bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in Dimethylsulfoniopropionate Demethylase Gene Assemblages in Response to an Induced Phytoplankton Bloom
Authors: Howard Erinn C. , Sun Shulei , Reisch Christopher R. , del Valle Daniela A. , Bürgmann Helmut , Kiene Ronald P. , Moran Mary Ann
Published: November 2010

Over half of the bacterioplankton cells in ocean surface waters are capable of carrying out a demethylation of the phytoplankton metabolite dimethylsulfoniopropionate (DMSP) that routes the sulfur moiety away from the climatically active gas dimethylsulfide (DMS). In this study, we tracked changes in
dmdA
, the gene responsible for DMSP demethylation, over the course of an induced phytoplankton bloom in Gulf of Mexico seawater microcosms. Analysis of >91,000 amplicon sequences indicated 578 different
dmdA
sequence clusters at a conservative clustering criterion of ≥90% nucleotide sequence identity over the 6-day study. The representation of the major clades of
dmdA
, several of which are linked to specific taxa through genomes of cultured marine bacterioplankton, remained fairly constant. However, the representation of clusters within these major clades shifted significantly in response to the bloom, including two
Roseobacter
-like clusters and a SAR11-like cluster, and the best correlate with shifts of the dominant
dmdA
clades was chlorophyll
a
concentration. Concurrent 16S rRNA amplification and sequencing indicated the presence of
Roseobacter
, SAR11, OM60, and marine
Rhodospirillales
populations, all of which are known to harbor
dmdA
genes, although the largest taxonomic change was an increase in
Flavobacteriaceae
, a group not yet demonstrated to have DMSP-demethylating capabilities. Sequence heterogeneity in
dmdA
and other functional gene populations is becoming increasingly evident with the advent of high-throughput sequencing technologies, and understanding the ecological implications of this heterogeneity is a major challenge for marine microbial ecology.
GeoChip-Based Analysis of the Functional Gene Diversity and Metabolic Potential of Microbial Communities in Acid Mine Drainage
Authors: Xie Jianping , He Zhili , Liu Xinxing , Liu Xueduan , Van Nostrand Joy D. , Deng Ye , Wu Liyou , Zhou Jizhong , Qiu Guanzhou
Published: November 2010

Acid mine drainage (AMD) is an extreme environment, usually with low pH and high concentrations of metals. Although the phylogenetic diversity of AMD microbial communities has been examined extensively, little is known about their functional gene diversity and metabolic potential. In this study, a comprehensive functional gene array (GeoChip 2.0) was used to analyze the functional diversity, composition, structure, and metabolic potential of AMD microbial communities from three copper mines in China. GeoChip data indicated that these microbial communities were functionally diverse as measured by the number of genes detected, gene overlapping, unique genes, and various diversity indices. Almost all key functional gene categories targeted by GeoChip 2.0 were detected in the AMD microbial communities, including carbon fixation, carbon degradation, methane generation, nitrogen fixation, nitrification, denitrification, ammonification, nitrogen reduction, sulfur metabolism, metal resistance, and organic contaminant degradation, which suggested that the functional gene diversity was higher than was previously thought. Mantel test results indicated that AMD microbial communities are shaped largely by surrounding environmental factors (e.g., S, Mg, and Cu). Functional genes (e.g.,
narG
and
norB
) and several key functional processes (e.g., methane generation, ammonification, denitrification, sulfite reduction, and organic contaminant degradation) were significantly (
P
< 0.10) correlated with environmental variables. This study presents an overview of functional gene diversity and the structure of AMD microbial communities and also provides insights into our understanding of metabolic potential in AMD ecosystems.
Genomic and Functional Analyses of
Rhodococcus equi
Phages ReqiPepy6, ReqiPoco6, ReqiPine5, and ReqiDocB7
Authors: Summer E. J. , Liu M. , Gill J. J. , Grant M. , Chan-Cortes T. N. , Ferguson L. , Janes C. , Lange K. , Bertoli M. , Moore C. , Orchard R. C. , Cohen N. D. , Young R.
Published: November 2010

