bluuebunny/crossref_metadata_2025_split huggingface dataset:
Yeast Identification Algorithm Based on Use of the Vitek MS System Selectively Supplemented with Ribosomal DNA Sequencing: Proposal of a Reference Assay for Invasive Fungal Surveillance Programs in China
Authors: Zhang Li , Xiao Meng , Wang He , Gao Ran , Fan Xin , Brown Mitchell , Gray Timothy J. , Kong Fanrong , Xu Ying-Chun 
Published: December 2013

ABSTRACT

Sequence analysis of the internal transcribed spacer (ITS) region was employed as the gold standard method for yeast identification in the China Hospital Invasive Fungal Surveillance Net (CHIF-NET). It has subsequently been found that matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is potentially a more practical approach for this purpose. In the present study, the performance of the Vitek MS v2.0 system for the identification of yeast isolates collected from patients with invasive fungal infections in the 2011 CHIF-NET was evaluated. A total of 1,243 isolates representing 31 yeast species were analyzed, and the identification results by the Vitek MS v2.0 system were compared to those obtained by ITS sequence analysis. By the Vitek MS v2.0 system, 96.7% (
n
= 1,202) of the isolates were correctly assigned to the species level and 0.2% (
n
= 2) of the isolates were identified to the genus level, while 2.4% (
n
= 30) and 0.7% (
n
= 9) of the isolates were unidentified and misidentified, respectively. After retesting of the unidentified and misidentified strains, 97.3% (
n
= 1,209) of the isolates were correctly identified to the species level. Based on these results, a testing algorithm that combines the use of the Vitek MS system with selected supplementary ribosomal DNA (rDNA) sequencing was developed and validated for yeast identification purposes. By employing this algorithm, 99.7% (1,240/1,243) of the study isolates were accurately identified with the exception of two isolates of
Candida fermentati
and one isolate of
Cryptococcus gattii
. In conclusion, the proposed identification algorithm could be practically implemented in strategic programs of fungal infection surveillance.
High-Resolution Melt PCR Analysis for Genotyping of Ureaplasma parvum Isolates Directly from Clinical Samples
Authors: Payne Matthew S. , Tabone Tania , Kemp Matthew W. , Keelan Jeffrey A. , Spiller O. Brad , Newnham John P. 
Published: December 2013

ABSTRACT

Ureaplasma
sp. infection in neonates and adults underlies a variety of disease pathologies. Of the two human
Ureaplasma
spp.,
Ureaplasma parvum
is clinically the most common. We have developed a high-resolution melt (HRM) PCR assay for the differentiation of the four serovars of
U. parvum
in a single step. Currently
U. parvum
strains are separated into four serovars by sequencing the promoter and coding region of the multiple-banded antigen (MBA) gene. We designed primers to conserved sequences within this region for PCR amplification and HRM analysis to generate reproducible and distinct melt profiles that distinguish clonal representatives of serovars 1, 3, 6, and 14. Furthermore, our HRM PCR assay could classify DNA extracted from 74 known (MBA-sequenced) test strains with 100% accuracy. Importantly, HRM PCR was also able to identify
U. parvum
serovars directly from 16 clinical swabs. HRM PCR performed with DNA consisting of mixtures of combined known serovars yielded profiles that were easily distinguished from those for single-serovar controls. These profiles mirrored clinical samples that contained mixed serovars. Unfortunately, melt curve analysis software is not yet robust enough to identify the composition of mixed serovar samples, only that more than one serovar is present. HRM PCR provides a single-step, rapid, cost-effective means to differentiate the four serovars of
U. parvum
that did not amplify any of the known 10 serovars of
Ureaplasma urealyticum
tested in parallel. Choice of reaction reagents was found to be crucial to allow sufficient sensitivity to differentiate
U. parvum
serovars directly from clinical swabs rather than requiring cell enrichment using microbial culture techniques.
Characterization of Human Astrovirus Cell Entry
Authors: Méndez Ernesto , Muñoz-Yañez Claudia , Sánchez-San Martín Claudia , Aguirre-Crespo Gabriela , Baños-Lara M. del Rocio , Gutierrez Michelle , Espinosa Rafaela , Acevedo Yunuén , Arias Carlos F. , López Susana 
Published: December 2013

