bluuebunny/crossref_metadata_2025 huggingface dataset:
Evolutionary Formation of New Centromeres in Macaque
Authors: Ventura Mario , Antonacci Francesca , Cardone Maria Francesca , Stanyon Roscoe , D'Addabbo Pietro , Cellamare Angelo , Sprague L. James , Eichler Evan E. , Archidiacono Nicoletta , Rocchi Mariano
Published: April 2007

A systematic fluorescence in situ hybridization comparison of macaque and human synteny organization disclosed five additional macaque evolutionary new centromeres (ENCs) for a total of nine ENCs. To understand the dynamics of ENC formation and progression, we compared the ENC of macaque chromosome 4 with the human orthologous region, at 6q24.3, that conserves the ancestral genomic organization. A 250-kilobase segment was extensively duplicated around the macaque centromere. These duplications were strictly intrachromosomal. Our results suggest that novel centromeres may trigger only local duplication activity and that the absence of genes in the seeding region may have been important in ENC maintenance and progression.
Time-Kill and Synergism Studies of Ceftobiprole against
Enterococcus faecalis
, Including β-Lactamase-Producing and Vancomycin-Resistant Isolates
Authors: Arias Cesar A. , Singh Kavindra V. , Panesso Diana , Murray Barbara E.
Published: April 2007

Ceftobiprole (BAL9141) is an investigational cephalosporin with broad in vitro activity against gram-positive cocci, including enterococci. Ceftobiprole MICs were determined for 93 isolates of
Enterococcus faecalis
(including 16 β-lactamase [Bla] producers and 17 vancomycin-resistant isolates) by an agar dilution method following the Clinical and Laboratory Standards Institute recommendations. Ceftobiprole MICs were also determined with a high inoculum concentration (10
7
CFU/ml) for a subset of five Bla producers belonging to different previously characterized clones by a broth dilution method. Time-kill and synergism studies (with either streptomycin or gentamicin) were performed with two β-lactamase-producing isolates (TX0630 and TX5070) and two vancomycin-resistant isolates (TX2484 [VanB] and TX2784 [VanA]). The MICs of ceftobiprole for 50 and 90% of the isolates tested were 0.25 and 1 μg/ml, respectively. All Bla producers and vancomycin-resistant isolates were inhibited by concentrations of ≤1 and ≤4 μg/ml, respectively, at the standard inoculum concentration. Ceftobiprole MICs at a high inoculum concentration for a subset of five Bla
+
E. faecalis
isolates were ≤1 μg/ml. Bactericidal activity was observed against four isolates tested at concentrations as low as 1 μg/ml regardless of the production of β-lactamase or vancomycin resistance. A combination of ceftobiprole (0.5 μg/ml) and streptomycin (25 μg/ml) was synergistic against Bla
+
TX0630 and TX5070. Ceftobiprole (0.5 μg/ml) plus gentamicin (10 μg/ml) was synergistic against VanB isolate TX2484 and showed enhanced killing, but not synergism, against TX2784 (VanA), despite the absence of high-level resistance to gentamicin. In conclusion, ceftobiprole exhibited good in vitro activity against
E. faecalis
, including Bla
+
and vancomycin-resistant strains, and exhibited synergism with aminoglycosides against selected isolates.
bla
VIM-2
-Harboring Integrons Isolated in India, Russia, and the United States Arise from an Ancestral Class 1 Integron Predating the Formation of the 3′ Conserved Sequence
Authors: Toleman Mark A. , Vinodh Hemalatha , Sekar Uma , Kamat Vijaylakshmi , Walsh Timothy R.
Published: April 2007

The metallo-β-lactamase gene
bla
VIM-2
was identified in a strain of
Pseudomonas aeruginosa
isolated in India. The integron encoding
bla
VIM-2
was virtually identical to those recently found in the United States and Russia. These unusual structures are likely to have arisen from an ancestral integron predating the formation of the 3′ conserved sequence.
The L80I Substitution in the Reverse Transcriptase Domain of the Hepatitis B Virus Polymerase Is Associated with Lamivudine Resistance and Enhanced Viral Replication In Vitro
Authors: Warner Nadia , Locarnini Stephen , Kuiper Michael , Bartholomeusz Angeline , Ayres Anna , Yuen Lilly , Shaw Tim
Published: April 2007

Long-term lamivudine (LMV) treatment of chronic hepatitis B almost inevitably engenders viral resistance. Mutations that result in the replacement of the methionine at position 204 of the deoxynucleoside triphosphate-binding site of the hepatitis B virus (HBV) reverse transcriptase (rt) by isoleucine, valine, or (rarely) serine (rtM204I/V/S) confer high-level resistance to LMV but reduce replication efficiency. The subsequent selection or coselection of secondary mutations that partially restore replication efficiency is common and may influence drug resistance. Genotyping has shown that LMV treatment can select for HBV rtL80V/I mutants, but their prevalence and phenotype have not been documented. Analysis of a large sequence database revealed that rtL80V/I occurred almost exclusively in association with LMV resistance, and 85% of these isolates encoded rtL80I. Coselection of rtL80V/I occurred in 46% of isolates in which LMV resistance was attributable to rtM204I but only 9% of those in which resistance was attributable to rtM204V. Moreover, rtL80V/I did not occur in HBV genotype A isolates but occurred at similar frequencies in genotype B, C, and D isolates. In vitro phenotyping showed that although the rtL80I mutant by itself replicated less efficiently and was hypersensitive to LMV compared to the replication efficiency and sensitivity of its wild-type parent, the presence of rtL80I enhanced the replication efficiency of rt204I/V mutants without significantly affecting LMV resistance. Molecular modeling revealed that rt80 does not interact directly with the enzyme's substrates. Collectively, these results suggest that coselection of rtL80V/I and rtM204I/V occurs because the former compensates for the loss of replication efficiency associated with the acquisition of LMV resistance, particularly in the case of rtM204I.
Production and Evaluation of Reagents for Detection of
Histoplasma capsulatum
Antigenuria by Enzyme Immunoassay
Authors: Lindsley Mark D. , Holland Heather L. , Bragg Sandra L. , Hurst Steven F. , Wannemuehler Kathleen A. , Morrison Christine J.
Published: April 2007

