bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fine Tuning of Craniofacial Morphology by Distant-Acting Enhancers
Authors: Attanasio Catia , Nord Alex S. , Zhu Yiwen , Blow Matthew J. , Li Zirong , Liberton Denise K. , Morrison Harris , Plajzer-Frick Ingrid , Holt Amy , Hosseini Roya , Phouanenavong Sengthavy , Akiyama Jennifer A. , Shoukry Malak , Afzal Veena , Rubin Edward M. , FitzPatrick David R. , Ren Bing , Hallgrímsson Benedikt , Pennacchio Len A. , Visel Axel 
Published: October 2013

Introduction
The shape of the face is one of the most distinctive features among humans, and differences in facial morphology have substantial implications in areas such as social interaction, psychology, forensics, and clinical genetics. Craniofacial shape is highly heritable, including the normal spectrum of morphological variation as well as susceptibility to major craniofacial birth defects. In this study, we explored the role of transcriptional enhancers in the development of the craniofacial complex. Our study is based on the rationale that such enhancers, which can be hundreds of kilobases away from their target genes, regulate the spatial patterns, levels, and timing of gene expression in normal development.

Methods
To identify distant-acting enhancers active during craniofacial development, we used chromatin immunoprecipitation on embryonic mouse face tissue followed by sequencing to identify noncoding genome regions bound by the enhancer-associated p300 protein. We used LacZ reporter assays in transgenic mice and optical projection tomography (OPT) to determine three-dimensional expression patterns of a subset of these candidate enhancers. Last, we deleted three of the craniofacial enhancers from the mouse genome to assess their effect on gene expression and craniofacial morphology during development.

Results
We identified more than 4000 candidate enhancer sequences predicted to be active in the developing craniofacial complex. The majority of these sequences are at least partially conserved between humans and mice, and many are located in chromosomal regions associated with normal facial morphology or craniofacial birth defects. Characterization of more than 200 candidate enhancer sequences in transgenic mice revealed a remarkable spatial complexity of in vivo expression patterns. Targeted deletions of three craniofacial enhancers near genes with known roles in craniofacial development resulted in changes of expression of those genes as well as quantitatively subtle but definable alterations of craniofacial shape.

Discussion
Our analysis identifies enhancers that fine tune expression of genes during craniofacial development in mice. These results support that variation in the sequence or copy number of craniofacial enhancers may contribute to the spectrum of facial variation we find in human populations. Because many craniofacial enhancers are located in genome regions associated with craniofacial birth defects, such as clefts of the lip and palate, our results also offer a starting point for exploring the contribution of noncoding sequences to these disorders.
Significant Reduction in Rate of Indeterminate Results of the QuantiFERON-TB Gold In-Tube Test by Shortening Incubation Delay
Authors: Yun Jae Won , Chung Hae-Sun , Koh Won-Jung , Chung Doo Ryeon , Kim Yae-Jean , Kang Eun-Suk 
Published: October 2013

ABSTRACT

The QuantiFERON-TB Gold In-Tube (QFT-G IT) test (Cellestis Inc., Valencia, CA) is one of the gamma interferon release assays (IGRAs) that are promising tools for diagnosing active or latent
Mycobacterium tuberculosis
infections. We investigated the clinical and laboratory factors that affect the rate of indeterminate QFT-G IT test results. We also suggest a workflow strategy for achieving optimized test results using the QFT-G IT test for the diagnosis of active tuberculosis (TB) or latent TB infection. We performed statistical analysis using data from a retrospective review of medical records. The first phase included 683 QFT-G IT test results from 676 patients tested between January 2008 and May 2008, and the second phase included an additional 663 QFT-G IT test results from 653 patients tested between January 2008 and December 2008 at Samsung Medical Center, a tertiary care hospital in South Korea. Immunosuppressive drug therapy, underlying diseases, bedridden status, and hypoalbuminemia were significantly associated with indeterminate QFT-G IT test results. With reduction of the incubation delay during the test procedure from an average of 9.82 h to an average of 2.70 h with changes in the workflow, the frequency of indeterminate QFT-G IT test results was significantly reduced from 11.4% to 2.7%. With >6 h of incubation delay, however, the frequency of indeterminate QFT-G IT test results was increased in a statistically significant manner. This study demonstrates that not only clinicopathological factors but also laboratory factors, such as incubation delay, significantly affect the rate of indeterminate QFT-G IT test results; therefore, optimization of the test procedure may contribute to reductions in the rate of indeterminate QFT-G IT test results, which delay the diagnosis of TB.
Rapid Identification and Subtyping of Helicobacter cinaedi Strains by Intact-Cell Mass Spectrometry Profiling with the Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry
Authors: Taniguchi Takako , Sekiya Ayumi , Higa Mariko , Saeki Yuji , Umeki Kazumi , Okayama Akihiko , Hayashi Tetsuya , Misawa Naoaki 
Published: October 2013

