bluuebunny/crossref_metadata_2025_split huggingface dataset:
Impact of the F508del mutation on ovine CFTR, a Cl− channel with enhanced conductance and ATP‐dependent gating
Authors: Cai Zhiwei , Palmai‐Pallag Timea , Khuituan Pissared , Mutolo Michael J. , Boinot Clément , Liu Beihui , Scott‐Ward Toby S. , Callebaut Isabelle , Harris Ann , Sheppard David N. 
Published: March 2015

Key points
Malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), a gated pathway for chloride movement, causes the common life‐shortening genetic disease cystic fibrosis (CF).
Towards the development of a sheep model of CF, we have investigated the function of sheep CFTR.
We found that sheep CFTR was noticeably more active than human CFTR, while the most common CF mutation, F508del, had reduced impact on sheep CFTR function.
Our results demonstrate that subtle changes in protein structure have marked effects on CFTR function and the consequences of the CF mutation F508del.
AbstractCross‐species comparative studies are a powerful approach to understanding the epithelial Cl− channel cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF). Here, we investigate the single‐channel behaviour of ovine CFTR and the impact of the most common CF mutation, F508del‐CFTR, using excised inside‐out membrane patches from transiently transfected CHO cells. Like human CFTR, ovine CFTR formed a weakly inwardly rectifying Cl− channel regulated by PKA‐dependent phosphorylation, inhibited by the open‐channel blocker glibenclamide. However, for three reasons, ovine CFTR was noticeably more active than human CFTR. First, single‐channel conductance was increased. Second, open probability was augmented because the frequency and duration of channel openings were increased. Third, with enhanced affinity and efficacy, ATP more strongly stimulated ovine CFTR channel gating. Consistent with these data, the CFTR modulator phloxine B failed to potentiate ovine CFTR Cl− currents. Similar to its impact on human CFTR, the F508del mutation caused a temperature‐sensitive folding defect, which disrupted ovine CFTR protein processing and reduced membrane stability. However, the F508del mutation had reduced impact on ovine CFTR channel gating in contrast to its marked effects on human CFTR. We conclude that ovine CFTR forms a regulated Cl− channel with enhanced conductance and ATP‐dependent channel gating. This phylogenetic analysis of CFTR structure and function demonstrates that subtle changes in structure have pronounced effects on channel function and the consequences of the CF mutation F508del.
Salmonella Serotype Determination Utilizing High-Throughput Genome Sequencing Data
Authors: Zhang Shaokang , Yin Yanlong , Jones Marcus B. , Zhang Zhenzhen , Deatherage Kaiser Brooke L. , Dinsmore Blake A. , Fitzgerald Collette , Fields Patricia I. , Deng Xiangyu 
Published: March 2015

ABSTRACT

Serotyping forms the basis of national and international surveillance networks for
Salmonella
, one of the most prevalent foodborne pathogens worldwide (1
–
3). Public health microbiology is currently being transformed by whole-genome sequencing (WGS), which opens the door to serotype determination using WGS data. SeqSero (
www.denglab.info/SeqSero
) is a novel Web-based tool for determining
Salmonella
serotypes using high-throughput genome sequencing data. SeqSero is based on curated databases of
Salmonella
serotype determinants (
rfb
gene cluster,
fliC
and
fljB
alleles) and is predicted to determine serotype rapidly and accurately for nearly the full spectrum of
Salmonella
serotypes (more than 2,300 serotypes), from both raw sequencing reads and genome assemblies. The performance of SeqSero was evaluated by testing (i) raw reads from genomes of 308
Salmonella
isolates of known serotype; (ii) raw reads from genomes of 3,306
Salmonella
isolates sequenced and made publicly available by GenomeTrakr, a U.S. national monitoring network operated by the Food and Drug Administration; and (iii) 354 other publicly available draft or complete
Salmonella
genomes. We also demonstrated
Salmonella
serotype determination from raw sequencing reads of fecal metagenomes from mice orally infected with this pathogen. SeqSero can help to maintain the well-established utility of
Salmonella
serotyping when integrated into a platform of WGS-based pathogen subtyping and characterization.
Molecular Diagnosis of Toxoplasmosis in Immunocompromised Patients: a 3-Year Multicenter Retrospective Study
Authors: Robert-Gangneux Florence , Sterkers Yvon , Yera Hélène , Accoceberry Isabelle , Menotti Jean , Cassaing Sophie , Brenier-Pinchart Marie-Pierre , Hennequin Christophe , Delhaes Laurence , Bonhomme Julie , Villena Isabelle , Scherer Emeline , Dalle Frédéric , Touafek Feriel , Filisetti Denis , Varlet-Marie Emmanuelle , Pelloux Hervé , Bastien Patrick 
Published: March 2015