The isolation and results of genomic and functional analyses of
Rhodococcus equi
phages ReqiPepy6, ReqiDocB7, ReqiPine5, and ReqiPoco6 (hereafter referred to as Pepy6, DocB7, Pine5, and Poco6, respectively) are reported. Two phages, Pepy6 and Poco6, more than 75% identical, exhibited genome organization and protein sequence likeness to
Lactococcus lactis
phage 1706 and clostridial prophage elements. An unusually high fraction, 27%, of Pepy6 and Poco6 proteins were predicted to possess at least one transmembrane domain, a value much higher than the average of 8.5% transmembrane domain-containing proteins determined from a data set of 36,324 phage protein entries. Genome organization and protein sequence comparisons place phage Pine5 as the first nonmycobacteriophage member of the large Rosebush cluster. DocB7, which had the broadest host range among the four isolates, was not closely related to any phage or prophage in the database, and only 23 of 105 predicted encoded proteins could be assigned a functional annotation. Because of the relationship of
Rhodococcus
to
Mycobacterium
, it was anticipated that these phages should exhibit some of the features characteristic of mycobacteriophages. Traits that were identified as shared by the
Rhodococcus
phages and mycobacteriophages include the prevalent long-tailed morphology and the presence of genes encoding LysB-like mycolate-hydrolyzing lysis proteins. Application of DocB7 lysates to soils amended with a host strain of
R. equi
reduced recoverable bacterial CFU, suggesting that phage may be useful in limiting
R. equi
load in the environment while foals are susceptible to infection.
l
-Malate Production by Metabolically Engineered
Escherichia
coli
Authors: Zhang X. , Wang X. , Shanmugam K. T. , Ingram L. O.
Published: November 2010

Escherichia
coli
strains (KJ060 and KJ073) that were previously developed for succinate production have now been modified for malate production. Many unexpected changes were observed during this investigation. The initial strategy of deleting fumarase isoenzymes was ineffective, and succinate continued to accumulate. Surprisingly, a mutation in fumarate reductase alone was sufficient to redirect carbon flow into malate even in the presence of fumarase. Further deletions were needed to inactivate malic enzymes (typically gluconeogenic) and prevent conversion to pyruvate. However, deletion of these genes (
sfcA
and
maeB
) resulted in the unexpected accumulation of
d
-lactate despite the prior deletion of
mgsA
and
ldhA
and the absence of apparent lactate dehydrogenase activity. Although the metabolic source of this
d
-lactate was not identified, lactate accumulation was increased by supplementation with pyruvate and decreased by the deletion of either pyruvate kinase gene (
pykA
or
pykF
) to reduce the supply of pyruvate. Many of the gene deletions adversely affected growth and cell yield in minimal medium under anaerobic conditions, and volumetric rates of malate production remained low. The final strain (XZ658) produced 163 mM malate, with a yield of 1.0 mol (mol glucose
−1
), half of the theoretical maximum. Using a two-stage process (aerobic cell growth and anaerobic malate production), this engineered strain produced 253 mM malate (34 g liter
−1
) within 72 h, with a higher yield (1.42 mol mol
−1
) and productivity (0.47 g liter
−1
h
−1
). This malate yield and productivity are equal to or better than those of other known biocatalysts.
Promising Multiple-Epitope Recombinant Vaccine against Foot-and-Mouth Disease Virus Type O in Swine
Authors: Shao Jun-Jun , Wong Chung Kai , Lin Tong , Lee Shuk Kwan , Cong Guo-Zheng , Sin Fion Wai Yee , Du Jun-Zheng , Gao Shan-Dian , Liu Xiang-Tao , Cai Xue-Peng , Xie Yong , Chang Hui-Yun , Liu Ji-Xing
Published: November 2010

