bluuebunny/crossref_metadata_2025_split huggingface dataset:
Whole-Genome Sequencing for High-Resolution Investigation of Methicillin-Resistant Staphylococcus aureus Epidemiology and Genome Plasticity
Authors: SenGupta Dhruba J. , Cummings Lisa A. , Hoogestraat Daniel R. , Butler-Wu Susan M. , Shendure Jay , Cookson Brad T. , Salipante Stephen J. 
Published: May 2014

ABSTRACT

Methicillin-resistant
Staphylococcus aureus
(MRSA) infections pose a major challenge in health care, yet the limited heterogeneity within this group hinders molecular investigations of related outbreaks. Pulsed-field gel electrophoresis (PFGE) has been the gold standard approach but is impractical for many clinical laboratories and is often replaced with PCR-based methods. Regardless, both approaches can prove problematic for identifying subclonal outbreaks. Here, we explore the use of whole-genome sequencing for clinical laboratory investigations of MRSA molecular epidemiology. We examine the relationships of 44 MRSA isolates collected over a period of 3 years by using whole-genome sequencing and two PCR-based methods, multilocus variable-number tandem-repeat analysis (MLVA) and
spa
typing. We find that MLVA offers higher resolution than
spa
typing, as it resolved 17 versus 12 discrete isolate groups, respectively. In contrast, whole-genome sequencing reproducibly cataloged genomic variants (131,424 different single nucleotide polymorphisms and indels across the strain collection) that uniquely identified each MRSA clone, recapitulating those groups but enabling higher-resolution phylogenetic inferences of the epidemiological relationships. Importantly, whole-genome sequencing detected a significant number of variants, thereby distinguishing between groups that were considered identical by both
spa
typing (minimum, 1,124 polymorphisms) and MLVA (minimum, 193 polymorphisms); this suggests that these more conventional approaches can lead to false-positive identification of outbreaks due to inappropriate grouping of genetically distinct strains. An analysis of the distribution of variants across the MRSA genome reveals 47 mutational hot spots (comprising ∼2.5% of the genome) that account for 23.5% of the observed polymorphisms, and the use of this selected data set successfully recapitulates most epidemiological relationships in this pathogen group.
Genome-Wide Small Interfering RNA Screens Reveal VAMP3 as a Novel Host Factor Required for Uukuniemi Virus Late Penetration
Authors: Meier Roger , Franceschini Andrea , Horvath Peter , Tetard Marilou , Mancini Roberta , von Mering Christian , Helenius Ari , Lozach Pierre-Yves 
Published: May 2014

ABSTRACT

The
Bunyaviridae
constitute a large family of enveloped animal viruses, many of which are important emerging pathogens. How bunyaviruses enter and infect mammalian cells remains largely uncharacterized. We used two genome-wide silencing screens with distinct small interfering RNA (siRNA) libraries to investigate host proteins required during infection of human cells by the bunyavirus Uukuniemi virus (UUKV), a late-penetrating virus. Sequence analysis of the libraries revealed that many siRNAs in the screens inhibited infection by silencing not only the intended targets but additional genes in a microRNA (miRNA)-like manner. That the 7-nucleotide seed regions in the siRNAs can cause a perturbation in infection was confirmed by using synthetic miRNAs (miRs). One of the miRs tested, miR-142-3p, was shown to interfere with the intracellular trafficking of incoming viruses by regulating the v-SNARE VAMP3, a strong hit shared by both siRNA screens. Inactivation of VAMP3 by the tetanus toxin led to a block in infection. Using fluorescence-based techniques in fixed and live cells, we found that the viruses enter VAMP3
+
endosomal vesicles 5 min after internalization and that colocalization was maximal 15 min thereafter. At this time, LAMP1 was associated with the VAMP3
+
virus-containing endosomes. In cells depleted of VAMP3, viruses were mainly trapped in LAMP1-negative compartments. Together, our results indicated that UUKV relies on VAMP3 for penetration, providing an indication of added complexity in the trafficking of viruses through the endocytic network.