ABSTRACT

Toxoplasmosis is a life-threatening infection in immunocompromised patients (ICPs). The definitive diagnosis relies on parasite DNA detection, but little is known about the incidence and burden of disease in HIV-negative patients. A 3-year retrospective study was conducted in 15 reference laboratories from the network of the French National Reference Center for Toxoplasmosis, in order to record the frequency of
Toxoplasma gondii
DNA detection in ICPs and to review the molecular methods used for diagnosis and the prevention measures implemented in transplant patients. During the study period, of 31,640 PCRs performed on samples from ICPs, 610 were positive (323 patients). Blood (
n
= 337 samples), cerebrospinal fluid (
n
= 101 samples), and aqueous humor (
n
= 100 samples) were more frequently positive. Chemoprophylaxis schemes in transplant patients differed between centers. PCR follow-up of allogeneic hematopoietic stem cell transplant (allo-HSCT) patients was implemented in 8/15 centers. Data from 180 patients (13 centers) were further analyzed regarding clinical setting and outcome. Only 68/180 (38%) patients were HIV
+
; the remaining 62% consisted of 72 HSCT, 14 solid organ transplant, and 26 miscellaneous immunodeficiency patients. Cerebral toxoplasmosis and disseminated toxoplasmosis were most frequently observed in HIV and transplant patients, respectively. Of 72 allo-HSCT patients with a positive PCR result, 23 were asymptomatic; all were diagnosed in centers performing systematic blood PCR follow-up, and they received specific treatment. Overall survival of allo-HSCT patients at 2 months was better in centers with PCR follow-up than in other centers (
P
< 0.01). This study provides updated data on the frequency of toxoplasmosis in HIV-negative ICPs and suggests that regular PCR follow-up of allo-HSCT patients could guide preemptive treatment and improve outcome.
Subtyping Novel Zoonotic Pathogen Cryptosporidium Chipmunk Genotype I
Authors: Guo Yaqiong , Cebelinski Elizabeth , Matusevich Christine , Alderisio Kerri A. , Lebbad Marianne , McEvoy John , Roellig Dawn M. , Yang Chunfu , Feng Yaoyu , Xiao Lihua 
Published: March 2015

ABSTRACT

Cryptosporidium
chipmunk genotype I is an emerging zoonotic pathogen in humans. The lack of subtyping tools makes it impossible to determine the role of zoonotic transmission in epidemiology. To identify potential subtyping markers, we sequenced the genome of a human chipmunk genotype I isolate. Altogether, 9,509,783 bp of assembled sequences in 853 contigs were obtained, with an N50 of 117,886 bp and >200-fold coverage. Based on the whole-genome sequence data, two genetic markers encoding the 60-kDa glycoprotein (gp60) and a mucin protein (ortholog of cgd1_470) were selected for the development of a subtyping tool. The tool was used for characterizing chipmunk genotype I in 25 human specimens from four U.S. states and Sweden, one specimen each from an eastern gray squirrel, a chipmunk, and a deer mouse, and 4 water samples from New York. At the gp60 locus, although different subtypes were seen among the animals, water, and humans, the 15 subtypes identified differed mostly in the numbers of trinucleotide repeats (TCA, TCG, or TCT) in the serine repeat region, with only two single nucleotide polymorphisms in the nonrepeat region. Some geographic differences were found in the subtype distribution of chipmunk genotype I from humans. In contrast, only two subtypes were found at the mucin locus, which differed from each other in the numbers of a 30-bp minisatellite repeat. Thus,
Cryptosporidium
chipmunk genotype I isolates from humans and wildlife are genetically similar, and zoonotic transmission might play a potential role in human infections.
Viral Infection of the Central Nervous System and Neuroinflammation Precede Blood-Brain Barrier Disruption during Japanese Encephalitis Virus Infection
Authors: Li Fang , Wang Yueyun , Yu Lan , Cao Shengbo , Wang Ke , Yuan Jiaolong , Wang Chong , Wang Kunlun , Cui Min , Fu Zhen F. 
Published: March 2015