The detection of urinary
Histoplasma capsulatum
polysaccharide antigen (HPA) by enzyme immunoassay (EIA) has proven useful for the presumptive diagnosis of histoplasmosis in AIDS patients. Assay limitations include (i) detection of a largely uncharacterized antigen and (ii) difficulty in reproducibly generating antibodies for use in the EIA. To improve antibody production for use in this test and to better understand the antigen being detected, we compared rabbit antibodies elicited using various immunization schedules, routes, and
H. capsulatum
-derived antigens. Antibodies were evaluated by EIA for their ability to detect purified
H. capsulatum
C antigen (C-Ag) and antigenuria. Reported as enzyme immunoassay (EI) units (the
A
450
with antigen divided by the
A
450
without antigen), results demonstrated that intravenous immunization of rabbits with whole, killed yeast-phase cells (yeast-i.v. regimen) produced antibodies giving the highest EI values in the C-Ag EIA (mean EI units ± standard deviation, 14.9 ± 0.6 versus 6.4 ± 0.4 for rabbits immunized with C-Ag versus 2.4 ± 0.3 for all other regimens combined). Yeast-i.v. antibodies were highly sensitive for the detection of antigenuria in patients with histoplasmosis, as shown by the following results: 12/12 patients compared to 10/12, 6/12, 3/12, and 3/12, respectively, for antibodies from rabbits immunized with (i) C-Ag; (ii) whole, killed yeast-phase cells administered subcutaneously and intramuscularly; (iii) yeast-phase culture filtrates; and (iv) HPA-positive urine. Rabbits immunized using the yeast-i.v. regimen also gave higher peak antibody titers than rabbits immunized by any other regimen (
P
< 0.03), and their antibodies were most comparable in reactivity to antibodies produced for use in the standard HPA-EIA test (
P
< 0.001). Therefore, rabbits immunized using the yeast-i.v. regimen produced the most sensitive antibodies with the highest titers for detection of C-Ag and antigenuria in histoplasmosis patients.
Performance of Two Commercial Immunochromatographic Assays for Rapid Detection of Antibodies Specific to Human Immunodeficiency Virus Types 1 and 2 in Serum and Urine Samples in a Rural Community-Based Research Setting (Rakai, Uganda)
Authors: Kagulire S. C. , Stamper P. D. , Opendi P. , Nakavuma J. L. , Mills L. A. , Makumbi F. , Gray R. H. , Serwadda D. , Reynolds S. J.
Published: April 2007

Rapid detection of human immunodeficiency virus (HIV) antibodies is of great importance in developing and developed countries to diagnose HIV infections quickly and at low cost. In this study, two new immunochromatographic rapid tests for the detection of HIV antibodies (Aware HIV-1/2 BSP and Aware HIV-1/2 U; Calypte Biomedical Corporation) were evaluated in rural Africa to determine the tests' performance and comparability to commercially available conventional enzyme immunoassay (EIA) and Western blot (WB) tests. This prospective study was conducted from March 2005 through May 2005 using serum and urine from respondents in the Rakai Community Cohort Survey. Nine hundred sixty-three serum samples were tested with the Aware blood rapid assay (Aware-BSP) and compared to two independent EIAs for HIV plus confirmatory Calypte WB for any positive EIAs. The sensitivity of Aware-BSP was 98.2%, and the specificity was 99.8%. Nine hundred forty-two urine samples were run using the Aware urine assay (Aware-U) and linked to blood sample results for analysis. The sensitivity of Aware-U was 88.7% and specificity was 99.9% compared to blood EIAs confirmed by WB analysis. These results support the adoption of the Aware-BSP rapid test as an alternative to EIA and WB assays for the diagnosis of HIV in resource-limited settings. However, the low sensitivity of the Aware-U assay with its potential for falsely negative HIV results makes the urine assay less satisfactory.
Noninferiority of Antibody Response to Human Papillomavirus Type 16 in Subjects Vaccinated with Monovalent and Quadrivalent L1 Virus-Like Particle Vaccines
Authors: Garland Suzanne M. , Steben Marc , Hernandez-Avila Mauricio , Koutsky Laura A. , Wheeler Cosette M. , Perez Gonzalo , Harper Diane M. , Leodolter Sepp , Tang Grace W. K. , Ferris Daron G. , Esser Mark T. , Vuocolo Scott C. , Nelson Micki , Railkar Radha , Sattler Carlos , Barr Eliav
Published: April 2007

The incorporation of multiple antigens into a single human papillomavirus (HPV) vaccine may induce immune interference. To evaluate whether interference occurs when HPV type 16 (HPV16) virus-like particles are combined in a multivalent vaccine, we conducted a study to evaluate anti-HPV16 responses among subjects receiving three-dose regimens of either a monovalent HPV16 vaccine or a quadrivalent HPV (types 6, 11, 16, and 18) vaccine.
Evaluation of an Enzyme Immunoassay for Detection of Immunoglobulin M Antibodies to West Nile Virus and the Importance of Background Subtraction in Detecting Nonspecific Reactivity
Authors: Rawlins Mindy L. , Swenson Erica M. , Hill Harry R. , Litwin Christine M.
Published: April 2007

