bluuebunny/crossref_metadata_2025_split huggingface dataset:
Monitoring of Vaccine-Specific Gamma Interferon Induction in Genital Mucosa of Mice by Real-Time Reverse Transcription-PCR
Authors: Revaz Véronique , Debonneville Anne , Bobst Martine , Nardelli-Haefliger Denise 
Published: March 2008

ABSTRACTMonitoring of T-cell responses in genital mucosa has remained a major challenge because of the absence of lymphoid aggregates and the low abundance of T cells. Here we have adapted to genital tissue a sensitive real-time reverse transcription-PCR (TaqMan) method to measure induction of gamma interferon (IFN-γ) mRNA transcription after 3 h of antigen-specific activation of CD8 T cells. For this purpose, we vaccinated C57BL/6 mice subcutaneously with human papillomavirus type 16 L1 virus-like particles and monitored the induction of CD8 T cells specific to the L1165-173H-2Db-restricted epitope. Comparison of the responses induced in peripheral blood mononuclear cells and lymph nodes (LN) by L1-specific IFN-γ enzyme-linked immunospot assay and TaqMan determination of the relative increase in L1-specific IFN-γ mRNA induction normalized to the content of CD8b mRNA showed a significant correlation, despite the difference in the readouts. Most of the cervicovaginal tissues could be analyzed by the TaqMan method if normalization to glyceraldehyde-3-phosphate dehydrogenase mRNA was used and a significant L1-specific IFN-γ induction was found in one-third of the immunized mice. This local response did not correlate with the immune responses measured in the periphery, with the exception of the sacral LN, an LN draining the genital mucosa, where a significant correlation was found. Our data show that the TaqMan method is sensitive enough to detect antigen-specific CD8 T-cell responses in the genital mucosa of individual mice, and this may contribute to elaborate effective vaccines against genital pathogens.
Reciprocal Expression of
ospA
and
ospC
in Single Cells of
Borrelia burgdorferi
Authors: Srivastava Siddharth Y. , de Silva Aravinda M. 
Published: March 2008

ABSTRACT

Outer surface proteins (Osp) A and C of the Lyme disease spirochete (
Borrelia burgdorferi
) are selectively produced and of functional significance in the tick vector and mammalian host, respectively. Some studies indicate a simple, reciprocal relationship where the signals and pathways that turn on
ospC
also turn off
ospA
. Other studies indicate a more complex regulation where many spirochetes produce both proteins and others produce one of the proteins or neither protein. Here, we have used flow cytometry to characterize
ospA
and
ospC
transcript and protein levels in individual bacterial cells grown in culture. The results support a simple, reciprocal model where, at the level of single cells, the transcription of
ospC
is linked to the repression of
ospA
. We also demonstrate that under conditions conducive for OspC production, spirochetes display an “all or none” response, with some cells displaying high levels of
ospC
transcription and others demonstrating little or no transcription. Despite the reciprocal regulation of
ospA
and
ospC
at the single-cell level, we propose that spirochetes display an array of phenotypes due to stochasticity in the pathways that regulate
osp
expression and the slow turnover of outer surface proteins.
An Ortholog of OxyR in
Legionella pneumophila
Is Expressed Postexponentially and Negatively Regulates the Alkyl Hydroperoxide Reductase (
ahpC2D
) Operon
Authors: LeBlanc Jason J. , Brassinga Ann Karen C. , Ewann Fanny , Davidson Ross J. , Hoffman Paul S. 
Published: March 2008

ABSTRACT

Legionella pneumophila
expresses two peroxide-scavenging alkyl hydroperoxide reductase systems (AhpC1 and AhpC2D) that are expressed differentially during the bacterial growth cycle. Functional loss of the postexponentially expressed AhpC1 system is compensated for by increased expression of the exponentially expressed AhpC2D system. In this study, we used an acrylamide capture of DNA-bound complexes (ACDC) technique and mass spectrometry to identify proteins that bind to the promoter region of the
ahpC2D
operon. The major protein captured was an ortholog of OxyR (OxyR
Lp
). Genetic studies indicated that

oxyR
Lp

was an essential gene expressed postexponentially and only partially complemented an
Escherichia coli oxyR
mutant (GS077). Gel shift assays confirmed specific binding of OxyR
Lp
to
ahpC2D
promoter sequences, but not to promoters of
ahpC1
or

