bluuebunny/crossref_metadata_2025_split huggingface dataset:
Use of Phenotypic and Molecular Serotype Identification Methods To Characterize Previously Nonserotypeable Group B Streptococci
Authors: Kong Fanrong , Lambertsen Lotte Munch , Slotved Hans-Christian , Ko Danny , Wang Hui , Gilbert Gwendolyn L. 
Published: June 2008

ABSTRACT

Among 1,762 isolates of
Streptococcus agalactiae
(group B streptococcus [GBS]), 207 (12%) initially nonserotypeable isolates were tested by improved conventional serotyping methods (Lancefield antigen extraction with 0.1 and 0.2 N HCl, latex agglutination assays, and use of antisera against all known serotypes [Ia, Ib, and II to IX]) and a molecular serotype identification system (multiplex PCR-based reverse line blot [mPCR/RLB] assays targeting serotype-specific sites in the region spanning
cpsH
to
cpsM
). Serotypes were assigned to 71 (34%) of the 207 isolates by using antisera and to 204 (98.5%) of them by mPCR/RLB. Sequencing of a portion of the
cpsE
-
cpsF
-
cpsG
region of 141 persistently nonserotypeable isolates and 1 with discrepant conventional and molecular serotyping results was attempted. Major mutations were identified in 34 isolates (24%), including 11 (8%) from which no amplicons were obtained and 23 (16%) with sequence variation compared with published sequences; of the latter, 21 (15%) were associated with amino acid changes. By contrast, mutations were identified in only 12 (2.3%) of 516 serotypeable isolates for which this region has been sequenced previously. In summary, an improved serotyping scheme allowed serotype identification of more than one-third of the previously nonserotypeable GBS isolates. Molecular serotypes were assigned to almost all of the isolates by mPCR/RLB. Significant mutations (with no amplicons or with associated amino acid changes) were found in the
cpsE
-
cpsF
-
cspG
region of a higher proportion of nonserotypeable than of serotypeable isolates (32/141 versus 8/516;
P
< 0.001), but further investigation is needed to determine the genetic basis for most nonserotypeable GBS isolates.
Optimal Probe Length and Target Location for Electrochemical Detection of Selected Uropathogens at Ambient Temperature
Authors: Mastali Mitra , Babbitt Jane T. , Li Yang , Landaw Elliot M. , Gau Vincent , Churchill Bernard M. , Haake David A. 
Published: June 2008

ABSTRACT

We have previously demonstrated the clinical validity of the rapid detection of uropathogens by use of a DNA biosensor. This assay involves the hybridization of capture and detector probe pairs with bacterial 16S rRNA target molecules to form a DNA-RNA sandwich on the sensor surface. Horseradish peroxidase-conjugated antibody binds to the detector probe to enzymatically amplify the hybridization signal. These previous studies involved the hybridization of bacterial 16S rRNA target sequences with 35-mer oligonucleotide probe pairs at 65°C. Achievement of point-of-care technology will be greatly facilitated by ambient-temperature detection. The purpose of this study was to examine the effects of probe length and target location on signal intensity using hybridization temperatures of 20 to 25°C. Signal intensity was found to vary dramatically with hybridization location in the species-specific bulge region of 16S rRNA helix 18. Probe pairs of as short as 10 nucleotides in length were able to produce a significant electrochemical signal, and signal intensity was correlated with probe length for probes of 10 to 20 nucleotides in length. The sensitivity of the
Escherichia coli
-specific 15-mer probe pairs was approximately 330 cells. These shorter probes allowed differentiation of
Klebsiella pneumoniae
from
Proteus mirabilis
16S rRNA target sequences differing by a single nucleotide. A panel of oligonucleotide probe pairs ranging from 11 to 23 nucleotides in length was able to distinguish among seven groups of urinary tract pathogens. In conclusion, we have developed short oligonucleotide probe pairs for the species-specific identification of uropathogens at ambient temperature by use of an electrochemical sensor.
Universal Primer Set for Amplification and Sequencing of HA
0
Cleavage Sites of All Influenza A Viruses
Authors: Gall Astrid , Hoffmann Bernd , Harder Timm , Grund Christian , Beer Martin 
Published: June 2008