ABSTRACT
Human astroviruses (HAstV) are a frequent cause of gastroenteritis in young children and immunocompromised patients. To understand the early steps of HAstV infection in the highly permissive Caco-2 cell line, the binding and entry processes of the virus were characterized. The half-time of virus binding to the cell surface was about 10 min, while virus decapsidation took around 130 min. Drugs affecting clathrin-mediated endocytosis, endosome acidification, and actin filament polymerization, as well as those that reduce the presence of cholesterol in the cell membrane, decreased the infectivity of the virus. The infection was also reduced by silencing the expression of the clathrin heavy chain (CHC) by RNA interference or by overexpression of dominant-negative mutants of dynamin 2 and Eps15. Furthermore, the entry of HAstV apparently depends on the maturation of endosomes, since the infection was reduced by silencing the expression of Rab7, a small GTPase involved in the early- to late-endosome maturation. Altogether, our results suggest that HAstV enters Caco-2 cells using a clathrin-dependent pathway and reaches late endosomes to enter cells. Here, we have characterized the mechanism used by human astroviruses, important agents of gastroenteritis in children, to gain entry into their host cells. Using a combination of biochemical and genetic tools, we found that these viruses enter Caco-2 cells using a clathrin-dependent endocytic pathway, where they most likely need to travel to late endosomes to reach the cytoplasm and begin their replication cycle.
In Vivo
SELEX of Single-Stranded Domains in the HIV-1 Leader RNA
Authors: van Bel Nikki , Das Atze T. , Berkhout Ben 
Published: December 2013

ABSTRACT

The 5′ untranslated leader region of the human immunodeficiency virus type 1 (HIV-1) RNA genome is a strongly conserved sequence that encodes several regulatory motifs important for viral replication. Most of these motifs are exposed as hairpin structures, including the dimerization initiation signal (DIS), the major splice donor site (SD), and the packaging signal (Ψ), which are connected by short single-stranded regions. Mutational analysis revealed many functions of these hairpins, but only a few studies have focused on the single-stranded purine-rich sequences. Using the
in vivo
SELEX (systematic evolution of ligands by exponential enrichment) approach, we probed the sequence space in these regions that is compatible with efficient HIV-1 replication and analyzed the impact on the RNA secondary structure of the leader RNA. Our results show a strong sequence requirement for the DIS hairpin flanking regions. We postulate that these sequences are important for the binding of specific protein factors that support leader RNA-mediated functions. The sequence between the SD and Ψ hairpins seems to have a less prominent role, despite the strong conservation of the stretch of 5 A residues in natural isolates. We hypothesize that this may reflect the subtle evolutionary pressure on HIV-1 to acquire an A-rich RNA genome.
In silico
analyses indicate that sequences are avoided in all 3 single-stranded domains that affect the local or overall leader RNA folding.

IMPORTANCE
Many regulatory RNA sequences are clustered in the untranslated leader domain of the HIV-1 RNA genome. Several RNA hairpin structures in this domain have been proposed to fulfill specific roles, e.g., mediating RNA dimer formation to facilitate HIV-1 recombination. We now focus on the importance of a few well-conserved single-stranded sequences that connect these hairpins. We created libraries of HIV-1 variants in which these segments were randomized and selected the best-replicating variants. For two segments we document the selection of the (nearly) wild-type sequence, thus demonstrating the importance of these primary nucleotide sequences and the power of the
in vivo
SELEX approach. However, for the third segment a large variety of sequences is compatible with efficient HIV-1 replication. Interestingly, the A-rich sequence of this segment is highly conserved among HIV-1 isolates, which likely reflects the evolutionary tendency of HIV-1 to adopt A-rich sequences.
Genetic Variation
In Vitro
and
In Vivo
of an Attenuated Lassa Vaccine Candidate
Authors: Zapata Juan C. , Goicochea Marco , Nadai Yuka , Eyzaguirre Lindsay M. , Carr Jean K. , Tallon Luke J. , Sadzewicz Lisa , Myers Garry , Fraser Claire M. , Su Qi , Djavani Mahmoud , Lukashevich Igor S. , Salvato Maria S. 
Published: December 2013

