bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of Novel Real-Time PCR Assays for Detection and Differentiation of Eleven Medically Important
Aspergillus
and
Candida
Species in Clinical Specimens
Authors: Schabereiter-Gurtner Claudia , Selitsch Brigitte , Rotter Manfred L. , Hirschl Alexander M. , Willinger Birgit 
Published: January 2007

ABSTRACT

In the present study, novel real-time PCR assays targeting the fungal ITS2 region were developed for the detection and differentiation of medically important
Aspergillus
species (
Aspergillus fumigatus
,
Aspergillus flavus
,
Aspergillus nidulans
,
Aspergillus niger
, and
Aspergillus terreus
) and
Candida
species (
Candida albicans
,
Candida dubliniensis
,
Candida glabrata
,
Candida krusei
,
Candida parapsilosis
, and
Candida tropicalis
) using a LightCycler instrument. The combination of a group-specific and a universal primer with five
Aspergillus
or six
Candida
species-specific biprobes in one reaction mixture facilitated rapid screening and species differentiation by the characteristic peak melting temperatures of the biprobes. Both assays can be performed either as single assays or simultaneously in the same LightCycler run. The analytical sensitivity using pure cultures and EDTA-anticoagulated blood, cerebrospinal fluid (CSF), and tissue samples spiked with
A. fumigatus
and
C. albicans
cell suspensions was shown to be at least 1 CFU per PCR, corresponding to 5 to 10 CFU/ml blood and 10 CFU/200 μl CSF or 0.02 g tissue. To assess the clinical applicability, 26 respiratory samples, 4 tissue samples from the maxillary sinus, and 1 blood sample were retrospectively tested and real-time PCR results were compared with results from culture, histology, or a galactomannan enzyme-linked immunosorbent assay (ELISA). Twenty samples (64.5%) were both culture positive and positive by real-time PCR. Six samples (19.4%) showed no growth of fungi but were positive by real-time PCR. However, all of the tissue samples were positive by both PCR and histology. The blood sample showed no growth of
Aspergillus
, but aspergillosis was confirmed by positive galactomannan ELISA, histology, and PCR results. The remaining samples (16.1%) were culture and PCR negative; also, no other signs indicating fungal infection were observed. Our data suggest that the
Aspergillus
and
Candida
assays may be appropriate for use in clinical laboratories as simple and rapid screening tests for the most frequently encountered
Aspergillus
and
Candida
species and might become an important tool in the early diagnosis of fungal infections in the future.
Use of Dried Spots of Whole Blood, Plasma, and Mother's Milk Collected on Filter Paper for Measurement of Human Immunodeficiency Virus Type 1 Burden
Authors: Ayele Workenesh , Schuurman Rob , Messele Tsehaynesh , Dorigo-Zetsma Wendelien , Mengistu Yohannes , Goudsmit Jaap , Paxton William A. , de Baar Michel P. , Pollakis Georgios 
Published: January 2007

ABSTRACTWe studied the use of dried spots of bodily fluids (plasma, whole blood, and mother's milk) on filter paper as a means of sample collection and storage for human immunodeficiency virus type 1 (HIV-1) viral load testing under stringent field conditions. Plasma placed directly in lysis buffer, which is customarily used for viral load assays, was used for comparison in all our experiments. Utilizing reconstruction experiments, we demonstrate no statistical differences between viral loads determined for plasma and mother's milk spotted on filter paper and those for the same fluids placed directly in lysis buffer. We found that the addition of whole blood directly to lysis buffer was unreliable and could not be considered a feasible option. However, viral load measurements for whole blood spotted onto filter paper correlated with plasma viral load values for both filter spots and lysis buffer (Pearson correlation coefficients, 0.7706 and 0.8155, respectively). In conclusion, dried spots of plasma, whole blood, or mother's milk provide a feasible means for the collection, storage, and shipment of samples for subsequent viral load measurement and monitoring. Virus material spotted and dried on filter paper is a good inexpensive alternative for collecting patient material to monitor the HIV-1 viral load. Measuring the HIV-1 burden from whole blood dried on filter paper provides a suitable alternative for low-technology settings with limited access to refrigeration, as can be found in sub-Saharan Africa.
Improvement of Hepatitis C Virus (HCV) Genotype Determination with the New Version of the INNO-LiPA HCV Assay
Authors: Bouchardeau Françoise , Cantaloube Jean François , Chevaliez Stéphane , Portal Christine , Razer Annie , Lefrère Jean-Jacques , Pawlotsky Jean Michel , De Micco Philippe , Laperche Syria 
Published: January 2007