In order to develop a completely safe immunogen to replace the traditional inactivated vaccine, a tandem-repeat multiple-epitope recombinant vaccine against foot-and-mouth disease (FMD) virus (FMDV) type O was developed. It contained three copies each of residues 141 to 160 and 200 to 213 of VP1 of the O/China/99 strain of FMDV coupled with a swine immunoglobulin G heavy-chain constant region (scIgG). The data showed that the multiple-epitope recombinant vaccine elicited high titers of anti-FMDV specific antibodies in swine at 30 days postvaccination (dpv) and conferred complete protection against a challenge with 10350% swine infective doses of the O/China/99 strain. The anti-FMDV specific antibody titers were not significantly different between the multiple-epitope recombinant vaccine and the traditional vaccine (ttest,P> 0.05). The number of 50% pig protective doses was 6.47, which is higher than the number recommended by the World Organization for Animal Health. The multiple-epitope recombinant vaccine resulted in a duration of immunity of at least 6 months. We speculate that the multiple-epitope recombinant vaccine is a promising vaccine that may replace the traditional inactivated vaccine for the prevention and control of FMD in swine in the future.
Human Leukocyte Antigens A*3001 and A*3002 Show Distinct Peptide-Binding Patterns of theMycobacterium tuberculosisProtein TB10.4: Consequences for Immune Recognition
Authors: Axelsson-Robertson Rebecca , Ahmed Raija K. , Weichold Frank F. , Ehlers Marthie M. , Kock Marleen M. , Sizemore Donata , Sadoff Jerry , Maeurer Markus
Published: November 2010

High-tuberculosis (TB)-burden countries are located in sub-Saharan Africa. We examined the frequency of human leukocyte antigen (HLA) alleles, followed by recombinant expression of the most frequent HLA-A alleles, i.e., HLA-A*3001 and HLA-A*3002, to study differences in mycobacterial peptide presentation and CD8+T-cell recognition. We screened a peptide library (9-mer peptides with an 8-amino-acid overlap) for binding, affinity, and off-rate of theMycobacterium tuberculosis-associated antigen TB10.4 and identified only three TB10.4 peptides with considerable binding to HLA-A*3001. In contrast, 22 peptides bound to HLA-A*3002. This reflects a marked difference in the binding preference between the two alleles, with A*3002 tolerating a more promiscuous peptide-binding pattern and A*3001 accommodating only a very selective peptide repertoire. Subsequent analysis of the affinity and off-rate of the binding peptides revealed a strong affinity (8 nM to 7 μM) and moderate off-rate (20 min to 3 h) for both alleles. Construction of HLA-A*3001 and HLA-A*3002 tetramers containing selected binding peptides from TB10.4, including a peptide which was shared among both alleles, QIMYNYPAM (TB10.43-11), allowed us to enumerate epitope-specific T cells in HLA-A*3001- and HLA-A*3002-typed patients with active TB. HLA-A*3001 and HLA-A*3002 major histocompatibility complex-peptide complexes were recognized in individuals with active TB, irrespective of their homozygous HLA-A*3001 or HLA-A*3002 genetic background. The antigen-specific T cells exhibited the CD45RA+CCR7+precursor phenotype and the interleukin-7 receptor (CD127), which were different from the phenotype and receptor exhibited by the parental CD8+T-cell population.
Sublingual Administration of an Adenovirus Serotype 5 (Ad5)-Based Vaccine Confirms Toll-Like Receptor Agonist Activity in the Oral Cavity and Elicits Improved Mucosal and Systemic Cell-Mediated Responses against HIV Antigens despite Preexisting Ad5 Immunity
Authors: Appledorn Daniel M. , Aldhamen Yasser A. , Godbehere Sarah , Seregin Sergey S. , Amalfitano Andrea
Published: November 2010