IMPORTANCE
Bunyaviruses represent a growing threat to humans and livestock globally. Unfortunately, relatively little is known about these emerging pathogens. We report here the first human genome-wide siRNA screens for a bunyavirus. The screens resulted in the identification of 562 host cell factors with a potential role in cell entry and virus replication. To demonstrate the robustness of our approach, we confirmed and analyzed the role of the v-SNARE VAMP3 in Uukuniemi virus entry and infection. The information gained lays the basis for future research into the cell biology of bunyavirus infection and new antiviral strategies. In addition, by shedding light on serious caveats in large-scale siRNA screening, our experimental and bioinformatics procedures will be valuable in the comprehensive analysis of past and future high-content screening data.
Productive Replication of Human Papillomavirus 31 Requires DNA Repair Factor Nbs1
Authors: Anacker Daniel C. , Gautam Dipendra , Gillespie Kenric A. , Chappell William H. , Moody Cary A. 
Published: May 2014

ABSTRACT
Activation of the ATM (ataxia telangiectasia-mutated kinase)-dependent DNA damage response (DDR) is necessary for productive replication of human papillomavirus 31 (HPV31). We previously found that DNA repair and homologous recombination (HR) factors localize to sites of HPV replication, suggesting that ATM activity is required to recruit factors to viral genomes that can productively replicate viral DNA in a recombination-dependent manner. The Mre11-Rad50-Nbs1 (MRN) complex is an essential component of the DDR that is necessary for ATM-mediated HR repair and localizes to HPV DNA foci. In this study, we demonstrate that the HPV E7 protein is sufficient to increase levels of the MRN complex and also interacts with MRN components. We have found that Nbs1 depletion blocks productive viral replication and results in decreased localization of Mre11, Rad50, and the principal HR factor Rad51 to HPV DNA foci upon differentiation. Nbs1 contributes to the DDR by acting as an upstream activator of ATM in response to double-strand DNA breaks (DSBs) and as a downstream effector of ATM activity in the intra-S-phase checkpoint. We have found that phosphorylation of ATM and its downstream target Chk2, as well as SMC1 (structural maintenance of chromosome 1), is maintained upon Nbs1 knockdown in differentiating cells. Given that ATM and Chk2 are required for productive replication, our results suggest that Nbs1 contributes to viral replication outside its role as an ATM activator, potentially through ensuring localization of DNA repair factors to viral genomes that are necessary for efficient productive replication.

IMPORTANCE
The mechanisms that regulate human papillomavirus (HPV) replication during the viral life cycle are not well understood. Our finding that Nbs1 is necessary for productive replication even in the presence of ATM (ataxia telangiectasia-mutated kinase) and Chk2 phosphorylation offers evidence that Nbs1 contributes to viral replication downstream of facilitating ATM activation. Nbs1 is required for the recruitment of Mre11 and Rad50 to viral genomes, suggesting that the MRN complex plays a direct role in facilitating productive viral replication, potentially through the processing of substrates that are recognized by the key homologous recombination (HR) factor Rad51. The discovery that E7 increases levels of MRN components, and MRN complex formation, identifies a novel role for E7 in facilitating productive replication. Our study not only identifies DNA repair factors necessary for HPV replication but also provides a deeper understanding of how HPV utilizes the DNA damage response to regulate viral replication.
MDA5 and LGP2: Accomplices and Antagonists of Antiviral Signal Transduction
Authors: Rodriguez Kenny R. , Bruns Annie M. , Horvath Curt M. 
Published: May 2014

ABSTRACT
Mammalian cells have the ability to recognize virus infection and mount a powerful antiviral transcriptional response that provides an initial barrier to replication and impacts both innate and adaptive immune responses. Retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) proteins mediate intracellular virus recognition and are activated by viral RNA ligands to induce antiviral signal transduction. While the mechanisms of RIG-I regulation are already well understood, less is known about the more enigmatic melanoma differentiation-associated 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2). Emerging evidence suggests that these two RLRs are intimately associated as both accomplices and antagonists of antiviral signal transduction.
Evaluation of the Contributions of Individual Viral Genes to Newcastle Disease Virus Virulence and Pathogenesis
Authors: Paldurai Anandan , Kim Shin-Hee , Nayak Baibaswata , Xiao Sa , Shive Heather , Collins Peter L. , Samal Siba K. 
Published: May 2014