ABSTRACT

Sequence analysis of the endoproteolytic cleavage site within the hemagglutinin (HA) precursor protein HA
0
is fundamental for studies of the molecular biology of influenza A viruses, in particular, for molecular pathotyping of subtype H5 and H7 isolates. A current problem for routine diagnostics is the emergence of new strains of the H5 or H7 subtype or even other subtypes which escape detection by commonly used reverse transcription-PCR (RT-PCR) protocols. Here, the first pan-HA (PanHA) RT-PCR assay targeting the HA
0
cleavage site of influenza A viruses of all 16 HA subtypes is reported. The assay was assessed in comparison to H5 and H7 subtype-specific RT-PCRs for the HA
0
cleavage site and a real-time RT-PCR detecting the M gene. A panel of 92 influenza A viruses was used for validation. Sequence data for influenza A viruses from 32 allantoic fluid samples and 11 diagnostic swab samples of all 16 HA subtypes were generated by direct sequencing of the PanHA RT-PCR products. The results demonstrate that the new PanHA RT-PCR assay—followed by cycle sequencing—can complement existing methods and strengthen the reliability of influenza A virus diagnostics, allowing both molecular pathotyping (H5 and H7) and subtyping (non-H5 or -H7) within a single approach.
Streptobacillus moniliformis
as the Causative Agent in Spondylodiscitis and Psoas Abscess after Rooster Scratches
Authors: Dubois Damien , Robin Frédéric , Bouvier Damien , Delmas Julien , Bonnet Richard , Lesens Olivier , Hennequin Claire 
Published: June 2008

ABSTRACT

We report a case of
Streptobacillus moniliformis
spondylodiscitis accompanied by a psoas abscess in an 80-year-old man scratched by a rooster.
S. moniliformis
was identified from abscess fluid by use of 16S rRNA gene sequencing. After 18 weeks of antimicrobial therapy, the clinical condition of the patient improved.
Geographic Distribution and Antifungal Susceptibility of the Newly Described Species
Candida orthopsilosis
and
Candida metapsilosis
in Comparison to the Closely Related Species
Candida parapsilosis
Authors: Lockhart Shawn R. , Messer Shawn A. , Pfaller Michael A. , Diekema Daniel J. 
Published: June 2008

ABSTRACT

Candida orthopsilosis
and
Candida metapsilosis
are recently described species, having previously been grouped with the more prevalent species
Candida parapsilosis
. Current literature contains very little data pertaining to the distributions and antifungal susceptibilities of these
Candida
species. We determined the species and antifungal susceptibilities of 1,929 invasive clinical isolates from the ARTEMIS antifungal surveillance program collected between 2001 and 2006 and identified as
C. parapsilosis
using Vitek and conventional methods. Of the 1,929 isolates of presumed
C. parapsilosis
tested, 117 (6.1%) were identified as
C. orthopsilosis
and 34 (1.8%) as
C. metapsilosis
. The percentage of presumed
C. parapsilosis
isolates found to be
C. orthopsilosis
varied greatly by region, with the highest percentage (10.9%) from South America and the lowest (0.7%) from Africa. The MIC distributions of the
C. orthopsilosis
and
C. metapsilosis
isolates were statistically significantly lower than those of
C. parapsilosis
for all drugs except fluconazole, for which they were significantly higher (
P
< 0.001 for all). No
C. orthopsilosis
or
C. metapsilosis
isolates were fluconazole resistant, and all were susceptible to caspofungin, anidulafungin, and micafungin.
Effect of Swab Composition and Use of Swabs versus Swab-Containing Skim Milk-Tryptone-Glucose-Glycerol (STGG) on Culture- or PCR-Based Detection of
Streptococcus pneumoniae
in Simulated and Clinical Respiratory Specimens in STGG Transport Medium
Authors: Rubin Lorry G. , Rizvi Atqia , Baer Aryeh 
Published: June 2008

ABSTRACT

To detect
Streptococcus pneumoniae
colonization, the nasopharynx is sampled using a swab placed in skim milk-tryptone-glucose-glycerol (STGG) transport medium, and then the swab specimen or STGG medium is cultured or subjected to PCR. We evaluated the effect of swab composition and compared the sensitivities of detection of culture and PCR using swabs and swab-containing medium. Calcium alginate, Dacron polyester, or rayon-tipped swabs were inoculated with pneumococci or were immersed in nasal wash specimens from children and then placed in STGG medium. Swabs and medium inoculated with pneumococci were cultured. Swabs grew significantly more colonies than medium. The number of colonies cultured from rayon swabs or medium was significantly higher than the number cultured from the calcium alginate swab or medium. The number of colonies from both the Dacron polyester swabs and medium were significantly lower than with either calcium alginate or rayon swabs. When DNA was separately extracted from the calcium alginate swab and medium and subjected to PCR for pneumococcal detection from either
S. pneumoniae
-inoculated swabs or clinical specimens that grew
S. pneumoniae
, the sensitivity was at least 10 times higher using the swab. With Dacron polyester or rayon-tipped swabs, there was no consistent difference between the sensitivity of PCR using swabs and that of PCR using medium. Thus, calcium alginate swabs may be superior to STGG medium for the culture and PCR-based detection of
S. pneumoniae
. For culture, rayon swabs are superior and Dacron polyester swabs are inferior. The sensitivity of the swab and swab-containing medium for culture or PCR detection of
S. pneumoniae
varies with swab composition.
Structure of the Main Protease from a Global Infectious Human Coronavirus, HCoV-HKU1
Authors: Zhao Qi , Li Shuang , Xue Fei , Zou Yilong , Chen Cheng , Bartlam Mark , Rao Zihe 
Published: June 2008