oxyR
Lp

; however, OxyR
Lp
weakly bound to
E. coli
OxyR-regulated promoters (
katG
,
oxyR
, and
ahpCF
). DNase I protection studies showed that the OxyR
Lp
binding motif spanned the promoter and transcriptional start sequences of
ahpC2
and that the protected region was unchanged by treatments with reducing agents or hydrogen peroxide (H
2
O
2
). Moreover, the OxyR
Lp
(pBADLp
oxyR
)-mediated repression of an
ahpC2
-
gfp
reporter construct in
E. coli
GS077 (the
oxyR
mutant) was not reversed by H
2
O
2
challenge. Alignments with other OxyR proteins revealed several amino acid substitutions predicted to ablate thiol oxidation or conformational changes required for activation. We suggest these mutations have locked OxyR
Lp
in an active DNA-binding conformation, which has permitted a divergence of function from a regulator of oxidative stress to a cell cycle regulator, perhaps controlling gene expression during postexponential differentiation.
RNase E Regulates the
Yersinia
Type 3 Secretion System
Authors: Yang Jing , Jain Chaitanya , Schesser Kurt 
Published: March 2008

ABSTRACT

Yersinia
spp. use a type 3 secretion system (T3SS) to directly inject six proteins into macrophages, and any impairment of this process results in a profound reduction in virulence. We previously showed that the exoribonuclease polynucleotide phosphorylase (PNPase) was required for optimal T3SS functioning in
Yersinia pseudotuberculosis
and
Yersinia pestis
. Here we report that
Y. pseudotuberculosis
cells with reduced RNase E activity are likewise impaired in T3SS functioning and that phenotypically they resemble Δ
pnp
cells. RNase E does not affect expression levels of the T3SS substrates but instead, like PNPase, regulates a terminal event in the secretion pathway. This similarity, together with the fact that RNase E and PNPase can be readily copurified from
Y. pseudotuberculosis
cell extracts, suggests that these two RNases regulate T3SS activity through a common mechanism. This is the first report that RNase E activity impacts the T3SS as well as playing a more general role in infectivity.
Differential Regulation by Magnesium of the Two MsbB Paralogs ofShigella flexneri
Authors: Goldman Seth R. , Tu Yupeng , Goldberg Marcia B. 
Published: March 2008

ABSTRACTShigella flexneri, a gram-negative enteric pathogen, is unusual in that it contains two nonredundant paralogous genes that encode the myristoyl transferase MsbB (LpxM) that catalyzes the final step in the synthesis of the lipid A moiety of lipopolysaccharide. MsbB1 is encoded on the chromosome, and MsbB2 is encoded on the large virulence plasmid present in all pathogenic shigellae. We demonstrate that myristoyl transferase activity due to MsbB2 is detected in limited magnesium medium, but not in replete magnesium medium, whereas that due to MsbB1 is detected under both conditions. MsbB2 increases overall hexa-acylation of lipid A under limited magnesium conditions. Regulation of MsbB2 by magnesium occurs at the level of transcription and is dependent on the conserved magnesium-inducible PhoPQ two-component regulatory pathway. Direct hexanucleotide repeats within the promoter upstream ofmsbB2were identified as a putative PhoP binding site, and mutations within the repeats led to diminished PhoP-dependent expression of a transcriptional fusion oflacZto this promoter. Thus, the virulence plasmid-encoded paralog ofmsbBis induced under limited magnesium in a PhoPQ-dependent manner. PhoPQ regulates the response of manyEnterobacteriaceaeto environmental signals, which include modifications of lipid A that confer increased resistance of the organism to stressful environments and antimicrobial peptides. The findings reported here are the first example of gene duplication in which one paralog has selectively acquired the mechanism for differential regulation by PhoPQ. Our findings provide molecular insight into the mechanisms by which each of the two MsbB proteins ofS. flexnerilikely contributes to pathogenesis.
Differential Mechanisms of Binding of Anti-Sigma Factors
Escherichia coli
Rsd and Bacteriophage T4 AsiA to
E. coli
RNA Polymerase Lead to Diverse Physiological Consequences
Authors: Sharma Umender K. , Chatterji Dipankar 
Published: March 2008