HIV/AIDS continue to devastate populations worldwide. Recent studies suggest that vaccines that induce beneficial immune responses in the mucosal compartment may improve the efficacy of HIV vaccines. Adenovirus serotype 5 (Ad5)-based vectors remain a promising platform for the development of effective vaccines. In an effort to improve the efficacy of Ad5-based vaccines, even in the presence of preexisting Ad5 immunity, we evaluated the potential for an Ad5-based HIV vaccine to induce antigen-specific immune responses following sublingual (s.l.) administration, a route not previously tested in regard to Ad-based vaccines. s.l. vaccination with an Ad5-based HIV-Gag vaccine resulted in a significant induction of Gag-specific cytotoxic T-lymphocyte (CTL) responses in both the systemic and the mucosal compartment. We also show that s.l. immunization not only avoided preexisting Ad5 immunity but also elicited a broad repertoire of antigen-specific CTL clones. Additionally, we confirm for the first time that oral delivery of a vaccine expressing a potent Toll-like receptor (TLR) agonist can stimulate innate immune responses through induction of cytokines and chemokines and activation of NK cells, NKT cells, and macrophages
in vivo
. These results positively correlated with improved antigen-specific CTL responses. These results could be achieved both in Ad5-naïve mice and in mice with preexisting immunity to Ad5. The simplicity of the s.l. vaccination regimen coupled with augmentation of TLR-dependent pathways active in the oral cavity makes s.l. delivery a promising method for HIV vaccine development specifically, as well as for many other vaccine applications in general.
Differentiation between Human Coronaviruses NL63 and 229E Using a Novel Double-Antibody Sandwich Enzyme-Linked Immunosorbent Assay Based on Specific Monoclonal Antibodies
Authors: Sastre Patricia , Dijkman Ronald , Camuñas Ana , Ruiz Tamara , Jebbink Maarten F. , van der Hoek Lia , Vela Carmen , Rueda Paloma
Published: November 2010

Human coronaviruses (HCoVs) are responsible for respiratory tract infections ranging from common colds to severe acute respiratory syndrome. HCoV-NL63 and HCoV-229E are two of the four HCoVs that circulate worldwide and are close phylogenetic relatives. HCoV infections can lead to hospitalization of children, elderly individuals, and immunocompromised patients. Globally, approximately 5% of all upper and lower respiratory tract infections in hospitalized children are caused by HCoV-229E and HCoV-NL63. The latter virus has recently been associated with the childhood disease croup. Thus, differentiation between the two viruses is relevant for epidemiology studies. The aim of this study was to develop a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) as a potential tool for identification and differentiation between HCoV-NL63 and HCoV-229E. The nucleocapsid (N) proteins of HCoV-NL63 and HCoV-229E were expressed in anEscherichia colisystem and used to immunize mice in order to obtain monoclonal antibodies (MAbs) specific for each virus. Three specific MAbs to HCoV-NL63, one MAb specific to HCoV-229E, and four MAbs that recognized both viruses were obtained. After their characterization, three MAbs were selected in order to develop a differential DAS-ELISA. The described assay could detect up to 3 ng/ml of N protein and 50 50% tissue culture infective doses/ml of virus stock. No cross-reactivity with other human coronaviruses or closely related animal coronaviruses was found. The newly developed DAS-ELISA was species specific, and therefore, it could be considered a potential tool for detection and differentiation of HCoV-NL63 and HCoV-229E infections.
Multilaboratory Comparison of
Streptococcus pneumoniae
Opsonophagocytic Killing Assays and Their Level of Agreement for the Determination of Functional Antibody Activity in Human Reference Sera
Authors: Rose Charles E. , Romero-Steiner Sandra , Burton Robert L. , Carlone George M. , Goldblatt David , Nahm Moon H. , Ashton Lindsey , Haston Mitch , Ekström Nina , Haikala Raili , Käyhty Helena , Henckaerts Isabelle , Durant Nathalie , Poolman Jan T. , Fernsten Phil , Yu Xinhong , Hu Branda T. , Jansen Kathrin U. , Blake Milan , Simonetti Elles R. , Hermans Peter W. M. , Plikaytis Brian D.
Published: November 2010

Antibody-mediated killing of
Streptococcus pneumoniae
(pneumococcus) by phagocytes is an important mechanism of protection of the human host against pneumococcal infections. Measurement of opsonophagocytic antibodies by use of a standardized opsonophagocytic assay (OPA) is important for the evaluation of candidate vaccines and required for the licensure of new pneumococcal conjugate vaccine formulations. We assessed agreement among six laboratories that used their own optimized OPAs on a panel of 16 human reference sera for 13 pneumococcal serotypes. Consensus titers, estimated using an analysis-of-variance (ANOVA) mixed-effects model, provided a common reference for assessing agreement among these laboratories. Agreement was evaluated in terms of assay accuracy, reproducibility, repeatability, precision, and bias. We also reviewed four acceptance criterion intervals for assessing the comparability of protocols when assaying the same reference sera. The precision, accuracy, and concordance results among laboratories and the consensus titers revealed acceptable agreement. The results of this study indicate that the bioassays evaluated in this study are robust, and the resultant OPA values are reproducible for the determination of functional antibody titers specific to 13 pneumococcal serotypes when performed by laboratories using highly standardized but not identical assays. The statistical methodologies employed in this study may serve as a template for evaluating future multilaboratory studies.
Promoter Discrimination at Class I MarA Regulon Promoters Mediated by Glutamic Acid 89 of the MarA Transcriptional Activator ofEscherichia coli
Authors: Martin Robert G. , Rosner Judah L.
Published: November 2010