ABSTRACT

The newly emergent human coronavirus HKU1 (HCoV-HKU1) was first identified in Hong Kong in 2005. Infection by HCoV-HKU1 occurs worldwide and causes syndromes such as the common cold, bronchitis, and pneumonia. The CoV main protease (M
pro
), which is a key enzyme in viral replication via the proteolytic processing of the replicase polyproteins, has been recognized as an attractive target for rational drug design. In this study, we report the structure of HCoV-HKU1 M
pro
in complex with a Michael acceptor, inhibitor N3. The structure of HCoV-HKU1 provides a high-quality model for group 2A CoVs, which are distinct from group 2B CoVs such as severe acute respiratory syndrome CoV. The structure, together with activity assays, supports the relative conservation at the P1 position that was discovered by sequencing the HCoV-HKU1 genome. Combined with structural data from other CoV M
pro
s, the HCoV-HKU1 M
pro
structure reported here provides insights into both substrate preference and the design of antivirals targeting CoVs.
Intramembrane Processing by Signal Peptide Peptidase Regulates the Membrane Localization of Hepatitis C Virus Core Protein and Viral Propagation
Authors: Okamoto Kiyoko , Mori Yoshio , Komoda Yasumasa , Okamoto Toru , Okochi Masayasu , Takeda Masatoshi , Suzuki Tetsuro , Moriishi Kohji , Matsuura Yoshiharu 
Published: June 2008

ABSTRACT

Hepatitis C virus (HCV) core protein has shown to be localized in the detergent-resistant membrane (DRM), which is distinct from the classical raft fraction including caveolin, although the biological significance of the DRM localization of the core protein has not been determined. The HCV core protein is cleaved off from a precursor polyprotein at the lumen side of Ala
191
by signal peptidase and is then further processed by signal peptide peptidase (SPP) within the transmembrane region. In this study, we examined the role of SPP in the localization of the HCV core protein in the DRM and in viral propagation. The C terminus of the HCV core protein cleaved by SPP in 293T cells was identified as Phe
177
by mass spectrometry. Mutations introduced into two residues (Ile
176
and Phe
177
) upstream of the cleavage site of the core protein abrogated processing by SPP and localization in the DRM fraction. Expression of a dominant-negative SPP or treatment with an SPP inhibitor, L685,458, resulted in reductions in the levels of processed core protein localized in the DRM fraction. The production of HCV RNA in cells persistently infected with strain JFH-1 was impaired by treatment with the SPP inhibitor. Furthermore, mutant JFH-1 viruses bearing SPP-resistant mutations in the core protein failed to propagate in a permissive cell line. These results suggest that intramembrane processing of HCV core protein by SPP is required for the localization of the HCV core protein in the DRM and for viral propagation.
Effects of Hemagglutinin-Neuraminidase Protein Mutations on Cell-Cell Fusion Mediated by Human Parainfluenza Type 2 Virus
Authors: Tsurudome Masato , Nishio Machiko , Ito Morihiro , Tanahashi Shunsuke , Kawano Mitsuo , Komada Hiroshi , Ito Yasuhiko 
Published: June 2008