Since the introduction of West Nile virus (WNV) in the United States in 1999, several assays have become commercially available to detect antibodies against WNV. Capture enzyme-linked immunosorbent assays (ELISAs) for the detection of WNV-specific immunoglobulin M (IgM) have been approved by the Food and Drug Administration for clinical testing and are available from Focus Diagnostics and PanBio, Inc. The Focus Diagnostics IgM capture ELISA utilizes a background subtraction protocol in order to detect nonspecific reactivity due to rheumatoid factor, heterophile antibodies, or other interfering substances. A background subtraction procedure is not currently recommended for the PanBio IgM capture ELISA. In previous experiments, we determined the agreement, sensitivity, and specificity of the PanBio first-generation IgM capture ELISA compared to an immunofluorescence assay and the Centers for Disease Control and Prevention's IgM capture ELISA. The PanBio assay has since been reformulated to improve the specificity of the assay. We evaluated the reformulated PanBio assay with and without an antigen subtraction procedure and compared the results to the Focus IgM capture ELISA. Agreement, sensitivity, and specificity of the PanBio assay were, respectively, 85%, 95%, and 76% without the subtraction protocol and 94%, 95%, and 93% with the subtraction protocol. In general, when the subtraction protocol was applied to the PanBio IgM capture ELISA, there was a reduction in some, but not all, false-positive results. We suggest that all WNV IgM assays be standardized with a procedure such as background subtraction to eliminate nonspecific reactivity that may cause false-positive results.
The HlyU Protein Is a Positive Regulator of
rtxA1
, a Gene Responsible for Cytotoxicity and Virulence in the Human Pathogen
Vibrio vulnificus
Authors: Liu Moqing , Alice Alejandro F. , Naka Hiroaki , Crosa Jorge H.
Published: April 2007

Vibrio vulnificus
is an opportunistic human pathogen that preferentially infects compromised iron-overloaded patients, causing a fatal primary septicemia with very rapid progress, resulting in a high mortality rate. In this study we determined that the HlyU protein, a virulence factor in
V. vulnificus
CMCP6, up-regulates the expression of VV20479, a homologue of the
Vibrio cholerae
RTX (repeats in toxin) toxin gene that we named
rtxA1
. This gene is part of an operon together with two other open reading frames, VV20481 and VV20480, that encode two predicted proteins, a peptide chain release factor 1 and a hemolysin acyltransferase, respectively. A mutation in
rtxA1
not only contributes to the loss of cytotoxic activity but also results in a decrease in virulence, whereas a deletion of VV20481 and VV20480 causes a slight decrease in virulence but with no effect in cytotoxicity. Activation of the expression of the
rtxA1
operon by HlyU occurs at the transcription initiation level by binding of the HlyU protein to a region upstream of this operon.
RyhB, an Iron-Responsive Small RNA Molecule, Regulates
Shigella dysenteriae
Virulence
Authors: Murphy Erin R. , Payne Shelley M.
Published: April 2007

Regulation of bacterial gene expression by small RNA (sRNA) molecules is an increasingly recognized phenomenon but one that is not yet fully understood. We show that the sRNA RyhB suppresses several virulence-associated phenotypes of
Shigella dysenteriae
, a causative agent of bacillary dysentery in humans. The virulence genes repressed by
S. dysenteriae
RyhB include those encoding the type III secretion apparatus, its secreted effectors, and specific chaperones. Suppression of
Shigella
virulence occurs via RyhB-dependent repression of the transcriptional activator VirB, leading to reduced expression of genes within the VirB regulon. Efficient repression of
virB
is mediated by a single-stranded region of RyhB that is distinct from the region required for repression of
Shigella sodB
. Regulation of
virB
by RyhB implicates iron as an environmental factor contributing to the complex regulation of
Shigella
virulence determinants.
Heterogeneity in the Activity of MexicanHelicobacter pyloriStrains in Gastric Epithelial Cells and Its Association with Diversity in thecagAGene
Authors: Reyes-Leon Adriana , Atherton John C. , Argent Richard H. , Puente J. L. , Torres J.
Published: April 2007

Helicobacter pyloriCagA is translocated into gastric epithelial cells by a type IV secretion system and interacts with the Src homology 2 phosphatase, altering cell morphology. Multiple EPIYA motifs in CagA are associated with increased activity in cells and with gastric cancer. The aim of this work was to study the heterogeneity in activity in cells of multipleH. pylorisingle colonies isolated from a single patient and its association with polymorphism incagA. The presence ofcagA,cagE,cagT, andcag10was studied with 318H. pyloriisolates from the antra and corpora of 18 patients. AGS gastric epithelial cells were infected with 75 isolates, and interleukin-8 (IL-8) secretion, cytoskeletal changes, CagA translocation, and tyrosine phosphorylation were measured. ThecagA3′-variable region was sequenced for 30 isolates to determine the number and types of EPIYA motifs. Isolates from an individual stomach were usually genetically related and had quantitatively similar phenotypic effects on cells (IL-8 induction and cytoskeletal changes). However, strains from different patients with similar CagA EPIYA motif patterns varied widely in these phenotypes. Among isolates with an EPIYA-ABC pattern, the phenotype was variable: IL-8 induction ranged from 200 to 1,200 pg/ml, and morphological changes occurred in 20 to 70% of cells. In several cases,cagAsequence diversity appeared to explain the lack of CagA activity, as isolates with an EPIYA-ACC pattern or a modified B motif had reduced cell activity.cagpathogenicity island-positiveH. pyloriisolates displayed a high level of heterogeneity in the capacity to induce IL-8 secretion and morphological changes; an absent or modified B motif was associated with low activity.
Inhibition of
Enterococcus faecium
Adherence to Collagen by Antibodies against High-Affinity Binding Subdomains of Acm
Authors: Nallapareddy Sreedhar R. , Sillanpää Jouko , Ganesh Vannakambadi K. , Höök Magnus , Murray Barbara E.
Published: April 2007