ABSTRACTHepatitis C virus (HCV) isolates have been classified into six main genotypes. Genotyping methods, and especially the widely used line probe assay (LiPA), are frequently based on the 5′-untranslated region (5′UTR). However, this region is not appropriate for discriminating HCV strains at the subtype level and for distinguishing many genotype 6 samples from genotype 1. We investigated the capacity of a novel LiPA (Versant HCV Genotype 2.0 assay) based on the simultaneous detection of 5′UTR and Core regions for genotypes 1 and 6 to provide correct HCV genotypes (characterized with a phylogenetic analysis) in a set of HCV strains mainly encountered in Western countries. The improvement was assessed by comparing the results to those obtained with the previous version of the assay. Of the 135 tested samples, 64.7% were concordant for genotype group and subtype with sequencing reference results using the Versant HCV Genotype 2.0 assay versus 37.5% with the previous version. The yield was mainly related to a better characterization of genotype 1, since the accuracy, tested in 62 genotype 1 samples, increased from 45.2% with the first version to 96.8% with the new one. However, this new version necessitates a specific PCR and could no longer be used after 5′UTR PCR used for current HCV infection diagnosis. Moreover, the information provided by 5′UTR hybridization is not reliable for correctly identifying the diversity within genotypes 2 and 4. Thus, the Versant HCV Genotype 2.0 assay remains a useful tool for clinical practice when only the discrimination between major HCV genotypes is necessary.
Kodamaea ohmeri
Isolates from Patients in a University Hospital: Identification, Antifungal Susceptibility, and Pulsed-Field Gel Electrophoresis Analysis
Authors: Lee Jin Sol , Shin Jong Hee , Kim Mi-Na , Jung Sook-In , Park Kyung Hwa , Cho Duck , Kee Seung Jung , Shin Myung Geun , Suh Soon Pal , Ryang Dong Wook 
Published: January 2007

ABSTRACT

Data on clinical isolates of
Kodamaea
(
Pichia
)
ohmeri
, an emerging fungal pathogen, are scarce. Over the past 5 years, we identified yeast isolates from nine patients with fungemia as
K. ohmeri
by using the API 20C system. Here, we reanalyzed these isolates first by sequencing the internal transcribed spacer 2 (ITS2) regions and then by growing the isolates on CHROMagar
Candida
medium and subjecting them to pulsed-field gel electrophoresis (PFGE). Based on their ITS2 sequences, six of the nine isolates were confirmed as
K. ohmeri
, while the others were identified as
Candida haemulonii
(
n
= 2) and
Candida parapsilosis
(
n
= 1). PFGE karyotyping of the
K. ohmeri
isolates revealed similar major bands, and their colonies showed a characteristic color change from pink to blue when grown on CHROMagar
Candida
medium for more than 48 h. For
K. ohmeri
, the ranges of MICs of fluconazole, voriconazole, caspofungin, and micafungin were 2 to 32 μg/ml, 0.03 to 0.5 μg/ml, 0.125 to 0.25 μg/ml, and 0.03 to 0.06 μg/ml, respectively. Restriction endonuclease analysis of genomic NotI-digested DNA (REAG-N) from isolates from different patients produced unique patterns, suggesting that the fungemia had occurred sporadically. This study determined that ITS2 sequence data, PFGE karyotypes, and CHROMagar
Candida
chromogenic culture medium are reliable diagnostic tools for identifying
K. ohmeri
while REAG-N is useful for genotyping the clinical isolates of
K. ohmeri
.
Frequent Intrapatient Recombination between Human Immunodeficiency Virus Type 1 R5 and X4 Envelopes: Implications for Coreceptor Switch
Authors: Mild Mattias , Esbjörnsson Joakim , Fenyö Eva Maria , Medstrand Patrik 
Published: January 2007