ABSTRACTThe monoclonal antibody M1-1A, specific for the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 2 virus (HPIV2), blocks virus-induced cell-cell fusion without affecting the hemagglutinating and neuraminidase activities. F13 is a neutralization escape variant selected with M1-1A and contains amino acid mutations N83Y and M186I in the HN protein, with no mutation in the fusion protein. Intriguingly, F13 exhibits reduced ability to induce cell-cell fusion despite its multistep replication. To investigate the potential role of HPIV2 HN protein in the regulation of cell-cell fusion, we introduced these mutations individually or in combination to the HN protein in the context of recombinant HPIV2. Following infection at a low multiplicity, Vero cells infected with the mutant virus H-83/186, which carried both the N83Y and M186I mutations, remained as nonfused single cells at least for 24 h, whereas most of the cells infected with wild-type virus mediated prominent cell-cell fusion within 24 h. On the other hand, the cells infected with the mutant virus, carrying either the H-83 or H-186 mutation, mediated cell-cell fusion but less efficiently than those infected with wild-type virus. Irrespective of the ability to cause cell-cell fusion, however, every virus could infect all the cells in the culture within 48 h after the initial infection. These results indicated that both the N83Y and M186I mutations in the HN protein are involved in the regulation of cell-cell fusion. Notably, the limited cell-cell fusion by H-83/186 virus was greatly promoted by lysophosphatidic acid, a stimulator of the Ras and Rho family GTPases.
Genetic Characterization of Human Immunodeficiency Virus Type 1 in Elite Controllers: Lack of Gross Genetic Defects or Common Amino Acid Changes
Authors: Miura Toshiyuki , Brockman Mark A. , Brumme Chanson J. , Brumme Zabrina L. , Carlson Jonathan M. , Pereyra Florencia , Trocha Alicja , Addo Marylyn M. , Block Brian L. , Rothchild Alissa C. , Baker Brett M. , Flynn Theresa , Schneidewind Arne , Li Bin , Wang Yaoyu E. , Heckerman David , Allen Todd M. , Walker Bruce D. 
Published: June 2008

ABSTRACTDespite reports of viral genetic defects in persons who control human immunodeficiency virus type 1 (HIV-1) in the absence of antiviral therapy, the extent to which such defects contribute to the long-term containment of viremia is not known. Most previous studies examining for such defects have involved small numbers of subjects, primarily focused on subjects expressing HLA-B57, or have examined single viral genes, and they have focused on cellular proviral DNA rather than plasma viral RNA sequences. Here, we attempted viral sequencing from 95 HIV-1 elite controllers (EC) who maintained plasma viral loads of <50 RNA copies/ml in the absence of therapy, the majority of whom did not express HLA-B57. HIV-1 gene fragments were obtained from 94% (89/95) of the EC, and plasma viral sequences were obtained from 78% (61/78), the latter indicating the presence of replicating virus in the majority of EC. Of 63 persons for whomnefwas sequenced, only three cases ofnefdeletions were identified, and gross genetic defects were rarely observed in other HIV-1 coding genes. In a codon-by-codon comparison between EC and persons with progressive infection, correcting for HLA bias and coevolving secondary mutations, a significant difference was observed at only three codons in Gag, all three of which represented the historic population consensus amino acid at the time of infection. These results indicate that the spontaneous control of HIV replication is not attributable to shared viral genetic defects or shared viral polymorphisms.
Nuclear Import of the Varicella-Zoster Virus Latency-Associated Protein ORF63 in Primary Neurons Requires Expression of the Lytic Protein ORF61 and Occurs in a Proteasome-Dependent Manner
Authors: Walters Matthew S. , Kyratsous Christos A. , Wan Shilin , Silverstein Saul 
Published: June 2008

ABSTRACT
Varicella-zoster virus (VZV) open reading frame (ORF) 63 protein (ORF63p) is one of six VZV ORFs shown to be transcribed and translated in latently infected human dorsal root ganglia. ORF63p accumulates exclusively in the cytoplasm of latently infected sensory neurons, whereas it is both nuclear and cytoplasmic during lytic infection and following reactivation from latency. Here, we demonstrate that infection of primary guinea pig enteric neurons (EN) with an adenovirus expressing ORF63p results in the exclusive cytoplasmic localization of the protein reminiscent of its distribution during latent VZV infection in humans. We show that the addition of the simian virus 40 large-T-antigen nuclear localization signal (NLS) results in the nuclear import of ORF63p in EN and that the ORF63p endogenous NLSs are functional in EN when fused to a heterologous protein. These data suggest that the cytoplasmic localization of ORF63p in EN results from the masking of the NLSs, thus blocking nuclear import. However, the coexpression of ORF61p, a strictly lytic VZV protein, and ORF63p in EN results in the nuclear import of ORF63p in a proteasome-dependent manner, and both ORF63p NLSs are required for this event. We propose that the cytoplasmic localization of ORF63p in neurons results from NLS masking and that the expression of ORF61p removes this block, allowing nuclear import to proceed.
Development of Reverse Genetics Systems for Bluetongue Virus: Recovery of Infectious Virus from Synthetic RNA Transcripts
Authors: Boyce Mark , Celma Cristina C. P. , Roy Polly 
Published: June 2008