Strains of
Enterococcus faecium
express a cell wall-anchored protein, Acm, which mediates adherence to collagen. Here, we (i) identify the minimal and high-affinity binding subsegments of Acm and (ii) show that anti-Acm immunoglobulin Gs (IgGs) purified against these subsegments reduced
E. faecium
TX2535 strain collagen adherence up to 73 and 50%, respectively, significantly more than the total IgGs against the full-length Acm A domain (28%) (
P
< 0.0001). Blocking Acm adherence with functional subsegment-specific antibodies raises the possibility of their use as therapeutic or prophylactic agents.
Multiplexed Identification of Blood-Borne Bacterial Pathogens by Use of a Novel 16S rRNA Gene PCR-Ligase Detection Reaction-Capillary Electrophoresis Assay
Authors: Pingle Maneesh R. , Granger Kathleen , Feinberg Philip , Shatsky Rebecca , Sterling Bram , Rundell Mark , Spitzer Eric , Larone Davise , Golightly Linnie , Barany Francis
Published: April 2007

We have developed a novel high-throughput PCR-ligase detection reaction-capillary electrophoresis (PCR-LDR-CE) assay for the multiplexed identification of 20 blood-borne pathogens (Staphylococcus epidermidis,Staphylococcus aureus,Bacillus cereus,Enterococcus faecalis,Enterococcus faecium,Listeria monocytogenes,Streptococcus pneumoniae,Streptococcus pyogenes,Streptococcus agalactiae,Escherichia coli,Klebsiella pneumoniae,Haemophilus influenzae,Pseudomonas aeruginosa,Acinetobacter baumannii,Neisseria meningitidis,Bacteroides fragilis,Bacillus anthracis,Yersinia pestis,Francisella tularensis, andBrucella abortus), the last four of which are biothreat agents. The method relies on the amplification of two regions within the bacterial 16S rRNA gene, using universal PCR primers and querying the identity of specific single-nucleotide polymorphisms within the amplified regions in a subsequent LDR. The ligation products vary in color and size and are separated by CE. Each organism generates a specific pattern of ligation products, which can be used to distinguish the pathogens using an automated software program we developed for that purpose. The assay has been verified on 315 clinical isolates and demonstrated a detection sensitivity of 98%. Additionally, 484 seeded blood cultures were tested, with a detection sensitivity of 97.7%. The ability to identify geographically variant strains of the organisms was determined by testing 132 isolates obtained from across the United States. In summary, the PCR-LDR-CE assay can successfully identify, in a multiplexed fashion, a panel of 20 blood-borne pathogens with high sensitivity and specificity.
Evaluation of Moxalactam with the BD Phoenix System for Detection of Methicillin Resistance in Coagulase-Negative Staphylococci
Authors: Pupin H. , Renaudin H. , Join-Lambert O. , Bébéar C. , Mégraud F. , Lehours P.
Published: April 2007

The performance of moxalactam with the BD Phoenix system for the detection of methicillin resistance in coagulase-negative staphylococci was evaluated by use of a collection of 186 strains. Moxalactam was a better drug as an indicator of methicillin resistance for
mecA
-positive strains than oxacillin and cefoxitin were. For strains other than
Staphylococcus saprophyticus
, a moxalactam MIC >16 μg/ml was indicative of methicillin resistance.
Nocardial Cerebral Abscess Associated with Mycetoma, Pneumonia, and Membranoproliferative Glomerulonephritis
Authors: Elmaci Ilhan , Senday Durmus , Silav Gökalp , Ekenel Figen , Balak Naci , Ayan Erdogan , Akinci Murat , Işik Nejat , Yazici Saadet
Published: April 2007

Nocardial brain abscesses remain a clinical challenge. We successfully treated a patient with nocardial brain abscess, mycetoma, pneumonia, and glomerulonephritis. Nocardial soft tissue involvement, mycetoma, is well known. However, the fact that actinomycetoma can metastasize may not be as well appreciated. The association between nocardiosis and glomerulonephritis should be better clarified.
Evaluation of Molecular Typing Methods in Characterizing a European Collection of Epidemic Methicillin-Resistant
Staphylococcus aureus
Strains: the HARMONY Collection
Authors: Cookson Barry D. , Robinson D. Ashley , Monk Alastair B. , Murchan Stephen , Deplano Ariane , de Ryck Rafaël , Struelens Marc J. , Scheel Christina , Fussing Vivian , Salmenlinna Saara , Vuopio-Varkila Jaana , Cuny Christina , Witte Wolfgang , Tassios Panayotis T. , Legakis Nikolas J. , van Leeuwen Willem , van Belkum Alex , Vindel Anna , Garaizar Javier , Haeggman Sara , Olsson-Liljequist Barbro , Ransjo Ulrika , Muller-Premru Manica , Hryniewicz Waleria , Rossney Angela , O'Connell Brian , Short Benjamin D. , Thomas Jonathan , O'Hanlon Simon , Enright Mark C.
Published: April 2007