ABSTRACT

Helicobacter cinaedi
infection is recognized as an increasingly important emerging disease in humans. Although
H. cinaedi
-like strains have been isolated from a variety of animals, it is difficult to identify particular isolates due to their unusual phenotypic profiles and the limited number of biochemical tests for detecting helicobacters. Moreover, analyses of the 16S rRNA gene sequences are also limited due to the high levels of similarity among closely related helicobacters. This study was conducted to evaluate intact-cell mass spectrometry (ICMS) profiling using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) as a tool for the identification of
H. cinaedi
. A total of 68 strains of
H. cinaedi
isolated from humans, dogs, a cat, and hamsters were examined in addition to other
Helicobacter
species. The major ICMS profiles of
H. cinaedi
were identical and differed from those of
Helicobacter bilis
, which show >98% sequence similarity at the 16S rRNA sequence level. A phyloproteomic analysis of the
H. cinaedi
strains examined in this work revealed that human isolates formed a single cluster that was distinct from that of the animal isolates, with the exception of two strains from dogs. These phyloproteomic results agreed with those of the phylogenetic analysis based on the nucleotide sequences of the
hsp60
gene. Because they formed a distinct cluster in both analyses, our data suggest that animal strains may not be a major source of infection in humans. In conclusion, the ICMS profiles obtained using a MALDI-TOF MS approach may be useful for the identification and subtyping of
H. cinaedi
.
Real-Time Reverse Transcription-PCR Assay Panel for Middle East Respiratory Syndrome Coronavirus
Authors: Lu Xiaoyan , Whitaker Brett , Sakthivel Senthil Kumar K. , Kamili Shifaq , Rose Laura E. , Lowe Luis , Mohareb Emad , Elassal Emad M. , Al-sanouri Tarek , Haddadin Aktham , Erdman Dean D. 
Published: October 2013

ABSTRACT

A new human coronavirus (CoV), subsequently named Middle East respiratory syndrome (MERS)-CoV, was first reported in Saudi Arabia in September 2012. In response, we developed two real-time reverse transcription-PCR (rRT-PCR) assays targeting the MERS-CoV nucleocapsid (N) gene and evaluated these assays as a panel with a previously published assay targeting the region upstream of the MERS-CoV envelope gene (upE) for the detection and confirmation of MERS-CoV infection. All assays detected ≤10 copies/reaction of quantified RNA transcripts, with a linear dynamic range of 8 log units and 1.3 × 10
−3
50% tissue culture infective doses (TCID
50
)/ml of cultured MERS-CoV per reaction. All assays performed comparably with respiratory, serum, and stool specimens spiked with cultured virus. No false-positive amplifications were obtained with other human coronaviruses or common respiratory viral pathogens or with 336 diverse clinical specimens from non-MERS-CoV cases; specimens from two confirmed MERS-CoV cases were positive with all assay signatures. In June 2012, the U.S. Food and Drug Administration authorized emergency use of the rRT-PCR assay panel as an
in vitro
diagnostic test for MERS-CoV. A kit consisting of the three assay signatures and a positive control was assembled and distributed to public health laboratories in the United States and internationally to support MERS-CoV surveillance and public health responses.
Human T-Cell Leukemia/Lymphoma Virus Type 1 p30, but Not p12/p8, Counteracts Toll-Like Receptor 3 (TLR3) and TLR4 Signaling in Human Monocytes and Dendritic Cells
Authors: Fenizia Claudio , Fiocchi Martina , Jones Kathryn , Parks Robyn Washington , Ceribelli Michele , Chevalier Sebastien A. , Edwards Dustin , Ruscetti Francis , Pise-Masison Cynthia A. , Franchini Genoveffa 
Published: October 2013