ABSTRACT

Naturally occurring Newcastle disease virus (NDV) strains vary greatly in virulence. The presence of multibasic residues at the proteolytic cleavage site of the fusion (F) protein has been shown to be a primary determinant differentiating virulent versus avirulent strains. However, there is wide variation in virulence among virulent strains. There also are examples of incongruity between cleavage site sequence and virulence. These observations suggest that additional viral factors contribute to virulence. In this study, we evaluated the contribution of each viral gene to virulence individually and in different combinations by exchanging genes between velogenic (highly virulent) strain GB Texas (GBT) and mesogenic (moderately virulent) strain Beaudette C (BC). These two strains are phylogenetically closely related, and their F proteins contain identical cleavage site sequences,
112
RRQKR↓F
117
. A total of 20 chimeric viruses were constructed and evaluated
in vitro
, in 1-day-old chicks, and in 2-week-old chickens. The results showed that both the envelope-associated and polymerase-associated proteins contribute to the difference in virulence between rBC and rGBT, with the envelope-associated proteins playing the greater role. The F protein was the major individual contributor and was sometimes augmented by the homologous M and HN proteins. The dramatic effect of F was independent of its cleavage site sequence since that was identical in the two strains. The polymerase L protein was the next major individual contributor and was sometimes augmented by the homologous N and P proteins. The leader and trailer regions did not appear to contribute to the difference in virulence between BC and GBT.

IMPORTANCE
This study is the first comprehensive and systematic study of NDV virulence and pathogenesis. Genetic exchanges between a mesogenic and a velogenic strain revealed that the fusion glycoprotein is the major virulence determinant regardless of the identical virulence protease cleavage site sequence present in both strains. The contribution of the large polymerase protein to NDV virulence is second only to that of the fusion glycoprotein. The identification of virulence determinants is of considerable importance, because of the potential to generate better live attenuated NDV vaccines. It may also be possible to apply these findings to other paramyxoviruses.
Structural Insight into BH3 Domain Binding of Vaccinia Virus Antiapoptotic F1L
Authors: Campbell Stephanie , Thibault John , Mehta Ninad , Colman Peter M. , Barry Michele , Kvansakul Marc 
Published: May 2014

ABSTRACT
Apoptosis is a tightly regulated process that plays a crucial role in the removal of virus-infected cells, a process controlled by both pro- and antiapoptotic members of the Bcl-2 family. The proapoptotic proteins Bak and Bax are regulated by antiapoptotic Bcl-2 proteins and are also activated by a subset of proteins known as BH3-only proteins that perform dual functions by directly activating Bak and Bax or by sequestering and neutralizing antiapoptotic family members. Numerous viruses express proteins that prevent premature host cell apoptosis. Vaccinia virus encodes F1L, an antiapoptotic protein essential for survival of infected cells that bears no discernible sequence homology to mammalian cell death inhibitors. Despite the limited sequence similarities, F1L has been shown to adopt a novel dimeric Bcl-2-like fold that enables hetero-oligomeric binding to both Bak and the proapoptotic BH3-only protein Bim that ultimately prevents Bak and Bax homo-oligomerization. However, no structural data on the mode of engagement of F1L and its Bcl-2 counterparts are available. Here we solved the crystal structures of F1L in complex with two ligands, Bim and Bak. Our structures indicate that F1L can engage two BH3 ligands simultaneously via the canonical Bcl-2 ligand binding grooves. Furthermore, by structure-guided mutagenesis, we generated point mutations within the binding pocket of F1L in order to elucidate the residues responsible for both Bim and Bak binding and prevention of apoptosis. We propose that the sequestration of Bim by F1L is primarily responsible for preventing apoptosis during vaccinia virus infection.

IMPORTANCE
Numerous viruses have adapted strategies to counteract apoptosis by encoding proteins responsible for sequestering proapoptotic components. Vaccinia virus, the prototypical member of the family
Orthopoxviridae
, encodes a protein known as F1L that functions to prevent apoptosis by interacting with Bak and the BH3-only protein Bim. Despite recent structural advances, little is known regarding the mechanics of binding between F1L and the proapoptotic Bcl-2 family members. Utilizing three-dimensional structures of F1L bound to host proapoptotic proteins, we generated variants of F1L that neutralize Bim and/or Bak. We demonstrate that during vaccinia virus infection, engagement of Bim and Bak by F1L is crucial for subversion of host cell apoptosis.
Enhancement of Interferon Induction by ORF3 Product of Hepatitis E Virus
Authors: Nan Yuchen , Ma Zexu , Wang Rong , Yu Ying , Kannan Harilakshmi , Fredericksen Brenda , Zhang Yan-Jin 
Published: May 2014