ABSTRACT

Anti-sigma factors
Escherichia coli
Rsd and bacteriophage T4 AsiA bind to the essential housekeeping sigma factor, σ
70
, of
E. coli
. Though both factors are known to interact with the C-terminal region of σ
70
, the physiological consequences of these interactions are very different. This study was undertaken for the purpose of deciphering the mechanisms by which
E. coli
Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of
E. coli
RNA polymerase, which leads to the inhibition of
E. coli
cell growth to different amounts. It was found that AsiA is the more potent inhibitor of in vivo transcription and thus causes higher inhibition of
E. coli
cell growth. Measurements of affinity constants by surface plasmon resonance experiments showed that Rsd and AsiA bind to σ
70
with similar affinity. Data obtained from in vivo and in vitro binding experiments clearly demonstrated that the major difference between AsiA and Rsd is the ability of AsiA to form a stable ternary complex with RNA polymerase. The binding patterns of AsiA and Rsd with σ
70
studied by using the yeast two-hybrid system revealed that region 4 of σ
70
is involved in binding to both of these anti-sigma factors; however, Rsd interacts with other regions of σ
70
as well. Taken together, these results suggest that the higher inhibition of
E. coli
growth by AsiA expression is probably due to the ability of the AsiA protein to trap the holoenzyme RNA polymerase rather than its higher binding affinity to σ
70
.
The Gene Encoding RNase III in
Streptomyces coelicolor
Is Transcribed during Exponential Phase and Is Required for Antibiotic Production and for Proper Sporulation
Authors: Sello Jason K. , Buttner Mark J. 
Published: March 2008

ABSTRACT

Phenotypic analysis of a constructed RNase III null mutant of
Streptomyces coelicolor
revealed that RNase III is required for both antibiotic production and proper formation of sporulation septa. Transcriptional analysis of the gene encoding RNase III indicated that it is transcribed exclusively during exponential phase as part of a tricistronic message.
Extended Function of Plasmid Partition Genes: the Sop System of Linear Phage-Plasmid N15 Facilitates Late Gene Expression
Authors: Ravin Nikolai V. , Rech Jérôme , Lane David 
Published: March 2008

ABSTRACT

The mitotic stability of the linear plasmid-prophage N15 of
Escherichia coli
depends on a partition system closely related to that of the F plasmid SopABC. The two Sop systems are distinguished mainly by the arrangement of their centromeric SopB-binding sites, clustered in F (
sopC
) and dispersed in N15 (IR1 to IR4). Because two of the N15 inverted repeat (IR) sites are located close to elements presumed (by analogy with phage λ) to regulate late gene expression during the lytic growth of N15, we asked whether Sop partition functions play a role in this process. In N15, a putative Q antiterminator gene is located 6 kb upstream of the probable major late promoter and two intrinsic terminator-like sequences, in contrast to λ, where the Q gene is adjacent to the late promoter. Northern hybridization and
lacZ
reporter activity confirmed the identity of the N15 late promoter (
p52
), demonstrated antiterminator activity of the Q analogue, and located terminator sequences between
p52
and the first open reading frame. Following prophage induction, N15 mutated in IR2 (downstream from gene
Q
) or IR3 (upstream of
p52
) showed a pronounced delay in lysis relative to that for wild-type N15. Expression of
ir3
−
-
p52
::
lacZ
during N15 wild-type lytic growth was strongly reduced relative to the equivalent
ir3
+
fusion. The provision of Q protein and the IR2 and SopAB proteins in
trans
to
ir3
+
-
p52
::
lacZ
increased expression beyond that seen in the absence of any one of these factors. These results indicate that the N15 Sop system has a dual role: partition and regulation of late gene transcription during lytic growth.
Evaluation of a Commercial Direct Fluorescent-Antibody Assay for Human Metapneumovirus in Respiratory Specimens
Authors: Vinh Donald C. , Newby Diane , Charest Hugues , McDonald Jane 
Published: March 2008

ABSTRACT
The performance of a commercially manufactured direct fluorescent-antibody assay kit for human metapneumovirus was compared to that of reverse transcriptase PCR, the current “gold standard.” The kit demonstrated a sensitivity of 95.2%, a specificity of 100%, and an accuracy of 98.9%.
Diagnostic and Clinical Implications of a Nested PCR Specific for Ribosomal DNA of the Feline Lungworm
Aelurostrongylus abstrusus
(Nematoda, Strongylida)
Authors: Traversa Donato , Iorio Raffaella , Otranto Domenico 
Published: March 2008