ABSTRACT
Bluetongue virus (BTV), an insect-vectored emerging pathogen of both wild ruminants and livestock, has had a severe economic impact in agriculture in many parts of the world. The investigation of BTV replication and pathogenesis has been hampered by the lack of a reverse genetics system. Recovery of infectious BTV is possible by the transfection of permissive cells with the complete set of 10 purified viral mRNAs derived in vitro from transcribing cores (M. Boyce and P. Roy, J. Virol. 81:2179-2186, 2007). Here, we report that in vitro synthesized T7 transcripts, derived from cDNA clones, can be introduced into the genome of BTV using a mixture of T7 transcripts and core-derived mRNAs. The replacement of genome segment 10 and the simultaneous replacement of segments 2 and 5 encoding the two immunologically important outer capsid proteins, VP2 and VP5, are described. Further, we demonstrate the recovery of infectious BTV entirely from T7 transcripts, proving that synthetic transcripts synthesized in the presence of cap analogue can functionally substitute for viral transcripts at all stages of the BTV replication cycle. The generation of BTV with a fully defined genome permits the recovery of mutations in a defined genetic background. The ability to generate specific mutants provides a new tool to investigate the BTV replication cycle as well as permitting the generation of designer vaccine strains, which are greatly needed in many countries.
Differential Requirements of the C Terminus of Nbs1 in Suppressing Adenovirus DNA Replication and Promoting Concatemer Formation
Authors: Lakdawala Seema S. , Schwartz Rachel A. , Ferenchak Kevin , Carson Christian T. , McSharry Brian P. , Wilkinson Gavin W. , Weitzman Matthew D. 
Published: June 2008

ABSTRACT
Adenoviruses (Ad) with the early region E4 deleted (E4-deleted virus) are defective for DNA replication and late protein synthesis. Infection with E4-deleted viruses results in activation of a DNA damage response, accumulation of cellular repair factors in foci at viral replication centers, and joining together of viral genomes into concatemers. The cellular DNA repair complex composed of Mre11, Rad50, and Nbs1 (MRN) is required for concatemer formation and full activation of damage signaling through the protein kinases Ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR). The E4orf3 and E4orf6 proteins expressed from the E4 region of Ad type 5 (Ad5) inactivate the MRN complex by degradation and mislocalization, and prevent the DNA damage response. Here we investigated individual contributions of the MRN complex, concatemer formation, and damage signaling to viral DNA replication during infection with E4-deleted virus. Using virus mutants, short hairpin RNA knockdown and hypomorphic cell lines, we show that inactivation of MRN results in increased viral replication. We demonstrate that defective replication in the absence of E4 is not due to concatemer formation or DNA damage signaling. The C terminus of Nbs1 is required for the inhibition of Ad DNA replication and recruitment of MRN to viral replication centers. We identified regions of Nbs1 that are differentially required for concatemer formation and inhibition of Ad DNA replication. These results demonstrate that targeting of the MRN complex explains the redundant functions of E4orf3 and E4orf6 in promoting Ad DNA replication. Understanding how MRN impacts the adenoviral life cycle will provide insights into the functions of this DNA damage sensor.
Antitumor effect of a transducible fusogenic peptide releasing multiple proapoptotic peptides by caspase-3
Authors: Kwon Mi-Kyung , Nam Ju-Ock , Park Rang-Woon , Lee Byung-Heon , Park Jae-Yong , Byun Young-Ro , Kim Sang-Yoon , Kwon Ick-Chan , Kim In-San 
Published: June 2008

Abstract
We have designed a novel peptide, TK3, composed of three functional domains, a protein transduction domain, a TAT followed by three tandem repeats of a proapoptotic peptide, and a caspase-3 cleavage site, (KLAKLAK)2-DEVD. TK3 was able to transduce into cells and then activate caspase-3, which in turn cleaved TK3 to release additional (KLAKLAK)2 peptides. (KLAKLAK)2 was well transduced by TAT into tumor cells and was able to induce apoptosis in vitro and in vivo. TK3 also induced apoptosis and inhibited angiogenesis in endothelial cells. Further, direct injection of TK3 into established B16F10 melanoma tumors in C57BL/6 mice resulted in almost complete inhibition of the tumor growth. These results suggest that TK3 could be beneficial for the treatment of accessible tumors and used as an adjuvant for cancer therapy. [Mol Cancer Ther 2008;7(6):1514–22]