ABSTRACTJapanese encephalitis is an acute zoonotic, mosquito-borne disease caused by Japanese encephalitis virus (JEV). Japanese encephalitis is characterized by extensive inflammation in the central nervous system (CNS) and disruption of the blood-brain barrier (BBB). However, the pathogenic mechanisms contributing to the BBB disruption are not known. Here, using a mouse model of intravenous JEV infection, we show that virus titers increased exponentially in the brain from 2 to 5 days postinfection. This was accompanied by an early, dramatic increase in the level of inflammatory cytokines and chemokines in the brain. Enhancement of BBB permeability, however, was not observed until day 4, suggesting that viral entry and the onset of inflammation in the CNS occurred prior to BBB damage.In vitrostudies revealed that direct infection with JEV could not induce changes in the permeability of brain microvascular endothelial cell monolayers. However, brain extracts derived from symptomatic JEV-infected mice, but not from mock-infected mice, induced significant permeability of the endothelial monolayer. Consistent with a role for inflammatory mediators in BBB disruption, the administration of gamma interferon-neutralizing antibody ameliorated the enhancement of BBB permeability in JEV-infected mice. Taken together, our data suggest that JEV enters the CNS, propagates in neurons, and induces the production of inflammatory cytokines and chemokines, which result in the disruption of the BBB.IMPORTANCEJapanese encephalitis (JE) is the leading cause of viral encephalitis in Asia, resulting in 70,000 cases each year, in which approximately 20 to 30% of cases are fatal, and a high proportion of patients survive with serious neurological and psychiatric sequelae. Pathologically, JEV infection causes an acute encephalopathy accompanied by BBB dysfunction; however, the mechanism is not clear. Thus, understanding the mechanisms of BBB disruption in JEV infection is important. Our data demonstrate that JEV gains entry into the CNS prior to BBB disruption. Furthermore, it is not JEV infectionper se, but the inflammatory cytokines/chemokines induced by JEV infection that inhibit the expression of TJ proteins and ultimately result in the enhancement of BBB permeability. Neutralization of gamma interferon (IFN-γ) ameliorated the enhancement of BBB permeability in JEV-infected mice, suggesting that IFN-γ could be a potential therapeutic target. This study would lead to identification of potential therapeutic avenues for the treatment of JEV infection.
Dynamics of Human Cytomegalovirus Infection in CD34+Hematopoietic Cells and Derived Langerhans-Type Dendritic Cells
Authors: Coronel Roxanne , Takayama Sachiko , Juwono Timothy , Hertel Laura 
Published: March 2015

ABSTRACTAcquisition of human cytomegalovirus (CMV) usually occurs by contact between contaminated bodily fluids, such as urine and saliva, and host mucosal cells. Langerhans-type dendritic cells (LC) are the only type of immune cells found in the outermost layers of the oral mucosae, where they not only provide a first line of defense against CMV but can easily be targeted by orally administered vaccines, while their bone marrow resident progenitors are important sites of virus latency. In this work, we tracked the progress of infection in CD34+progenitor cells, immature LC (iLC), and mature LC (mLC) exposed to the clinical-like strain TB40-BAC4 or to the vaccine strain AD169varATCC, prior to their long-term maintenance under either immature or mature conditions. We show that the genomes of both strains are efficiently maintained in CD34+cells during their differentiation into iLC, although this requires the presence of larger amounts of input AD169varATCC DNA. Lipopolysaccharide- and CD40 ligand-induced maturation of iLC derived from latently infected progenitors was not associated with robust viral genome replication and progeny production, while maturation of directly infected iLC increased and prolonged expression of the viral immediate early proteins. While effective replication of viral genomes from both strains occurred only in mLC, both iLC and mLC produced viral progeny, suggesting that both types of LC may contribute to CMV horizontal transmissionin vivo.IMPORTANCEHuman CMV is usually acquired via the oral and nasal mucosae. Langerhans-type dendritic cells (LC) are the only type of immune cells found in the outermost layers of these tissues. Understanding how CMV interacts with LC and their hematopoietic progenitors is thus essential to develop innovative means of defense against this virus. Here we show that the genomes of a virulent and an attenuated strain of CMV are maintained in hematopoietic progenitor cells during their differentiation into immature LC and that maturation of these cells by exposure to lipopolysaccharide and CD40 ligand is not sufficient to trigger virus reactivation. While the extents of viral protein expression and genome replication were broadest in directly infected mature LC populations, similar amounts of viral progeny were detected in the supernatants of immature and mature LC, suggesting that these immune cells of the oral mucosa are likely to be important for CMV transmission within the human population.
Murine Gammaherpesvirus 68 ORF48 Is an RTA-Responsive Gene Product and Functions in both Viral Lytic Replication and Latency during
In Vivo
Infection
Authors: Qi Jing , Han Chuanhui , Gong Danyang , Liu Ping , Zhou Sheng , Deng Hongyu 
Published: March 2015