We analyzed a representative sample of methicillin-resistant
Staphylococcus aureus
(MRSA) from 11 European countries (referred to as the HARMONY collection) using three molecular typing methods used within the HARMONY group to examine their usefulness for large, multicenter MRSA surveillance networks that use these different laboratory methodologies. MRSA isolates were collected based on their prevalence in each center and their genetic diversity, assessed by pulsed-field gel electrophoresis (PFGE). PFGE groupings (≤3 bands difference between patterns) were compared to those made by sequencing of the variable repeats in the protein A gene
spa
and clonal designations based on multilocus sequence typing (MLST), combined with PCR analysis of the staphylococcal chromosome cassette containing the
mec
genes involved in methicillin resistance (SCC
mec
). A high level of discrimination was achieved using each of the three methodologies, with discriminatory indices between 89.5% and 91.9% with overlapping 95% confidence intervals. There was also a high level of concordance of groupings made using each method. MLST/SCC
mec
typing distinguished 10 groups containing at least two isolates, and these correspond to the majority of nosocomial MRSA clones described in the literature. PFGE and
spa
typing resolved 34 and 31 subtypes, respectively, within these 10 MRSA clones, with each subtype differing only slightly from the most common pattern using each method. The HARMONY group has found that the methods used in this study differ in their availability and affordability to European centers involved in MRSA surveillance. Here, we demonstrate that the integration of such technologies is achievable, although common protocols (such as we have developed for PFGE) may also be important, as is the use of centralized Internet sites to facilitate data analysis. PFGE and
spa
-typing data from analysis of MRSA isolates from the many centers that have access to the relevant equipment can be compared to reference patterns/sequences, and clonal designations can be made. In the majority of cases, these will correspond to those made by the (more expensive) method of choice—MLST/SCC
mec
typing—and these alternative methods can therefore be used as frontline typing systems for multicenter surveillance of MRSA.
Rapid and Reliable Method for Quantification of
Mycobacterium paratuberculosis
by Use of the BACTEC MGIT 960 System
Authors: Shin Sung Jae , Han Jun Hee , Manning Elizabeth J. B. , Collins Michael T.
Published: April 2007

A simple method for the enumeration of viable
Mycobacterium paratuberculosis
cells was developed and evaluated using the MGIT 960 culture system. For each of 12
M. paratuberculosis
strains isolated from either cattle or humans, single-cell suspensions of
M. paratuberculosis
cells were adjusted to an optical density at 600 nm of 1.00 (10
7.6
to 10
8.2
cells/ml), and serial dilutions were prepared. Standard curves were established by relating the MGIT time-to-detection data to the log
10
CFU for these suspensions using standard plate counting and BACTEC 460 results as reference methods. Universal and strain-specific standard quantification curves were generated. A one-phase exponential decay equation best fit the universal standard curve and strain-specific curves (
R
2
of 0.96 and >0.99, respectively). Two subgroups within the universal curves were distinguished: one for laboratory-adapted strains and the other for recently isolated low-passage bovine strains. The predictive errors for log
10
estimations using the universal standard curve, each subgroup's standard curve, and strain-specific curves were ±0.87, ±0.45, and ±0.31 log
10
units, respectively. CFU estimations by all three standard curves were highly reproducible, regardless of the
M. paratuberculosis
strain or inoculum volume. In comparison with the previously described BACTEC 460
M. paratuberculosis
counting method, quantification with MGIT 960 was less expensive, more rapid, more accurate, and more sensitive (<10 CFU). This MGIT counting method has broad applications for studies requiring the quantification of viable
M. paratuberculosis
cells, such as drug susceptibility testing or environmental survival studies.
Rotavirus Infection of Infant and Young Adult Nonobese Diabetic Mice Involves Extraintestinal Spread and Delays Diabetes Onset
Authors: Graham Kate L. , O'Donnell Joanne A. , Tan Yan , Sanders Natalie , Carrington Emma M. , Allison Janette , Coulson Barbara S.
Published: April 2007

Rotaviruses have been implicated as a possible viral trigger for exacerbations in islet autoimmunity, suggesting they might modulate type 1 diabetes development. In this study, the ability of rotavirus strain RRV to infect the pancreas and affect insulitis and diabetes was examined in nonobese diabetic (NOD) mice, an experimental model of type 1 diabetes. Mice were inoculated either orally or intraperitoneally as infants or young adults. In infant mice inoculated orally, rotavirus antigen was detected in pancreatic macrophages outside islets and infectious virus was found in blood cells, pancreas, spleen, and liver. Extraintestinal RRV spread and pancreatic presence of infectious virus also occurred in intraperitoneally inoculated infant and adult mice. The initiation of insulitis was unaltered by infection. The onset of diabetes was delayed in infant mice inoculated orally and infant and adult mice inoculated intraperitoneally. In contrast, adult mice inoculated orally showed no evidence of pancreatic RRV, the lowest rate of detectable RRV replication, and no diabetes modulation. Thus, the ability of RRV infection to modulate diabetes development in infant and young adult NOD mice was related to the overall extent of detectable virus replication and the presence of infectious virus extraintestinally, including in the pancreas. These studies show that RRV infection of infant and young adult NOD mice provides significant protection against diabetes. As these findings do not support the hypothesis that rotavirus triggers autoimmunity related to type 1 diabetes, further research is needed to resolve this issue.
Potent Rescue of Human Immunodeficiency Virus Type 1 Late Domain Mutants by ALIX/AIP1 Depends on Its CHMP4 Binding Site
Authors: Usami Yoshiko , Popov Sergei , Göttlinger Heinrich G.
Published: April 2007

The release of human immunodeficiency virus type 1 (HIV-1) and of other retroviruses from certain cells requires the presence of distinct regions in Gag that have been termed late assembly (L) domains. HIV-1 harbors a PTAP-type L domain in the p6 region of Gag that engages an endosomal budding machinery through Tsg101. In addition, an auxiliary L domain near the C terminus of p6 binds to ALIX/AIP1, which functions in the same endosomal sorting pathway as Tsg101. In the present study, we show that the profound release defect of HIV-1 L domain mutants can be completely rescued by increasing the cellular expression levels of ALIX and that this rescue depends on an intact ALIX binding site in p6. Furthermore, the ability of ALIX to rescue viral budding in this system depended on two putative surface-exposed hydrophobic patches on its N-terminal Bro1 domain. One of these patches mediates the interaction between ALIX and the ESCRT-III component CHMP4B, and mutations which disrupt the interaction also abolish the activity of ALIX in viral budding. The ability of ALIX to rescue a PTAP mutant also depends on its C-terminal proline-rich domain (PRD), but not on the binding sites for Tsg101, endophilin, CIN85, or for the newly identified binding partner, CMS, within the PRD. Our data establish that ALIX can have a dramatic effect on HIV-1 release and suggest that the ability to use ALIX may allow HIV-1 to replicate in cells that express only low levels of Tsg101.
Emergence of Mammalian Cell-Adapted Vesicular Stomatitis Virus from Persistent Infections of Insect Vector Cells
Authors: Novella Isabel S. , Ebendick-Corpus Bonnie E. , Zárate Selene , Miller Eric L.
Published: April 2007