ABSTRACT
Hepatitis E virus (HEV) causes both the endemic and epidemic spread of acute hepatitis in many parts of the world. HEV open reading frame 3 (ORF3) encodes a 13-kDa multifunctional protein (vp13) that is essential for HEV infection of animals. The exact role of vp13 in HEV infection remains unclear. In this study, vp13 was found to enhance interferon (IFN) production induced by poly(I · C), a synthetic analog of double-stranded RNA. Poly(I · C) treatment induced a higher level of IFN-β mRNA in HeLa cells stably expressing vp13 than in control cells. Using a luciferase reporter construct driven by the IFN-β promoter, we demonstrated that vp13 enhanced retinoic acid-inducible gene I (RIG-I)-dependent luciferase expression. This enhancement was found to be due to both an increased level of RIG-I protein and its activation. The levels of both endogenous and exogenous RIG-I were increased by vp13 by extension of the half-life of RIG-I. Additionally, vp13 interacts with the RIG-I N-terminal domain and enhances its K63-linked ubiquitination, which is essential for RIG-I activation. Analysis of vp13 deletion constructs suggested that the C-terminal domain of vp13 was essential for the enhancement of RIG-I signaling. In HEV-infected hepatoma cells, wild-type HEV led to a higher level of RIG-I and more poly(I · C)-induced IFN-β expression than did ORF3-null mutants. Analysis of vp13 from four HEV genotypes showed that vp13 from genotype I and III strains boosted RIG-I signaling, while vp13 from genotype II and IV strains had a minimal effect. These results indicate that vp13 enhances RIG-I signaling, which may play a role in HEV invasion.

IMPORTANCE
Hepatitis E virus (HEV) is a significant pathogen causing hepatitis in many parts of the world, yet it is understudied compared with other viral hepatitis pathogens. Here we found that the HEV open reading frame 3 product, vp13, enhances interferon induction stimulated by a synthetic analog of double-stranded RNA. This enhancement may play a role in HEV invasion, as vp13 is essential for HEV infection
in vivo
. The results of this study provide insights into virus-cell interactions during HEV infection. In addition to revealing its possible roles in HEV interference with cellular signaling, these results suggest that the second half of the vp13 sequence can be ligated into the genomes of attenuated live viruses to induce an innate immune response for better protective immunity, as well as a marker for differentiation of vaccinated animals from those infected with the corresponding wild-type viruses.
Protocol for a prospective neuroimaging study investigating the supraspinal control of lower urinary tract function in healthy controls and patients with non-neurogenic lower urinary tract symptoms
Authors: Walter Matthias , Michels Lars , Kollias Spyros , van Kerrebroeck Philip E , Kessler Thomas M , Mehnert Ulrich 
Published: May 2014

IntroductionLower urinary tract symptoms (LUTS) are highly prevalent, cause an enormous economic burden on healthcare systems and significantly impair the quality of life (QoL) of affected patients. The dependence of the LUT on complex central neuronal circuits makes it unique in comparison to other visceral functions, such as the gastrointestinal tract, but also more vulnerable to neurological diseases.Methods and analysisThis is a prospective neuroimaging study investigating the supraspinal control of LUT function in healthy controls and in patients with non-neurogenic LUTS. The clinical assessment will include medical history, neuro-urological examination, bladder diary, urine analysis, urodynamic investigations, as well as standardised questionnaires regarding LUTS and QoL. The acquisition of neuroimaging data will include structural assessments (T1-weighted imaging and diffusion tensor imaging) as well as functional investigations using blood-oxygen-level dependent sensitive functional MRI (fMRI) in a 3 T MR scanner. The fMRI will be performed during four different bladder tasks using an automated MR-compatible and MR-synchronised pump system. The first three task-related fMRIs will consist of automated, repetitive filling of 100 mL warm (37°C) saline starting with (1) an empty bladder, (2) a low prefilled bladder volume (100 mL) and (3) a high prefilled bladder volume (persistent desire to void). The fourth task-related fMRI will comprise of automated, repetitive filling of 100 mL cold (4–8°C) saline starting with an empty bladder.Ethics and disseminationThe local ethics committee approved this study (KEK-ZH-Nr. 2011–0346). The findings of the study will be published in peer-reviewed journals and presented at national and international scientific meetings.Trial registration numberThis study has been registered at clinicaltrials.gov (http://www.clinicaltrials.gov/ct2/show/NCT01768910).
Rosa Beta granite (Sardinian Pink Granite): a heritage stone of international significance from Italy
Authors: Careddu N. , Grillo S. 
Published: May 2014