ABSTRACT

Emergence of human immunodeficiency virus type 1 (HIV-1) populations that switch or broaden coreceptor usage from CCR5 to CXCR4 is intimately coupled to CD4
+
cell depletion and disease progression toward AIDS. To better understand the molecular mechanisms involved in the coreceptor switch, we determined the nucleotide sequences of 253 V1 to V3
env
clones from 27 sequential HIV-1 subtype B isolates from four patients with virus populations that switch coreceptor usage. Coreceptor usage of clones from dualtropic R5X4 isolates was characterized experimentally. Sequence analysis revealed that 9% of the clones from CXCR4-using isolates had originated by recombination events between R5 and X4 viruses. The majority (73%) of the recombinants used CXCR4. Furthermore, coreceptor usage of the recombinants was determined by a small region of the envelope, including V3. This is the first report demonstrating that intrapatient recombination between viruses with distinct coreceptor usage occurs frequently. It has been proposed that X4 viruses are more easily suppressed by the immune system than R5 viruses. We hypothesize that recombination between circulating R5 viruses and X4 viruses can result in chimeric viruses with the potential to both evade the immune system and infect CXCR4-expressing cells. The broadening in cell tropism of the viral population to include CXCR4-expressing cells would gradually impair the immune system and eventually allow the X4 population to expand. In conclusion, intrapatient recombination between viruses with distinct coreceptor usage may contribute to the emergence of X4 viruses in later stages of infection.
Modest Human Immunodeficiency Virus Coreceptor Function of CXCR3 Is Strongly Enhanced by Mimicking the CXCR4 Ligand Binding Pocket in the CXCR3 Receptor
Authors: Hatse Sigrid , Huskens Dana , Princen Katrien , Vermeire Kurt , Bridger Gary J. , De Clercq Erik , Rosenkilde Mette M. , Schwartz Thue W. , Schols Dominique 
Published: January 2007

ABSTRACT
The chemokine receptor CXCR3 can exhibit weak coreceptor function for several human immunodeficiency virus type 1 (HIV-1) and HIV-2 strains and clinical isolates. These viruses produced microscopically visible cytopathicity in U87.CD4.CXCR3 cell cultures, whereas untransfected (CXCR3-negative) U87.CD4 cells remained uninfected. Depending on the particular virus, the coreceptor efficiency of CXCR3 was 100- to >10,000-fold lower compared to that of CXCR4. A CXCR3 variant carrying the CXCR4 binding pocket was constructed by simultaneous lysine-to-alanine and serine-to-glutamate substitutions at positions 300 and 304 of the CXCR3 receptor. This mutant receptor (CXCR3[K300A, S304E]) showed markedly enhanced HIV coreceptor function compared to the wild-type receptor (CXCR3[WT]). Moreover, the CXCR4 antagonist AMD3100 exhibited antagonistic and anti-HIV activities in U87.CD4.CXCR3[K300A, S304E] cells but not in U87.CD4.CXCR3[WT] cells.
Crystal Structure of Poliovirus 3CD Protein: Virally Encoded Protease and Precursor to the RNA-Dependent RNA Polymerase
Authors: Marcotte Laura L. , Wass Amanda B. , Gohara David W. , Pathak Harsh B. , Arnold Jamie J. , Filman David J. , Cameron Craig E. , Hogle James M. 
Published: January 2007