Arboviruses (arthropod-borne viruses) represent quintessential generalists, with the ability to infect and perform well in multiple hosts. However, antagonistic pleiotropy imposed a cost during the adaptation to persistent replication of vesicular stomatitis virus in sand fly cells and resulted in strains that initially replicated poorly in hamster cells, even when the virus was allowed to replicate periodically in the latter. Once a debilitated strain started replicating continuously in mammalian cells, fitness increased significantly. Fitness recovery did not entail back mutations or compensatory mutations, but instead, we observed the replacement of persistence-adapted genomes by mammalian cell-adapted strains with a full set of new, unrelated sequence changes. These mammalian cell-adapted genomes were present at low frequencies in the populations with a history of persistence for up to a year and quickly became dominant during mammalian infection, but coexistence was not stable in the long term. Periodic acute replication in mammalian cells likely contributed to extending the survival of minority genomes, but these genomes were also found in strictly persistent populations.
Effect of Preexisting Immunity to Adenovirus Human Serotype 5 Antigens on the Immune Responses of Nonhuman Primates to Vaccine Regimens Based on Human- or Chimpanzee-Derived Adenovirus Vectors
Authors: McCoy Kimberly , Tatsis Nia , Korioth-Schmitz Birgit , Lasaro Marcio O. , Hensley Scott E. , Lin Shih-Wen , Li Yan , Giles-Davis Wynetta , Cun Ann , Zhou Dongming , Xiang Zhiquan , Letvin Norman L. , Ertl Hildegund C. J.
Published: April 2007

In this study we compared a prime-boost regimen with two serologically distinct replication-defective adenovirus (Ad) vectors derived from chimpanzee serotypes C68 and C1 expressing Gag, Pol, gp140, and Nef of human immunodeficiency virus type 1 with a regimen in which replication-defective Ad vectors of the human serotype 5 (AdHu5) were given twice. Experiments were conducted in rhesus macaques that had or had not been preexposed to antigens of AdHu5. There was no significant difference in T-cell responses tested from peripheral blood of the different groups, although responses were overall highest in nonpreexposed animals immunized with the chimpanzee Ad vectors. Preexisting immunity to AdHu5 completely inhibited induction of transgene product-specific antibodies by the AdHu5 vectors without affecting antibody responses to the chimpanzee vectors. Upon euthanasia, T-cell responses were tested from a number of tissues. Preexisting immunity to AdHu5, commonly found in humans, changed the homing pattern of vaccine-induced T cells. In AdHu5-preexposed animals vaccinated with the chimpanzee Ad vectors, frequencies of transgene-specific T cells were higher in spleens than in blood, and in most preexposed animals vaccinated either with AdHu5 vectors or chimpanzee adenovirus vectors, frequencies of such T cells were exceptionally high in livers. The latter results indicate that analysis of T-cell responses solely from blood mononuclear cells of vaccine recipients may not suffice to compare the potencies of different vaccine regimens.
Mutagenesis of the Herpesvirus Saimiri Terminal Repeat Region Reveals Important Elements for Virus Production
Authors: White Robert E. , Carline Lindsay , Allday Martin J.
Published: April 2007

Deletion of the terminal repeats (TR) from herpesvirus saimiri (HVS) renders it unable to produce infectious virus or generate plaques. However, a TR-deleted HVS bacterial artificial chromosome can form replication compartments. Complementation of this mutant shows that one copy of the TR, plus the right junction of the genome with the TR, is sufficient for efficient plaque formation and generation of infectious virus. Within the TR unit, the region around the cleavage site of the genome appears both necessary and sufficient for virus production. Analysis of episomes from productive cells indicates a propensity to amplify TR numbers during the lytic cycle.
Evaluation of Scalp and Auricular Acupuncture on EEG, HRV, and PRV
Authors: Hsu Chih-Chieh , Weng Ching-Sung , Sun Mao-Feng , Shyu Liang-Yu , Hu Wei-Chih , Chang Yung-Hsien
Published: April 2007

In this study, the EEG, ECG and blood-pressure-pulse recorder were employed to evaluate heart rate variability, pulse rate variability, and EEG of 10 adults after scalp (experimental test I) at Sishencong scalp acupoint and auricular (experimental test II) acupuncture at Shenmen auricular acupoint for about 10 min. Comparison of the results between the experimental tests and a control with no stimulation test showed that both the heart rate and pulse rate were decreased, and the blood pressure fell. The high and low frequency power of FFT analysis of heart rate was increased and decreased, respectively; indicating that the parasympathetic nerves were activated and the sympathetic nerves were inhibited. The analysis of the power spectrum of EEG showed that the number of low frequency waves was increased after acupuncture stimulation. Therefore, acupuncture on either Sishencong or Shenmen might calm the mind, slow down the heart rate, and activate the parasympathetic nerves.
Sho-Saiko-To (Xiao-Chai-Hu-Tang) and Crude Saikosaponins Inhibit Hepatitis B Virus in a Stable HBV-Producing Cell Line
Authors: Chang Jung-San , Wang Kuo-Chih , Liu Hong-Wen , Chen Mei-Chun , Chiang Lien-Chai , Lin Chun-Ching
Published: April 2007