ABSTRACT

The attenuated Lassa vaccine candidate ML29 is a laboratory-produced reassortant between Lassa and Mopeia viruses, two Old World arenaviruses that differ by 40% in nucleic acid sequence. In our previous studies, ML29 elicited sterilizing immunity against Lassa virus challenge in guinea pigs and marmosets and virus-specific cell-mediated immunity in both simian immunodeficiency virus (SIV)-infected and uninfected rhesus macaques. Here, we show that ML29 is stable after 12 passages
in vitro
without losing its plaque morphology or its attenuated phenotype in suckling mice. Additionally, we used deep sequencing to characterize the viral population comprising the original stock of ML29, the stock of ML29 after 12 passages in Vero cells, and the ML29 isolates obtained from vaccinated animals. Twenty-seven isolates bore approximately 77 mutations that exceeded 20% of the single-nucleotide polymorphism (SNP) changes at any single locus. Of these 77 mutations, 5 appeared to be host specific, for example, appearing in mice but not in primates. None of these mutations were reversions of ML29 to the sequences of the parental Lassa and Mopeia viruses. The host-specific mutations indicate viral adaptations to virus-host interactions, and such interactions make reasonable targets for antiviral approaches. Variants capable of chronic infection did not emerge from any of the primate infections, even in immune-deficient animals, indicating that the ML29 reassortant is reasonably stable
in vivo
. In conclusion, the preclinical studies of ML29 as a Lassa virus vaccine candidate have been advanced, showing high levels of protection in nonhuman primates and acceptable stability both
in vitro
and
in vivo
.
Utility of Humanized BLT Mice for Analysis of Dengue Virus Infection and Antiviral Drug Testing
Authors: Frias-Staheli Natalia , Dorner Marcus , Marukian Svetlana , Billerbeck Eva , Labitt Rachael N. , Rice Charles M. , Ploss Alexander 
Published: December 2013

ABSTRACT

Dengue virus (DENV) is the cause of a potentially life-threatening disease that affects millions of people worldwide. The lack of a small animal model that mimics the symptoms of DENV infection in humans has slowed the understanding of viral pathogenesis and the development of therapies and vaccines. Here, we investigated the use of humanized “bone marrow liver thymus” (BLT) mice as a model for immunological studies and assayed their applicability for preclinical testing of antiviral compounds. Human immune system (HIS) BLT-NOD
/SCID
mice were inoculated intravenously with a low-passage, clinical isolate of DENV-2, and this resulted in sustained viremia and infection of leukocytes in lymphoid and nonlymphoid organs. In addition, DENV infection increased serum cytokine levels and elicited DENV-2-neutralizing human IgM antibodies. Following restimulation with DENV-infected dendritic cells,
in vivo
-primed T cells became activated and acquired effector function. An adenosine nucleoside inhibitor of DENV decreased the circulating viral RNA when administered simultaneously or 2 days postinfection, simulating a potential treatment protocol for DENV infection in humans. In summary, we demonstrate that BLT mice are susceptible to infection with clinical DENV isolates, mount virus-specific adaptive immune responses, and respond to antiviral drug treatment. Although additional refinements to the model are required, BLT mice are a suitable platform to study aspects of DENV infection and pathogenesis and for preclinical testing of drug and vaccine candidates.

IMPORTANCE
Infection with dengue virus remains a major medical problem. Progress in our understanding of the disease and development of therapeutics has been hampered by the scarcity of small animal models. Here, we show that humanized mice, i.e., animals engrafted with components of a human immune system, that were infected with a patient-derived dengue virus strain developed clinical symptoms of the disease and mounted virus-specific immune responses. We further show that this mouse model can be used to test preclinically the efficacy of antiviral drugs.
Unraveling the Entry Mechanism of Baculoviruses and Its Evolutionary Implications
Authors: Wang Manli , Wang Jue , Yin Feifei , Tan Ying , Deng Fei , Chen Xinwen , Jehle Johannes A. , Vlak Just M. , Hu Zhihong , Wang Hualin 
Published: December 2013