ABSTRACT

Poliovirus 3CD is a multifunctional protein that serves as a precursor to the protease 3C
pro
and the viral polymerase 3D
pol
and also plays a role in the control of viral replication. Although 3CD is a fully functional protease, it lacks polymerase activity. We have solved the crystal structures of 3CD at a 3.4-Å resolution and the G64S fidelity mutant of 3D
pol
at a 3.0-Å resolution. In the 3CD structure, the 3C and 3D domains are joined by a poorly ordered polypeptide linker, possibly to facilitate its cleavage, in an arrangement that precludes intramolecular proteolysis. The polymerase active site is intact in both the 3CD and the 3D
pol
G64S structures, despite the disruption of a network proposed to position key residues in the active site. Therefore, changes in molecular flexibility may be responsible for the differences in fidelity and polymerase activities. Extensive packing contacts between symmetry-related 3CD molecules and the approach of the 3C domain's N terminus to the VPg binding site suggest how 3D
pol
makes biologically relevant interactions with the 3C, 3CD, and 3BCD proteins that control the uridylylation of VPg during the initiation of viral replication. Indeed, mutations designed to disrupt these interfaces have pronounced effects on the uridylylation reaction in vitro.
Clinical Resistance to Enfuvirtide Does Not Affect Susceptibility ofHuman Immunodeficiency Virus Type 1 to Other Classes of Entry Inhibitors
Authors: Ray Neelanjana , Harrison Jessamina E. , Blackburn Leslie A. , Martin Jeffrey N. , Deeks Steven G. , Doms Robert W. 
Published: January 2007

ABSTRACTThe clinical use of the human immunodeficiency virus (HIV) fusion inhibitor enfuvirtide (ENF) can select for drug-resistant HIV-1 strains bearing mutations in the HR1 region of the viral envelope (Env) protein. We analyzed the properties of multiple Env proteins isolated from five patients who experienced an initial decline in viral load after ENF therapy followed by subsequent rebound due to emergence of ENF-resistant HIV-1. Prior to ENF therapy, each patient harbored genetically and phenotypically diverse Env proteins that used CCR5 and/or CXCR4 to elicit membrane fusion. Coreceptor usage patterns of the Envs isolated from two patients underwent homogenization following ENF therapy, whereas in the other three patients, recombination appeared to allow the introduction of a single HR1 sequence with ENF resistance mutations into phenotypically distinct Env proteins. Analysis of individual Env clones also revealed that prior to ENF therapy, there was sometimes marked heterogeneity in the susceptibility of individual Env proteins to coreceptor inhibitors. After virologic failure, all Envs acquired resistance to ENF but exhibited no consistent change in their sensitivity to the fusion inhibitor T-1249 or to coreceptor inhibitors. In summary, using patient-derived Env proteins, we found that ENF failure was associated with emergence of high-level resistance to ENF due largely to mutations in HR1 but that susceptibility to other entry inhibitors was unaffected, that in these late-stage patients there was greater clonal variability to coreceptor than to fusion inhibitors, and that recombination events in vivo could sometimes restore Env genotypic and phenotypic heterogeneity by introducing drug-resistant gp41 sequences into heterologous gp120 backgrounds.
Spring-Loaded Heptad Repeat Residues Regulate the Expression and Activation of Paramyxovirus Fusion Protein
Authors: Luque Laura E. , Russell Charles J. 
Published: January 2007