ABSTRACT

Aelurostrongylus abstrusus
(Nematoda, Strongylida, Metastrongyloidea) is a cosmopolitan parasite of cats and causes severe respiratory distress. Information on the biology and epidemiology of feline aelurostrongylosis is fragmentary, mainly due to the limits inherent in the classical diagnosis. In the present work, a two-step nested PCR based on the use of genetic markers in the second internal transcribed spacer (ITS2) of ribosomal DNA was established for
A. abstrusus
in different biological samples. Characterization of the ITS2 (321 bp of length) revealed a G+C content of 39.5%. To exploit the sequence difference between the ITS2 of
A. abstrusus
and those of other common feline endoparasites, specific primers were designed and tested by PCR for their specificities and sensitivities. The PCR assay was validated on a panel of fecal (i.e., feces, flotation supernatant, and Baermann sediment) and pharyngeal swab samples from cats with known histories of lungworm infections, and it showed a specificity of 100% and a sensitivity of up to 96.6%. Also, the nested PCR was able to identify cats that were actually infected but that tested negative by the classical diagnostic methods. This PCR method was shown to be a powerful tool for the molecular diagnosis of feline aelurostrongylosis, overcoming the constraints of the classical diagnosis. The implications of such a molecular tool for further bioepidemiological studies of both intermediate and definitive hosts have been discussed.
Assessment of
Echinococcus granulosus
Somatic Protoscolex Antigens for Serological Follow-Up of Young Patients Surgically Treated for Cystic Echinococcosis
Authors: Nouir Nadia Ben , Nuñez Sandra , Gianinazzi Christian , Gorcii Mohamed , Müller Norbert , Nouri Abdellatif , Babba Hamouda , Gottstein Bruno 
Published: March 2008

ABSTRACT

Echinococcus granulosus
protoscolex soluble somatic antigens (PSSAs) were assessed for their prognostic value in the serological follow-up of young patients treated for cystic echinococcosis (CE), compared to conventional hydatid fluid (HF) antigen. Based on different clinical courses and outcome of infection, as well as imaging findings, patients were retrospectively classified into two different groups including either cured CE (CCE; i.e., absence of active cysts or presence of inactive cysts, respectively) and noncured CE (NCCE) patients still presenting active cysts at the end of an up to 5-year follow-up period. An immunoglobulin G (IgG)-PSSA enzyme-linked immunosorbent assay (ELISA) showed a gradual decrease in antibody levels in CCE cases, reaching seronegativity in 20% of the cases at least within 5 years postsurgery. In comparison, the conventional IgG-HF ELISA showed a significantly lower progressive decrease in antibody levels, serology becoming negative in only 15% of CCE patients at the endpoint of the follow-up period. Serological analysis of PSSA by immunoblotting yielded an interesting immunoreactive double band of 27 and 28 kDa that, in 15 (75%) of 20 CCE cases, exhibited a rapid decrease and subsequent disappearance of respective antibody reactivities within 3 years postsurgery. Conversely, anti-27- and -28-kDa antibody reactivity strongly persisted until the endpoint of the follow-up period in all of the five NCCE patients. Further analysis of the 27- and 28-kDa doublet by using affinity-purified antibodies showed that the double band was not detectable in HF. Furthermore, a predominantly IgG4 subclass-restricted humoral immune response against the 27- and 28-kDa antigens was demonstrated in seroreactive CE patients. Overall, an anti-27- and -28-kDa response appeared to correlate with cyst activity. In conclusion, PSSA represents a useful candidate to carry out a serologic follow-up of CE subsequent to treatment and deserves further respective evaluation for other age groups of CE patients.
Comparison of the Third Wave Invader Human Papillomavirus (HPV) Assay and the Digene HPV Hybrid Capture 2 Assay for Detection of High-Risk HPV DNA
Authors: Ginocchio C. C. , Barth D. , Zhang F. 
Published: March 2008

ABSTRACT
This study compared the clinical performance of the Digene Hybrid Capture 2 (HC2) assay to that of a prototype Third Wave Invader human papillomavirus (HPV) (IHPV) analyte-specific reagent-based assay for the detection of oncogenic or “high-risk” (HR) HPV DNA using liquid-based cytology specimens. In total, 821 ThinPrep vials were tested using both assays. In accordance with the type-specific probes contained within each test, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for the IHPV assay were 95.9%, 97.6%, 97.5%, and 96.1%, respectively, and those for the HC2 assay were 98.1%, 86.2%, 87.1%, and 97.9%. Overall, the sensitivity and NPV were comparable between the assays, but the IHPV assay demonstrated a better specificity and PPV, since the IHPV assay had fewer false-positive HR HPV results. The incorporation of an internal control to evaluate the cellularity of the test material is an important feature of the IHPV assay and should reduce the risk of false-negative results due to insufficient sample collection rather than the lack of HR HPV DNA. An additional benefit of the IHPV assay was the smaller sample volume required (1 ml versus 4 ml for the HC2 assay).
Characterization of “
Mycobacterium paraffinicum
” Associated with a Pseudo-Outbreak
Authors: Wang Shu-Hua , Mangino Julie E. , Stevenson Kurt , Yakrus Mitchell A. , Cooksey Robert , Butler W. Ray , Healy Mimi , Wise Mark G. , Schlesinger Larry S. , Pancholi Preeti 
Published: March 2008