ABSTRACT

Replication and transcription activator (RTA) of gammaherpesvirus is an immediate early gene product and regulates the expression of many downstream viral lytic genes. ORF48 is also conserved among gammaherpesviruses; however, its expression regulation and function remained largely unknown. In this study, we characterized the transcription unit of
ORF48
from murine gammaherpesvirus 68 (MHV-68) and analyzed its transcriptional regulation. We showed that RTA activates the
ORF48
promoter via an RTA-responsive element (48pRRE). RTA binds to 48pRRE directly
in vitro
and also associates with ORF48 promoter
in vivo
. Mutagenesis of 48pRRE in the context of the viral genome demonstrated that the expression of ORF48 is activated by RTA through 48pRRE during
de novo
infection. Through site-specific mutagenesis, we generated an ORF48-null virus and examined the function of ORF48
in vitro
and
in vivo
. The ORF48-null mutation remarkably reduced the viral replication efficiency in cell culture. Moreover, through intranasal or intraperitoneal infection of laboratory mice, we showed that ORF48 is important for viral lytic replication in the lung and establishment of latency in the spleen, as well as viral reactivation from latency. Collectively, our study identified
ORF48
as an RTA-responsive gene and showed that ORF48 is important for MHV-68 replication both
in vitro
and
in vivo
.

IMPORTANCE
The replication and transcription activator (RTA), conserved among gammaherpesviruses, serves as a molecular switch for the virus life cycle. It works as a transcriptional regulator to activate the expression of many viral lytic genes. However, only a limited number of such downstream genes have been uncovered for MHV-68. In this study, we identified
ORF48
as an RTA-responsive gene of MHV-68 and mapped the
cis
element involved. By constructing a mutant virus that is deficient in ORF48 expression and through infection of laboratory mice, we showed that ORF48 plays important roles in different stages of viral infection
in vivo
. Our study provides insights into the transcriptional regulation and protein function of MHV-68, a desired model for studying gammaherpesviruses.
The pH Stability of Foot-and-Mouth Disease Virus Particles Is Modulated by Residues Located at the Pentameric Interface and in the N Terminus of VP1
Authors: Caridi Flavia , Vázquez-Calvo Angela , Sobrino Francisco , Martín-Acebes Miguel A. 
Published: March 2015

ABSTRACT
The picornavirus foot-and-mouth disease virus (FMDV) is the etiological agent of a highly contagious disease that affects important livestock species. The FMDV capsid is highly acid labile, and viral particles lose infectivity due to their disassembly at pH values slightly below neutrality. This acid sensitivity is related to the mechanism of viral uncoating and genome penetration from endosomes. In this study, we have analyzed the molecular basis of FMDV acid-induced disassembly by isolating and characterizing a panel of novel FMDV mutants differing in acid sensitivity. Amino acid replacements altering virion stability were preferentially distributed in two different regions of the capsid: the N terminus of VP1 and the pentameric interface. Even more, the acid labile phenotype induced by a mutation located at the pentameric interface in VP3 could be compensated by introduction of an amino acid substitution in the N terminus of VP1. These results indicate that the acid sensitivity of FMDV can be considered a multifactorial trait and that virion stability is the fine-tuned product of the interaction between residues from different capsid proteins, in particular those located within the N terminus of VP1 or close to the pentameric interface.

IMPORTANCE
The viral capsid protects the viral genome from environmental factors and contributes to virus dissemination and infection. Thus, understanding of the molecular mechanisms that modulate capsid stability is of interest for the basic knowledge of the biology of viruses and as a tool to improve the stability of conventional vaccines based on inactivated virions or empty capsids. Using foot-and-mouth disease virus (FMDV), which displays a capsid with extreme acid sensitivity, we have performed a genetic study to identify the molecular determinants involved in capsid stability. A panel of FMDV mutants with differential sensitivity to acidic pH was generated and characterized, and the results showed that two different regions of FMDV capsid contribute to modulating viral particle stability. These results provide new insights into the molecular mechanisms of acid-mediated FMDV uncoating.
Degradation of SAMHD1 by Vpx Is Independent of Uncoating
Authors: Jáuregui Paula , Logue Eric C. , Schultz Megan L. , Fung Stephanie , Landau Nathaniel R. 
Published: March 2015