ABSTRACT
During viral entry, the paramyxovirus fusion (F) protein fuses the viral
envelope to a cellular membrane. Similar to other class I viral fusion
glycoproteins, the F protein has two heptad repeat regions (HRA and
HRB) that are important in membrane fusion and can be targeted by
antiviral inhibitors. Upon activation of the F protein, HRA refolds
from a spring-loaded, crumpled structure into a coiled coil that
inserts a hydrophobic fusion peptide into the target membrane and binds
to the HRB helices to form a fusogenic hairpin. To investigate how F
protein conformational changes are regulated, we mutated in the Sendai
virus F protein a highly conserved 10-residue sequence in HRA that
undergoes major structural changes during protein refolding. Nine of
the 15 mutations studied caused significant defects in F protein
expression, processing, and fusogenicity. Conversely, the remaining six
mutations enhanced the fusogenicity of the F protein, most likely by
helping spring the HRA coil. Two of the residues that were neither
located at “a” or “d” positions in the
heptad repeat nor conserved among the paramyxoviruses were key
regulators of the folding and fusion activity of the F protein, showing
that residues not expected to be important in coiled-coil formation may
play important roles in regulating membrane fusion. Overall, the data
support the hypothesis that regions in the F protein that undergo
dramatic changes in secondary and tertiary structure between the
prefusion and hairpin conformations regulate F protein expression and
activation.
Two Potentially Important Elements of the Hepatitis B Virus Large Envelope Protein Are Dispensable for the Infectivity of Hepatitis Delta Virus
Authors: Gudima Severin , Meier Anja , Dunbrack Roland , Taylor John , Bruss Volker 
Published: January 2007

ABSTRACT
Previous studies have attempted to clarify the roles of the pre-S1 and pre-S2 domains of the large envelope protein of hepatitis B virus (HBV) in attachment and entry into susceptible cells. Difficulties arise in that these domains contain regions involved in the nucleocapsid assembly of HBV and overlapping with the coding regions of the viral polymerase and RNA sequences required for reverse transcription. Such difficulties can be circumvented with hepatitis delta virus (HDV), which needs the HBV large envelope protein only for infectivity. Thus, mutated HBV envelope proteins were examined for their effects on HDV infectivity. Changing the C-terminal region of pre-S1 critical for HBV assembly allowed the envelopment of HDV and had no effect on infectivity in primary human hepatocytes. Similarly, a deletion of the 12 amino acids of a putative translocation motif (TLM) in pre-S2 had no effect. Thus, these two regions are not necessary for HDV infectivity and, by inference, are not needed for HBV attachment and entry into susceptible cells.
The Ubiquitous Cellular Transcriptional Factor USF Targets the Varicella-Zoster Virus Open Reading Frame 10 Promoter and Determines Virulence in Human Skin Xenografts in SCIDhu Mice In Vivo
Authors: Che Xibing , Berarducci Barbara , Sommer Marvin , Ruyechan William T. , Arvin Ann M. 
Published: January 2007

ABSTRACT
Varicella-zoster virus (VZV) open reading frame 10 (ORF10) is a determinant of virulence in SCIDhu skin xenografts but not in human T cells in vivo. In this analysis of the regulation of ORF10 transcription, we have identified four ORF10-related transcripts, including a major 1.3-kb RNA spanning ORF10 only and three other read-through transcripts. Rapid-amplification-of-cDNA-ends experiments indicated that the 1.3-kb transcript of ORF10 has single initiation and termination sites. In transient expression assays, the ORF10 promoter was strongly stimulated by the major VZV transactivator, IE62. Deletion analyses revealed approximate boundaries for the full ORF10 promoter activity between −75 and −45 and between +5 and −8, relative to the ORF10 transcription start site. The recombinant virus POKA10-Δpro, with the ORF10 promoter deletion, blocked transcription of ORF10 and also of ORF9A and ORF9 mRNAs, whereas expression of read-through ORF9A/9/10 and ORF9/10 transcripts was increased, compensating for the loss of the monocistronic mRNAs. The cellular factor USF bound specifically to its consensus site within the ORF10 promoter and was required for IE62 transactivation, whereas disrupting the predicted TATA boxes or Oct-1 binding elements had no effect. The USF binding site was disrupted in the recombinant virus, POKA10-proΔUSF, and no ORF10 protein was produced. Both ORF10 promoter mutants reduced VZV replication in SCIDhu skin xenografts. These observations provided further evidence of the contribution of the ORF10 protein to VZV pathogenesis in skin and demonstrated that VZV depends upon the cellular transcriptional factor USF to support its virulence in human skin in vivo.
Lumbar Bone Marrow Microcirculation Measurements from Dynamic Contrast-Enhanced Magnetic Resonance Imaging Is a Predictor of Event-Free Survival in Progressive Multiple Myeloma
Authors: Hillengass Jens , Wasser Klaus , Delorme Stefan , Kiessling Fabian , Zechmann Christian , Benner Axel , Kauczor Hans-Ulrich , Ho Anthony D. , Goldschmidt Hartmut , Moehler Thomas M. 
Published: January 2007