ABSTRACT

The human T-cell leukemia/lymphoma virus type 1 (HTLV-1) p30 protein, essential for virus infectivity
in vivo
, is required for efficient infection of human dendritic cells (DCs) but not B and T cells
in vitro
. We used a human monocytic cell line, THP-1, and dendritic cells to study the mechanism of p30 and p12/p8 requirements in these cell types. p30 inhibited the expression of interferon (IFN)-responsive genes (ISG) following stimulation by lipopolysaccharide (LPS) of Toll-like receptor 4 (TLR4) and by poly(I·C) of TLR3 but not of TLR7/8 with imiquimod. Results with THP-1 mirrored those for
ex vivo
human primary monocytes and monocyte-derived dendritic cells (Mo-mDC). The effect of p30 on TLR signaling was also demonstrated by ablating its expression within a molecular clone of HTLV-1. HTLV-1 infection of monocytes inhibited TLR3- and TLR4-induced ISG expression by 50 to 90% depending on the genes, whereas the isogenic clone p30 knockout virus was less effective at inhibiting TLR3 and TRL4 signaling and displayed lower infectivity. Viral expression and inhibition of ISG transcription was, however, rescued by restoration of p30 expression. A chromatin immunoprecipitation assay demonstrated that p30 inhibits initiation and elongation of PU.1-dependent transcription of IFN-α1, IFN-β, and TLR4 genes upon TLR stimulation. In contrast, experiments conducted with p12/p8 did not demonstrate an effect on ISG expression. These results provide a mechanistic explanation of the requirement of p30 for HTLV-1 infectivity
in vivo
, suggest that dampening interferon responses in monocytes and DCs is specific for p30, and represent an essential early step for permissive HTLV-1 infection and persistence.
Host Restriction of Influenza Virus Polymerase Activity by PB2 627E Is Diminished on Short Viral Templates in a Nucleoprotein-Independent Manner
Authors: Paterson Duncan , te Velthuis Aartjan J. W. , Vreede Frank T. , Fodor Ervin 
Published: October 2013

ABSTRACT
Most avian influenza viruses do not replicate efficiently in human cells. This is partly due to the low activity of the RNA polymerase of avian influenza viruses in mammalian cells. Nevertheless, this impediment can be overcome through an E→K adaptive mutation at residue 627 of the PB2 subunit of the polymerase. Accordingly, viral ribonucleoprotein (RNP) reconstitution assays show that a viral polymerase containing PB2 627E has impaired activity in mammalian cells compared to a viral polymerase that contains PB2 627K, characteristic of mammalian-adapted influenza viruses. In contrast, purified viral polymerases containing either PB2 627E or PB2 627K show comparable levels of activity in transcription assays that require no RNP assembly. We sought to reconcile these conflicting observations by using an NP-independent cell-based transcription/replication assay to assess viral polymerase activity. We found that PB2 627E polymerase restriction in mammalian cells is independent of NP expression but is dependent on the length of the viral RNA template. In addition, restriction of PB2 627E polymerase was overcome by mutations specific to the viral RNA template promoter sequence. Consequently, we propose that PB2 627E affects recruitment of the viral RNA promoter by the viral polymerase in mammalian cells.
Varicella-Zoster Virus ORF49 Functions in the Efficient Production of Progeny Virus through Its Interaction with Essential Tegument Protein ORF44
Authors: Sadaoka Tomohiko , Serada Satoshi , Kato Junko , Hayashi Mayuko , Gomi Yasuyuki , Naka Tetsuji , Yamanishi Koichi , Mori Yasuko 
Published: October 2013

ABSTRACT
The ORF49 tegument protein of varicella-zoster virus (VZV) is one of the core gene products that is conserved among herpesvirus family members. Although ORF49 is known to be a cell-tropic factor, its detailed functions remain elusive. ORF44 is another core gene product reported to be essential, although its characterization and detailed functional analysis have not been reported. These two core gene products form a complex in other herpesviruses beyond the host species and herpesvirus subfamilies. Here, we show that complex formation between ORF44 and ORF49 is conserved in VZV. We serendipitously found that binding is eliminated by an amino acid substitution at position 129 (phenylalanine 129), and four amino acids in the carboxyl-terminal half of the acidic cluster in ORF49 (i.e., aspartate-phenylalanine-aspartate-glutamate from positions 41 to 44 [41DFDE44]) were identified as its binding motif. Alanine substitutions in each domain rendered the ORF44F129A mutation lethal for VZV, similar to deletion of the entire ORF44. The phenotype of the ORF49-41AAAA44 mutation was comparable to that of the ORF49-defective virus, including small-plaque formation, impaired growth, and low infectious virus production. These results suggest that the interaction between ORF44 and ORF49 is essential for their role in VZV infection and that ORF49 is required for the efficient production of infectious progeny virus mediated by the conserved interaction between the two proteins.
Crystal Structure of the Nipah Virus Phosphoprotein Tetramerization Domain
Authors: Bruhn Jessica F. , Barnett Katherine C. , Bibby Jaclyn , Thomas Jens M. H. , Keegan Ronan M. , Rigden Daniel J. , Bornholdt Zachary A. , Saphire Erica Ollmann 
Published: October 2013