Abstract
‘Rosa Beta’ or ‘Sardinian Pink granite’, an Hercynian granitoid from the Italian island of Sardinia, is formally here nominated as a Global Heritage Stone Resource. Rosa Beta is sourced from the Gallura region of northern Sardinia and has been associated with human heritage since prehistoric times. Notably it can be found in construction ranging from prehistoric Tomba dei Giganti, the 1st century Pantheon in Rome and the Renaissance Cathedral in Pisa to early 20th century monuments in Brazil and Egypt. Most importantly, it has also been used very extensively in the late 20th century for significant construction in countries as wide ranging as the USA, Saudi Arabia, Hong Kong, Singapore and Australia.
The ‘Global Heritage Stone Resource’ designation: past, present and future
Authors: Cooper Barry J. 
Published: May 2014

Abstract
The proposal to designate those natural stones that have achieved widespread utilization in human culture was first mooted in late 2007, and first presented in a public forum at the 33rd International Geological Congress in July 2008. Over the next four years, a network of international correspondents was created, circulars were distributed, a website was established and formal rules were adopted, all with the focus of establishing the ‘Global Heritage Stone Resource’ designation. During the 34th International Geological Congress in July 2012, the Heritage Stone Task Group was formally established under the auspices of the International Union of Geological Sciences, in association with the International Association of Engineering Geology and the Environment, Commission C-10 Building Stones and Ornamental Rocks, and with a formal Board of Management. It has now been recognized that the new designation has value both in offering a mechanism to promote the safeguarding of stone resources, as well as in the formal definition of stone types within sufficiently tight parameters that it can facilitate name protection. For the future, the Board of Management needs to encourage and approve nominations for the new designation. As well, an annual conference needs to be organized.
Prostate MRI: Evaluating Tumor Volume and Apparent Diffusion Coefficient as Surrogate Biomarkers for Predicting Tumor Gleason Score
Authors: Donati Olivio F. , Afaq Asim , Vargas Hebert Alberto , Mazaheri Yousef , Zheng Junting , Moskowitz Chaya S. , Hricak Hedvig , Akin Oguz 
Published: May 2014

Abstract
Purpose: To investigate whether tumor volume derived from apparent diffusion coefficient (ADC) maps (VolumeADC) and tumor mean ADC value (ADCmean) are independent predictors of prostate tumor Gleason score (GS).
Experimental Design: Tumor volume and GS were recorded from whole-mount histopathology for 131 men (median age, 60 years) who underwent endorectal diffusion-weighted MRI for local staging of prostate cancer before prostatectomy. VolumeADC and ADCmean were derived from ADC maps and correlated with histopathologic tumor volume and GS. Univariate and multivariate analyses were performed to evaluate prediction of tumor aggressiveness. Areas under receiver-operating characteristics curves (AUC) were calculated to evaluate the performance of VolumeADC and ADCmean in discriminating tumors of GS 6 and GS ≥7.
Results: Histopathology identified 116 tumor foci &gt;0.5 mL. VolumeADC correlated significantly with histopathologic tumor volume (ρ = 0.683). The correlation increased with increasing GS (ρ = 0.453 for GS 6 tumors; ρ = 0.643 for GS 7 tumors; ρ = 0.980 for GS ≥8 tumors). Both VolumeADC (ρ = 0.286) and ADCmean (ρ = −0.309) correlated with GS. At univariate analysis, both VolumeADC (P = 0.0325) and ADCmean (P = 0.0033) could differentiate GS = 6 from GS ≥7 tumor foci. However, at multivariate analysis, only ADCmean (P = 0.0156) was a significant predictor of tumor aggressiveness (i.e., GS 6 vs. GS ≥7). For differentiating GS 6 from GS ≥7 tumors, AUCs were 0.644 and 0.704 for VolumeADC and ADCmean, respectively, and 0.749 for both parameters combined.
Conclusion: In patients with prostate cancer, ADCmean is an independent predictor of tumor aggressiveness, but VolumeADC is not. The latter parameter adds little to the ADCmean in predicting tumor GS. Clin Cancer Res; 20(14); 3705–11. ©2014 AACR.