Abstract
Purpose: Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) with high temporal resolution enables the detection of microcirculation variables amplitude A and exchange rate constant kep. In this study, the prognostic value of the DCE-MRI variables for overall survival and event-free survival in patients with progressive multiple myeloma was investigated.
Experimental Design: Between 1999 and 2001, 65 patients with progressive or relapse of multiple myeloma requiring therapy were investigated with DCE-MRI of the lumbar spine before start of therapy. The contrast uptake was quantified using a two-compartment model with the output variables amplitude A and exchange rate constant kep reflecting bone marrow microcirculation. The estimated median follow-up was 56 months. Event-free survival and overall survival were investigated for DCE-MRI variables and for established prognosis variables (β2-microglobulin, lactate dehydrogenase, albumin, and age).
Results: Using a multivariate Cox regression model, β2-microglobulin and amplitude A of DCE-MRI were identified as statistically significant prognostic variable of event-free survival with Ps of 0.01 and 0.02, respectively. A statistical correlation of DCE-MRI variables with overall survival could not be found. The multivariate analysis of β2-microglobulin, age, lactate dehydrogenase, and albumin revealed β2-microglobulin as statistically significant prognostic factor for overall survival in this group of patients (P &lt; 0.001).
Conclusions: This analysis identifies contrast-enhanced DCE-MRI variable amplitude A reflecting increased bone marrow microcirculation and angiogenesis as a novel and possibly useful prognostic factor in patients with multiple myeloma. Prospective studies are currently done to further investigate this functional variable for prognosis and stratification of myeloma patients.
Glucocorticoids Modulate Tumor Radiation Response through a Decrease in Tumor Oxygen Consumption
Authors: Crokart Nathalie , Jordan Bénédicte F. , Baudelet Christine , Cron Gregory O. , Hotton Julie , Radermacher Kim , Grégoire Vincent , Beghein Nelson , Martinive Philippe , Bouzin Caroline , Feron Olivier , Gallez Bernard 
Published: January 2007

AbstractPurpose: We hypothesized that glucocorticoids may enhance tumor radiosensitivity by increasing tumor oxygenation (pO2) through inhibition of mitochondrial respiration.Experimental Design: The effect of three glucocorticoids (hydrocortisone, dexamethasone, and prednisolone) on pO2 was studied in murine TLT liver tumors and FSaII fibrosarcomas. At the time of maximum pO2 (tmax, 30 min after administration), perfusion, oxygen consumption, and radiation sensitivity were studied. Local pO2 measurements were done using electron paramagnetic resonance. The oxygen consumption rate of tumor cells after in vivo glucocorticoid administration was measured using high-frequency electron paramagnetic resonance. Tumor perfusion and permeability measurements were assessed by dynamic contrast-enhanced magnetic resonance imaging.Results: All glucocorticoids tested caused a rapid increase in pO2. At tmax, tumor perfusion decreased, indicating that the increase in pO2 was not caused by an increase in oxygen supply. Also at tmax, global oxygen consumption decreased. When irradiation (25 Gy) was applied at tmax, the tumor radiosensitivity was enhanced (regrowth delay increased by a factor of 1.7).Conclusion: These results show the potential usefulness of the administration of glucocorticoids before irradiation.