ABSTRACT
The Nipah virus phosphoprotein (P) is multimeric and tethers the viral polymerase to the nucleocapsid. We present the crystal structure of the multimerization domain of Nipah virus P: a long, parallel, tetrameric, coiled coil with a small, α-helical cap structure. Across the paramyxoviruses, these domains share little sequence identity yet are similar in length and structural organization, suggesting a common requirement for scaffolding or spatial organization of the functions of P in the virus life cycle.
Cytomegalovirus Expresses the Chemokine Homologue vXCL1 Capable of Attracting XCR1
+
CD4
−
Dendritic Cells
Authors: Geyer Henriette , Hartung Evelyn , Mages Hans Werner , Weise Christoph , Belužić Robert , Vugrek Oliver , Jonjić Stipan , Kroczek Richard A. , Voigt Sebastian 
Published: October 2013

ABSTRACT

Cytomegaloviruses (CMV) have developed various strategies to escape the immune system of the host. One strategy involves the expression of virus-encoded chemokines to modulate the host chemokine network. We have identified in the English isolate of rat CMV (murid herpesvirus 8 [MuHV8]) an open reading frame encoding a protein homologous to the chemokine XCL1, the only known C chemokine. Viral XCL1 (vXCL1), a glycosylated protein of 96 amino acids, can be detected 13 h postinfection in the supernatant of MuHV8-infected rat embryo fibroblasts. vXCL1 exclusively binds to CD4
−
rat dendritic cells (DC), a subset of DC that express the corresponding chemokine receptor XCR1. Like endogenous rat XCL1, vXCL1 selectively chemoattracts XCR1
+
CD4
−
DC. Since XCR1
+
DC in mice and humans have been shown to excel in antigen cross-presentation and thus in the induction of cytotoxic CD8
+
T lymphocytes, the virus has apparently hijacked this gene to subvert cytotoxic immune responses. The biology of vXCL1 offers an interesting opportunity to study the role of XCL1 and XCR1
+
DC in the cross-presentation of viral antigens.
Human Cytomegalovirus UL50 and UL53 Recruit Viral Protein Kinase UL97, Not Protein Kinase C, for Disruption of Nuclear Lamina and Nuclear Egress in Infected Cells
Authors: Sharma Mayuri , Kamil Jeremy P. , Coughlin Margaret , Reim Natalia I. , Coen Donald M. 
Published: October 2013

ABSTRACT

Herpesvirus nucleocapsids traverse the nuclear envelope into the cytoplasm in a process called nuclear egress that includes disruption of the nuclear lamina. In several herpesviruses, a key player in nuclear egress is a complex of two proteins, whose homologs in human cytomegalovirus (HCMV) are UL50 and UL53. However, their roles in nuclear egress during HCMV infection have not been shown. Based largely on transfection studies, UL50 and UL53 have been proposed to facilitate disruption of the nuclear lamina by recruiting cellular protein kinase C (PKC), as occurs with certain other herpesviruses, and/or the viral protein kinase UL97 to phosphorylate lamins. To investigate these issues during HCMV infection, we generated viral mutants null for
UL50
or
UL53
. Correlative light electron microscopic analysis of null mutant-infected cells showed the presence of intranuclear nucleocapsids and the absence of cytoplasmic nucleocapsids. Confocal immunofluorescence microscopy revealed that UL50 and UL53 are required for disruption of the nuclear lamina. A subpopulation of UL97 colocalized with the nuclear rim, and this was dependent on UL50 and, to a lesser extent, UL53. However, PKC was not recruited to the nuclear rim, and its localization was not affected by the absence of UL50 or UL53. Immunoprecipitation from cells infected with HCMV expressing tagged UL53 detected UL97 but not PKC. In summary, HCMV UL50 and UL53 are required for nuclear egress and disruption of nuclear lamina during HCMV infection, and they recruit UL97, not PKC, for these processes. Thus, despite the strong conservation of herpesvirus nuclear egress complexes, a key function can differ among them.
Generation of a Complete Single-Gene Knockout Bacterial Artificial Chromosome Library of Cowpox Virus and Identification of Its Essential Genes
Authors: Xu Zhiyong , Zikos Dimitrios , Osterrieder Nikolaus , Tischer B. Karsten 
Published: October 2013