ABSTRACT

The entry of baculovirus budded virus into host cells is mediated by two distinct types of envelope fusion proteins (EFPs), GP64 and F protein. Phylogenetic analysis suggested that F proteins were ancestral baculovirus EFPs, whereas GP64 was acquired by progenitor group I alphabaculovirus more recently and may have stimulated the formation of the group I lineage. This study was designed to experimentally recapitulate a possible major step in the evolution of baculoviruses. We demonstrated that the infectivity of an F-null group II alphabaculovirus (
Helicoverpa armigera
nucleopolyhedrovirus [HearNPV]) can be functionally rescued by coinsertion of GP64 along with the nonfusogenic F
def
(furin site mutated HaF) from HearNPV. Interestingly, HearNPV enters cells by endocytosis and, less efficiently, by direct membrane fusion at low pH. However, this recombinant HearNPV coexpressing F
def
and GP64 mimicked group I virus not only in its EFP composition but also in its abilities to enter host cells via low-pH-triggered direct fusion pathway. Neutralization assays indicated that the nonfusogenic F proteins contribute mainly to binding to susceptible cells, while GP64 contributes to fusion. Coinsertion of GP64 with an F-like protein (Ac23) from group I virus led to efficient rescue of an F-null group II virus. In summary, these recombinant viruses and their entry modes are considered to resemble an evolutionary event of the acquisition of GP64 by an ancestral group I virus and subsequent adaptive inactivation of the original F protein. The study described here provides the first experimental evidence to support the hypothesis of the evolution of baculovirus EFPs.
A Novel DDB2-ATM Feedback Loop Regulates Human Cytomegalovirus Replication
Authors: E Xiaofei , Savidis George , Chin Christopher R. , Wang Shixia , Lu Shan , Brass Abraham L. , Kowalik Timothy F. 
Published: December 2013

ABSTRACT

Human cytomegalovirus (HCMV) genome replication requires host DNA damage responses (DDRs) and raises the possibility that DNA repair pathways may influence viral replication. We report here that a nucleotide excision repair (NER)-associated-factor is required for efficient HCMV DNA replication. Mutations in genes encoding NER factors are associated with xeroderma pigmentosum (XP). One of the XP complementation groups, XPE, involves mutation in
ddb2
, which encodes DNA damage binding protein 2 (DDB2). Infectious progeny virus production was reduced by >2 logs in XPE fibroblasts compared to levels in normal fibroblasts. The levels of immediate early (IE) (IE2), early (E) (pp65), and early/late (E/L) (gB55) proteins were decreased in XPE cells. These replication defects were rescued by infection with a retrovirus expressing DDB2 cDNA. Similar patterns of reduced viral gene expression and progeny virus production were also observed in normal fibroblasts that were depleted for DDB2 by RNA interference (RNAi). Mature replication compartments (RCs) were nearly absent in XPE cells, and there were 1.5- to 2.0-log reductions in viral DNA loads in infected XPE cells relative to those in normal fibroblasts. The expression of viral genes (
UL122
,
UL44
,
UL54
,
UL55
, and
UL84
) affected by DDB2 status was also sensitive to a viral DNA replication inhibitor, phosphonoacetic acid (PAA), suggesting that DDB2 affects gene expression upstream of or events associated with the initiation of DNA replication. Finally, a novel, infection-associated feedback loop between DDB2 and ataxia telangiectasia mutated (ATM) was observed in infected cells. Together, these results demonstrate that DDB2 and a DDB2-ATM feedback loop influence HCMV replication.
Can routinely collected ambulance data about assaults contribute to reduction in community violence?
Authors: Ariel Barak , Weinborn Cristobal , Boyle Adrian 
Published: December 2013