To search for an effective antiviral agent, this study tested the hypothesis that sho-saiko-to (Xiao-Chai-Hu-Tang) and crude saikosaponins possess the activity directly against HBV and could affect the expressions of viral antigens, HBeAg and HBsAg, in HepG2 2.2.15 cell model. The viral amount and viral antigens in the suspension were estimated by quantitative real time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. The results showed that sho-saiko-to could inhibit the production of HBV (p < 0.0001), 20 μg/ml sho-saiko-to was efficacious at day-3 of treatment and 10 μg/ml at day-6. The calculated IC50 and CC50 of sho-saiko-to were 55.76 μg/ml and 372 μg/ml, respectively, with a selectivity index of 6.67. Crude saponin of B. chinense could also inhibit the replication of HBV (p < 0.0001). Owing to the anti-neoplastic activity of sho-saiko-to and saikosaponin, their calculated CC50 and selectivity index might be under-estimated. Sho-saiko-to also decreased the expression of HBeAg with the minimal effective concentration of 20 μg/ml. Sho-saiko-to contained too little saikosaponin. Therefore, the anti-HBV activity of sho-saiko-to might not be mediated by saikosaponin. Sho-saiko-to could be supplementary to nucleotide analogues to minimize the recurrence of viremia after its discontinuation.
Application of sulphur isotopes to discriminate Cu–Zn VHMS mineralization from barren Fe sulphide mineralization in the greenschist to granulite facies Flin Flon–Snow Lake–Hargrave River region, Manitoba, Canada
Authors: Polito Paul , Kyser Kurt , Lawie David , Cook Steven , Oates Chris
Published: April 2007

The Flin Flon Belt in northern Canada is one of the largest Palaeoproterozoic volcanic-hosted massive sulphide (VHMS) districts in the world, but up to 20 000 km
2
of prospective Palaeoproterozoic basement south of this belt is buried beneath 10 to 100 m of Phanerozoic calcareous cover. The recent acquisition of airborne SPECTREM geophysics data south of the Flin Flon Belt has resulted in the discovery of Cu-Zn sulphide prospects comprising pyrrhotite, pyrite, chalcopyrite, sphalerite and galena, but numerous barren Fe sulphide occurrences comprising only pyrite and pyrrhotite have also been intersected. The problem for explorers is trying to determine whether a barren Fe sulphide intersection that has just been cored is part of a larger Cu-Zn mineralized system, or nothing more than a pyrite–pyrrhotite occurrence.

A sulphur isotope study of sulphides from the Flin Flon–Snow Lake–Hargrave River–Talbot area shows that sulphides from the Cu-Zn VHMS deposits have δ
34
S values that range between −1.4 and 6.4‰, with a mean δ
34
S value of 1.6 ± 1.7‰ (2σ error). More than 95% of these samples have δ
34
S values of <3.3‰. In contrast, pyrite and pyrrhotite separates from barren Fe sulphide deposits have δ
34
S values between 1.8 and 10.0‰, with a mean δ
34
S value of 4.3 ± 1.8‰ (2σ error). In this case, >84% of these samples have δ
34
S values of >3.3‰. The results imply that the barren Fe sulphide deposits can be statistically distinguished from Cu-Zn VHMS mineralization based on S isotopic composition, which should make future exploration drilling decisions easier.
Soil and biogeochemical signatures of the Aripuanã base metal deposit, Mato Grosso, Brazil
Authors: Cornelius M. , Porto C. G. , Dunn C. E. , Butt C. R. M. , Oates C. , Coelho R.
Published: April 2007

A geochemical survey was undertaken along two traverses at the Aripuanã base metal deposit in the Mato Grosso province of Brazil. Traverse 1 has residual and colluvial soils and intersects a 130-m-long gossan body near the top of a ridge. Traverse 2 intersects 10–20-m-wide base metal mineralization concealed by
c.
5–10 m of transported cover. Humic (A-horizon) and clay-rich and reddish (B-horizon) samples were collected on both traverses; saprolite was sampled on Traverse 1. The compositions of these regolith samples were compared with those of litter and bark samples from Traverse 1, and litter samples from Traverse 2. The soil samples were split into <75-μm and 75-250-μm fractions and, with saprolite samples, analysed for 68 elements following multi-acid, aqua regia or fusion digests. Samples of litter and bark from the imbauba tree (Moraceae family,
Cecropia
genus) were prepared by digesting dry tissue and ash in mixed acids.

Humic A-horizon soil samples exhibit the greatest anomaly contrast of all sample media. Fine-grained samples (<75 μm) are preferable for regional exploration, whereas coarser material (75–250 μm) is more suitable for prospect-scale surveys seeking gossan outcrops. Litter samples have similar contrasts to those in B-horizon samples, and delineate the gossan zones along both traverses. Bark samples from trees over the gossan are all anomalous and may indicate mineralization beneath barren regolith. The element suite that best identifies mineralization in soil at Aripuanã comprises the target elements Cu, Pb, Zn and Au, and associated pathfinder elements In, Mo and Hg. In dry litter, the best anomaly contrast is exhibited by Cu, Pb, Zn, As, Mo, Sb and In. In dry bark, the best indicators are Pb, Mo and Sb.