ABSTRACT

Cowpox virus (CPXV) belongs to the genus
Orthopoxvirus
in the
Poxviridae
family. It infects a broad range of vertebrates and can cause zoonotic infections. CPXV has the largest genome among the orthopoxviruses and is therefore considered to have the most complete set of genes of all members of the genus. Since CPXV has also become a model for studying poxvirus genetics and pathogenesis, we created and characterized a complete set of single gene knockout bacterial artificial chromosome (BAC) clones of the CPXV strain Brighton Red. These mutants allow a systematic assessment of the contribution of single CPXV genes to the outcome of virus infection and replication, as well as to the virus host range. A full-length BAC clone of CPXV strain Brighton Red (pBRF) harboring the gene expressing the enhanced green fluorescent protein under the control of a viral late promoter was modified by introducing the
mrfp1
gene encoding the monomeric red fluorescent protein driven by a synthetic early vaccinia virus promoter. Based on the modified BAC (pBRFseR), a library of targeted knockout mutants for each single viral open reading frame (ORF) was generated. Reconstitution of infectious virus was successful for 109 of the 183 mutant BAC clones, indicating that the deleted genes are not essential for virus replication. In contrast, 74 ORFs were identified as essential because no virus progeny was obtained upon transfection of the mutant BAC clones and in the presence of a helper virus. More than 70% of all late CPXV genes belonged to this latter group of essential genes.
Measles Virus C Protein Impairs Production of Defective Copyback Double-Stranded Viral RNA and Activation of Protein Kinase R
Authors: Pfaller Christian K. , Radeke Monte J. , Cattaneo Roberto , Samuel Charles E. 
Published: October 2013

ABSTRACT

Measles virus (MV) lacking expression of C protein (C
KO
) is a potent activator of the double-stranded RNA (dsRNA)-dependent protein kinase (PKR), whereas the isogenic parental virus expressing C protein is not. Here, we demonstrate that significant amounts of dsRNA accumulate during C
KO
mutant infection but not following parental virus infection. dsRNA accumulated during late stages of infection and localized with virus replication sites containing N and P proteins. PKR autophosphorylation and stress granule formation correlated with the timing of dsRNA appearance. Phospho-PKR localized to dsRNA-containing structures as revealed by immunofluorescence. Production of dsRNA was sensitive to cycloheximide but resistant to actinomycin D, suggesting that dsRNA is a viral product. Quantitative PCR (qPCR) analyses revealed reduced viral RNA synthesis and a steepened transcription gradient in C
KO
virus-infected cells compared to those in parental virus-infected cells. The observed alterations were further reflected in lower viral protein expression levels and reduced C
KO
virus infectious yield. RNA deep sequencing confirmed the viral RNA expression profile differences seen by qPCR between C
KO
mutant and parental viruses. After one subsequent passage of the C
KO
virus, defective interfering RNA (DI-RNA) with a duplex structure was obtained that was not seen with the parental virus. We conclude that in the absence of C protein, the amount of PKR activator RNA, including DI-RNA, is increased, thereby triggering innate immune responses leading to impaired MV growth.
Guiding the Immune Response against Influenza Virus Hemagglutinin toward the Conserved Stalk Domain by Hyperglycosylation of the Globular Head Domain
Authors: Eggink Dirk , Goff Peter H. , Palese Peter 
Published: October 2013

ABSTRACT
Influenza virus hemagglutinin consists of a highly variable and immunodominant head domain and a more conserved but immunosubdominant stalk domain. We introduced seven N-linked glycosylation sites in the hemagglutinin head domain to shield the immunodominant antigenic sites. The hyperglycosylated hemagglutinin enhanced stalk-directed seroreactivity while dampening the head response in immunized mice. Upon influenza virus challenge, mice vaccinated with the hyperglycosylated hemagglutinin were better protected against morbidity and mortality than mice receiving the wild-type hemagglutinin.