BackgroundThe ‘law of spatiotemporal concentrations of events’ introduced major preventative shifts in policing communities. ‘Hotspots’ are at the forefront of these developments yet somewhat understudied in emergency medicine. Furthermore, little is known about interagency ‘data-crossover’, despite some developments through the Cardiff Model. Can police-ED interagency data-sharing be used to reduce community-violence using a hotspots methodology?Methods12-month (2012) descriptive study and analysis of spatiotemporal clusters of police and emergency calls for service using hotspots methodology and assessing the degree of incident overlap. 3775 violent crime incidents and 775 assault incidents analysed using spatiotemporal clustering withk-means++algorithm and Spearman's rho.ResultsSpatiotemporal location of calls for services to the police and the ambulance service are equally highly concentrated in a small number of geographical areas, primarily within intra-agency hotspots (33% and 53%, respectively) but across agencies’ hotspots as well (25% and 15%, respectively). Datasets are statistically correlated with one another at the 0.57 and 0.34 levels, with 50% overlap when adjusted for the number of hotspots. At least one in every two police hotspots does not have an ambulance hotspot overlapping with it, suggesting half of assault spatiotemporal concentrations are unknown to the police. Data further suggest that more severely injured patients, as estimated by transfer to hospital, tend to be injured in the places with the highest number of police-recorded crimes.ConclusionsA hotspots approach to sharing data circumvents the problem of disclosing person-identifiable data between different agencies. Practically, at least half of ambulance hotspots are unknown to the police; if causal, it suggests that data sharing leads to both reduced community violence by way of prevention (such as through anticipatory patrols or problem-oriented policing), particularly of more severe assaults, and improved efficiency of resource deployment.
Estimating bias from loss to follow-up in a prospective cohort study of bicycle crash injuries
Authors: Tin Tin Sandar , Woodward Alistair , Ameratunga Shanthi 
Published: December 2013

BackgroundLoss to follow-up, if related to exposures, confounders and outcomes of interest, may bias association estimates. We estimated the magnitude and direction of such bias in a prospective cohort study of crash injury among cyclists.MethodsThe Taupo Bicycle Study involved 2590 adult cyclists recruited from New Zealand's largest cycling event in 2006 and followed over a median period of 4.6 years through linkage to four administrative databases. We resurveyed the participants in 2009 and excluded three participants who died prior to the resurvey. We compared baseline characteristics and crash outcomes of the baseline (2006) and follow-up (those who responded in 2009) cohorts by ratios of relative frequencies and estimated potential bias from loss to follow-up on seven exposure-outcome associations of interest by ratios of HRs.ResultsOf the 2587 cyclists in the baseline cohort, 1526 (60%) responded to the follow-up survey. The responders were older, more educated and more socioeconomically advantaged. They were more experienced cyclists who often rode in a bunch, off-road or in the dark, but were less likely to engage in other risky cycling behaviours. Additionally, they experienced bicycle crashes more frequently during follow-up. The selection bias ranged between −10% and +9% for selected associations.ConclusionsLoss to follow-up was differential by demographic, cycling and behavioural risk characteristics as well as crash outcomes, but did not substantially bias association estimates of primary research interest.
Correlation of thrombus formation on 7 T MRI with histology in a rat carotid artery side wall aneurysm model
Authors: Zhang Chao , Chaudhary Neeraj , Gemmete Joseph J , Elias Augusto , Song Jianping , Thompson Byron G , Galban Craig , Xi Guohua , Pandey Aditya S 
Published: December 2013

Background/purposeVarious aneurysm animal models have been utilized to study the histological reaction post coil embolization. Our aim was to evaluate the imaging findings at day 14 of a rat external carotid artery side wall aneurysm treated with coil embolization using a gradient echo sequence on 7 T MRI and to correlate this with the histological findings.Materials and methodsMale Sprague Drawley rats were utilized to create a sidewall external carotid artery blind pouch aneurysm. A 5 mm segment of hydrocoil or bare platinum coil was inserted into the created aneurysm. Five sham operated rats were used as controls. The arterial construct was harvested on day 14. The block of tissue was evaluated with histopathology and immunohistochemistry. Prior to sacrifice, the animal underwent 7 T MRI. Statistical analysis was then performed to assess the correlation of signal abnormality with intra-aneurysmal thrombus formation on histology.Results10 rats were used for the experiment. Five rats had implantation of hydrocoils and five of bare platinum coils. There was a statistically significant linear correlation between the intra-aneurysmal thrombus on histology and gradient echo 7 T MRI sequences. There was no correlation demonstrated in the hydrocoil implanted group. No thrombus or abnormal signal was seen in the sham group.ConclusionsIn our experiment, thrombus formation in aneurysms treated with bare platinum coils is well correlated with the presence of abnormal signal on 7 T MRI at 14 days. No correlation was appreciated in the hydrocoil implanted group due to the presence of intra-aneurysmal reactive tissue instead of thrombus.