ABSTRACT

We describe the first “
Mycobacterium paraffinicum
” (unofficial taxon) pseudo-outbreak in a tertiary-care medical center. Fifteen clinical nontuberculous mycobacterium isolates from 10 patients were initially identified by biochemical tests and high-performance liquid chromatography as
Mycobacterium scrofulaceum
. However, further testing by molecular analysis revealed “
M. paraffinicum
.” Epidemiological and environmental investigation determined that the ice machine was the source of the pseudo-outbreak.
Comparison of Serological and Real-Time PCR Assays To Diagnose
Bordetella pertussis
Infection in 2007
Authors: André Philippe , Caro Valerie , Njamkepo Elisabeth , Wendelboe Aaron M. , Van Rie Annelies , Guiso Nicole 
Published: March 2008

ABSTRACT

Bacterial culture for diagnosing pertussis infection has high specificity but poor sensitivity and is slow. Highly sensitive real-time PCR assays and single-serum pertussis serology have been developed to overcome these limitations, but there are few data available on the relative sensitivities and specificities of such assays for pertussis diagnosis. Using data on 195 participants (≥7 years old) from an epidemiological study, we assessed the sensitivity, specificity, and performance (Youden index) for pertussis diagnosis of the pertussis toxin enzyme-linked immunosorbent assay (using single and paired serology) and of real-time PCR assays (using the IS
481
and
ptxA
-Pr targets). All available diagnostic information (clinical and laboratory) was pooled to serve as the gold standard. Single serology was the most efficient diagnostic test (Youden index, 0.57 to 0.58), with relatively high sensitivity (>64%) and high specificity (>90%), independent of the cutoff level. IS
481
PCR performance was superior to that of
ptxA
-Pr PCR, and it was the second-most-efficient tool (Youden index, 0.30). Performing both
ptxA
-Pr and IS
481
PCRs did not improve diagnostic performance. The greatest test efficiency (Youden index, 0.69 to 0.74) was achieved when single-serum serology was used in combination with IS
481
or
ptxA
-Pr PCR or paired serology. Combining single serology with one PCR or paired serology increased the sensitivity with an associated limited decrease in specificity. The most specific tests for diagnosis of pertussis were single serology and
ptxA
-Pr PCR, and the most sensitive diagnostic tool was the combination of IS
481
PCR with single serology.
Genotypic Diversity of Coagulase-Negative Staphylococci Causing Endocarditis: a Global Perspective
Authors: Petti Cathy A. , Simmon Keith E. , Miro Jose M. , Hoen Bruno , Marco Francesc , Chu Vivian H. , Athan Eugene , Bukovski Suzana , Bouza Emilio , Bradley Suzanne , Fowler Vance G. , Giannitsioti Efthymia , Gordon David , Reinbott Porl , Korman Tony , Lang Selwyn , Garcia-de-la-Maria Cristina , Raglio Annibale , Morris Arthur J. , Plesiat Patrick , Ryan Suzanne , Doco-Lecompte Thanh , Tripodi Francesca , Utili Riccardo , Wray Dannah , Federspiel J. Jeffrey , Boisson K. , Reller L. Barth , Murdoch David R. , Woods Christopher W. 
Published: March 2008

ABSTRACT

Coagulase-negative staphylococci (CNS) are important causes of infective endocarditis (IE), but their microbiological profiles are poorly described. We performed DNA target sequencing and susceptibility testing for 91 patients with definite CNS IE who were identified from the International Collaboration on Endocarditis—Microbiology, a large, multicenter, multinational consortium. A hierarchy of gene sequences demonstrated great genetic diversity within CNS from patients with definite endocarditis that represented diverse geographic regions. In particular,
rpoB
sequence data demonstrated unique genetic signatures with the potential to serve as an important tool for global surveillance.
Molecular Characterization ofBartonella vinsoniisubsp.berkhoffiiGenotype III
Authors: Cadenas Maria B. , Bradley Julie , Maggi Ricardo G. , Takara Matt , Hegarty Barbara C. , Breitschwerdt Edward B. 
Published: March 2008

ABSTRACTThe molecular characterization of aBartonella vinsoniisubsp.berkhoffiigenotype III strain (NCSU strain 06-CO1) isolated from the blood of a military working dog diagnosed with endocarditis is reported in this study. Several genes were amplified and sequenced for comparative sequence similarity with other strains.