ABSTRACT
Sterile alpha motif domain and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) replication in myeloid and resting T cells. Lentiviruses such as HIV-2 and some simian immunodeficiency viruses (SIVs) counteract the restriction by encoding Vpx or Vpr, accessory proteins that are packaged in virions and which, upon entry of the virus into the cytoplasm, induce the proteasomal degradation of SAMHD1. As a tool to study these mechanisms, we generated HeLa cell lines that express a fusion protein termed NLS.GFP.SAM595 in which the Vpx binding domain of SAMHD1 is fused to the carboxy terminus of green fluorescent protein (GFP) and a nuclear localization signal is fused to the amino terminus of GFP. Upon incubation of Vpx-containing virions with the cells, the NLS.GFP.SAM595 fusion protein was degraded over several hours and the levels remained low over 5 days as the result of continued targeting of the CRL4 E3 ubiquitin ligase. Degradation of the fusion protein required that it contain a nuclear localization sequence. Fusion to the cytoplasmic protein muNS rendered the protein resistant to Vpx-mediated degradation, confirming that SAMHD1 is targeted in the nucleus. Virions treated with protease inhibitors failed to release Vpx, indicating that Gag processing was required for Vpx release from the virion. Mutations in the capsid protein that altered the kinetics of virus uncoating and the Gag binding drug PF74 had no effect on the Vpx-mediated degradation. These results suggest that Vpx is released from virions without a need for uncoating of the capsid, allowing Vpx to transit to the nucleus rapidly upon entry into the cytoplasm.

IMPORTANCE
SAMHD1 restricts lentiviral replication in myeloid cells and resting T cells. Its importance is highlighted by the fact that viruses such as HIV-2 encode an accessory protein that is packaged in the virion and is dedicated to inducing SAMHD1 degradation. Vpx needs to act rapidly upon infection to allow reverse transcription to proceed. The limited number of Vpx molecules in a virion also needs to clear the cell of SAMHD1 over a prolonged period of time. Using an engineered HeLa cell line that expresses a green fluorescent protein (GFP)-SAMHD1 fusion protein, we showed that the Vpx-dependent degradation occurs without a need for viral capsid uncoating. In addition, the fusion protein was degraded only when it was localized to the nucleus, confirming that SAMHD1 is targeted in the nucleus and thus explaining why Vpx also localizes to the nucleus.
Do small doses of atropine (<0.1 mg) cause bradycardia in young children?
Authors: Eisa Lara , Passi Yuvesh , Lerman Jerrold , Raczka Michelle , Heard Christopher 
Published: March 2015

ObjectiveTo determine the heart rate response to atropine (<0.1 mg) in anaesthetised young infants.DesignProspective, observational and controlled.SettingElective surgery.PatientsSixty unpremedicated healthy infants less than 15 kg were enrolled. Standard monitoring was applied. Anaesthesia was induced by mask with nitrous oxide (66%) and oxygen (33%) followed by sevoflurane (8%).InterventionsIntravenous (IV) atropine (5 µg/kg) was flushed into a fast flowing IV. The ECG was recorded continuously from 30 s before the atropine until 5 min afterwards.Main outcome measuresThe incidence of bradycardia and arrhythmias was determined from the ECGs by a blinded observer.ResultsThe median (IQR) age was 6.5 (4–12) months and the mean (95% CI) weight was 8.6 (8.1 to 9.1) kg. The mean (95% CI) dose of atropine was 40.9 (37.3 to 44) µg. Bradycardia did not occur. Two infants developed premature atrial contractions and one developed a premature ventricular contraction. When compared with baseline values, heart rate increased by 7% 30 s after atropine, 14% 1 min after atropine and 25% 5 min after atropine. Twenty-nine infants (48%) experienced tachycardia (>20% above baseline rate) after atropine lasting 222.7 s (range 27.9–286). The change in heart rate 5 min after atropine was inversely related to the baseline heart rate.ConclusionsThe upper 95% CI for the occurrence of bradycardia in the entire population of infants based on a zero incidence in this study is 5%. These results rebut the notion that atropine <0.1 mg IV causes bradycardia in young infants.Trial registration numberClinicalTrials.gov #NCT01819064.