mice from lethal infection with SARS-CoV MA15, suggesting that further optimization of the delivery and stability of PLpro inhibitors is needed. We extended the chimeric-virus platform to evaluate the papain-like protease/deISGylating activity of Middle East respiratory syndrome coronavirus (MERS-CoV) to provide a small-animal model to evaluate PLpro inhibitors of this recently emerged pathogen. This platform has the potential to be universally adaptable to other viral and cellular enzymes that have deISGylating activities.

IMPORTANCE
Evaluating viral protease inhibitors in a small-animal model is a critical step in the path toward antiviral drug development. We modified a biosafety level 2 chimeric virus system to facilitate evaluation of inhibitors directed against highly pathogenic coronaviruses. We used this system to demonstrate the
in vivo
efficacy of an inhibitor of the papain-like protease of severe acute respiratory syndrome coronavirus. Furthermore, we demonstrate that the chimeric-virus system can be adapted to study the proteases of emerging human pathogens, such as Middle East respiratory syndrome coronavirus. This system provides an important tool to rapidly assess the efficacy of protease inhibitors targeting existing and emerging human pathogens, as well as other enzymes capable of removing ISG15 from cellular proteins.
Effect of Formaldehyde Inactivation on Poliovirus
Authors: Wilton Thomas , Dunn Glynis , Eastwood David , Minor Philip D. , Martin Javier 
Published: August 2014

ABSTRACT

Inactivated polio vaccines, which have been used in many countries for more than 50 years, are produced by treating live poliovirus (PV) with formaldehyde. However, the molecular mechanisms underlying virus inactivation are not well understood. Infection by PV is initiated by virus binding to specific cell receptors, which results in viral particles undergoing sequential conformational changes that generate altered structural forms (135S and 80S particles) and leads to virus cell entry. We have analyzed the ability of inactivated PV to bind to the human poliovirus receptor (hPVR) using various techniques such as ultracentrifugation, fluorescence-activated cell sorting flow cytometry and real-time reverse transcription-PCR (RT-PCR). The results showed that although retaining the ability to bind to hPVR, inactivated PV bound less efficiently in comparison to live PV. We also found that inactivated PV showed resistance to structural conversion
in vitro
, as judged by measuring changes in antigenicity, the ability to bind to hPVR, and viral RNA release at high temperature. Furthermore, viral RNA from inactivated PV was shown to be modified, since cDNA yields obtained by RT-PCR amplification were severely reduced and no infectious virus was recovered after RNA transfection into susceptible cells.

IMPORTANCE
This study represents a novel insight into the molecular mechanisms responsible for poliovirus inactivation. We show that inactivation with formaldehyde has an effect on early steps of viral replication as it reduces the ability of PV to bind to hPVR, decreases the sensitivity of PV to convert to 135S particles, and abolishes the infectivity of its viral RNA. These changes are likely responsible for the loss of infectivity shown by PV following inactivation. Techniques used in this study represent new approaches for the characterization of inactivated PV products and could be useful in developing improved methods for the production and quality control testing of inactivated polio vaccines. Measuring the antigenicity, capsid stability, and RNA integrity of inactivated PV samples could help establishing the optimal balance between the loss of infectivity and the preservation of virus antigenicity during inactivation.
Coinfection with Streptococcus pneumoniae Modulates the B Cell Response to Influenza Virus
Authors: Wolf Amaya I. , Strauman Maura C. , Mozdzanowska Krystyna , Whittle James R. R. , Williams Katie L. , Sharpe Arlene H. , Weiser Jeffrey N. , Caton Andrew J. , Hensley Scott E. , Erikson Jan 
Published: August 2014

ABSTRACTPathogen-specific antibodies (Abs) protect against respiratory infection with influenza A virus (IAV) andStreptococcus pneumoniaeand are the basis of effective vaccines. Sequential or overlapping coinfections with both pathogens are common, yet the impact of coinfection on the generation and maintenance of Ab responses is largely unknown. We report here that the B cell response to IAV is altered in mice coinfected with IAV andS. pneumoniaeand that this response differs, depending on the order of pathogen exposure. In mice exposed toS. pneumoniaeprior to IAV, the initial virus-specific germinal center (GC) B cell response is significantly enhanced in the lung-draining mediastinal lymph node and spleen, and there is an increase in CD4+T follicular helper (TFH) cell numbers. In contrast, secondaryS. pneumoniaeinfection exaggerates early antiviral antibody-secreting cell formation, and at later times, levels of GCs, TFH cells, and antiviral serum IgG are elevated. Mice exposed toS. pneumoniaeprior to IAV do not maintain the initially robust GC response in secondary lymphoid organs and exhibit reduced antiviral serum IgG with diminished virus neutralization activity a month after infection. Our data suggest that the history of pathogen exposures can critically affect the generation of protective antiviral Abs and may partially explain the differential susceptibility to and disease outcomes from IAV infection in humans.IMPORTANCERespiratory tract coinfections, specifically those involving influenza A viruses andStreptococcus pneumoniae, remain a top global health burden. We sought to determine howS. pneumoniaecoinfection modulates the B cell immune response to influenza virus since antibodies are key mediators of protection.