In depositional terrain, such as Traverse 2, both soil and litter appear to identify the location of mineralization but the number of samples is insufficient to exclude the possibility (at a probability >95%) of obtaining the same results by chance.
Vertical variations and dispersion of elements in arid desertregolith: a case study from the Jinwozi gold deposit,northwestern China
Authors: Wang Xueqiu , Wen Xueqin , Rong Ye , Liu Zhanyuan , Sun Binbin , Zhao Shanding , Shi Shujuan , Wei Hualing
Published: April 2007

Desert terrains in northern China are covered by widespread regolith sediments which mask geochemical signals from ore bodies and are major obstacles to mineral exploration. There is a critical need to study the vertical distribution of elements in the regolith, and to determine potential mechanisms for transferring elements from the ore body upwards through regolith cover to the surface, and to establish appropriate sampling media and analytical methods. The Jinwozi gold deposit, which is covered by four to several tens of metres of regolith sediment, was selected for this study. Samples were taken from different regolith horizons in two vertical profiles over the deposit. Gold and associated elements showed crescent-shaped distribution patterns in the vertical profile, i.e. elements tend to be enriched in lower and upper parts of the profile over the ore body. Enrichment of elements in weathered rocks is derived from the ore body, whereas enrichment at or near surface is due to adsorption by clays or oxide coatings. For gold, the greatest contrast between anomalous and background concentration is in the fine fraction of soils, and the Au anomalies are located directly over the ore body. Gold appears to migrate vertically to the surface to be trapped in clays and oxide coatings to produce superimposed anomalies in the regolith near or at the surface. Fine fraction samples (−120 mesh; 125 μm) from clay-rich horizons or selective leaching of elements adsorbed on clays or oxide coatings are effective for locating buried deposits.
Application of lithogeochemistry to gold exploration in the St Ives goldfield, Western Australia
Authors: Prendergast Kylie
Published: April 2007

Lithogeochemistry is being routinely applied to gold exploration programmes at St Ives, Kambalda. Sampling of fresh rock for multi-element analysis is undertaken in all ongoing exploration drilling, and supplementary sampling of archived drill hole pulp samples has also been implemented. Samples are digested by a four-acid digest (hydrofluoric, nitric, perchloric and hydrochloric acids) in Teflon tubes using a specially designed microprocessor controlled digester and analysed by ICP-MS/OES for 36 elements (Ag, As, Ba, Bi, Ce, Cs, Hf, La, Li, Mo, Nb, Pb, Rb, Sb, Ta, Te, Th, W, Zr, Al, Ca, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, P, S, Sc, Sr, Ti, V, Zn). Multi-element results undergo two stages of assessment: (a) utilization of trace elements to characterize lithological and stratigraphic domains, and (b) utilization of major and pathfinder elements to characterize samples that are altered or enriched above background levels.

Bi-plots of trace elements are used to constrain rock type and stratigraphy. Diagnostic inter-element ratios are apparent, which enable clear differentiation of Kambalda Komatiite, Devon Consols Basalt and Paringa Basalt. Condenser Dolerite can be differentiated from other mafic units except Lunnon Basalt. Fractionated dolerite sills are highly prospective host rocks for gold mineralization in the St Ives field. In all cases the fractionated portion of dolerite sills is readily identified by a Ti concentration greater than 6000 ppm.

Unaltered and altered ultramafic, mafic, felsic–intermediate rocks have been assessed using molar Na/Al and K/Al alteration indices to define areas of sodic, potassic and strong potassic alteration. The distribution of potassic and sodic altered samples favourably correlates with the distribution of observed biotite and albite alteration and known gold mineralization.

Pathfinder elements (As, Bi, Sb, W, Mo) have been assessed at the scale of the whole field. Antimony is decoupled from Mo and Bi. Bismuth displays good association with existing gold deposits and areas of known intrusions. Areas of As enrichment correlate spatially with Sb although they are not as widely distributed. Tungsten is the most constrained of the pathfinders and displays excellent correlation with major structures and existing gold deposits.
Bending Mechanics and Molecular Organization in Biological Membranes
Authors: Groves Jay T.
Published: April 2007

The underlying structure of cell membranes consists of a highly heterogeneous fluid lipid bilayer. Within this milieu, complexes of proteins transiently assemble and dissolve in the performance of the functions of life. The length scales of these coordinated spatial rearrangements can approach the size of the cell, itself, enabling direct visualization in some cases with tantalizing clarity. There has been much interest in the physical driving forces responsible for the assembly of organized structures in cell membranes. Cholesterol-mediated miscibility phase separation within the lipid bilayer has attracted enormous attention over the past decade. This, however, is not the only ordering principle at play. In the following sections, I review recent experimental observations of bending-mediated force transduction and molecular organization in lipid membranes. These results have emerged largely from new experimental methodologies, which are discussed in parallel.
Human NADH:ubiquinone oxidoreductase deficiency: radical changes in mitochondrial morphology?
Authors: Koopman Werner J. H. , Verkaart Sjoerd , Visch Henk Jan , van Emst-de Vries Sjenet , Nijtmans Leo G. J. , Smeitink Jan A. M. , Willems Peter H. G. M.
Published: April 2007

Malfunction of NADH:ubiquinone oxidoreductase or complex I (CI), the first and largest complex of the mitochondrial oxidative phosphorylation system, has been implicated in a wide variety of human disorders. To demonstrate a quantitative relationship between CI amount and activity and mitochondrial shape and cellular reactive oxygen species (ROS) levels, we recently combined native electrophoresis and confocal and video microscopy of dermal fibroblasts of healthy control subjects and children with isolated CI deficiency. Individual mitochondria appeared fragmented and/or less branched in patient fibroblasts with a severely reduced CI amount and activity (class I), whereas patient cells in which these latter parameters were only moderately reduced displayed a normal mitochondrial morphology (class II). Moreover, cellular ROS levels were significantly more increased in class I compared with class II cells. We propose a mechanism in which a mutation-induced decrease in the cellular amount and activity of CI leads to enhanced ROS levels, which, in turn, induce mitochondrial fragmentation when not appropriately counterbalanced by the cell's antioxidant defense systems.