Three paralogous transcriptional activators MarA, SoxS, and Rob, activate >40Escherichia colipromoters. To understand why MarA does not activate certain promoters as strongly as SoxS, we compared MarA, MarA mutants, and SoxS for their abilities to activate 16 promoters and to bind their cognate marbox binding sites. Replacement of the MarA glutamic acid residue 89 with alanine greatly increased the marbox binding and activation of many class I promoters. Like cells constitutive for SoxS, cells expressing the MarA with the E89A mutation were more resistant to superoxides than those harboring WT MarA. The activities of several other E89 substitutions ranked as follows: E89A > E89G > E89V > WT > E89D. Increased binding and activation occurred only at class I promoters when the 12th base of the promoter's marbox (a position at which there is no known interaction between the marbox and MarA) was not a T residue. Furthermore, WT MarA binding to a synthetic marboxin vitrowas enhanced when the phosphate group between positions 12 and 13 was eliminated on one strand. The results demonstrate that relatively minor changes in a single amino acid side chain (e.g., alanine to valine or glutamic acid to aspartic acid) can strongly influence activity despite any evidence that the side chain is involved in positive interactions with either DNA or RNA polymerase. We present a model which attributes the differences in binding and activation to the interference between the β- and γ-carbons of the amino acid at position 89 and the phosphate group between positions 12 and 13.
Spatial Regulation of Histidine Kinases Governing Biofilm Formation inBacillus subtilis
Authors: McLoon Anna L. , Kolodkin-Gal Ilana , Rubinstein Shmuel M. , Kolter Roberto , Losick Richard
Published: November 2010

Bacillus subtilisis able to form architecturally complex biofilms on solid medium due to the production of an extracellular matrix. A master regulator that controls the expression of the genes involved in matrix synthesis is Spo0A, which is activated by phosphorylation via a phosphorelay involving multiple histidine kinases. Here we report that four kinases, KinA, KinB, KinC, and KinD, help govern biofilm formation but that their contributions are partially masked by redundancy. We show that the kinases fall into two categories and that the members of each pair (one pair comprising KinA and KinB and the other comprising KinC and KinD) are partially redundant with each other. We also show that the kinases are spatially regulated: KinA and KinB are active principally in the older, inner regions of the colony, and KinC and KinD function chiefly in the younger, outer regions. These conclusions are based on the morphology of kinase mutants, real-time measurements of gene expression using luciferase as a reporter, and confocal microscopy using a fluorescent protein as a reporter. Our findings suggest that multiple signals from the older and younger regions of the colony are integrated by the kinases to determine the overall architecture of the biofilm community.
Altered Regulation of the OmpF Porin by Fis inEscherichia coliduring an Evolution Experiment and between B and K-12 Strains
Authors: Crozat Estelle , Hindré Thomas , Kühn Lauriane , Garin Jérome , Lenski Richard E. , Schneider Dominique
Published: November 2010

The phenotypic plasticity of global regulatory networks provides bacteria with rapid acclimation to a wide range of environmental conditions, while genetic changes in those networks provide additional flexibility as bacteria evolve across long time scales. We previously identified mutations in the global regulator-encoding genefisthat enhanced organismal fitness during a long-term evolution experiment withEscherichia coli. To gain insight into the effects of these mutations, we produced two-dimensional protein gels with strains carrying differentfisalleles, including a beneficial evolved allele and one with an in-frame deletion. We found that Fis controls the expression of the major porin-encoding geneompFin theE. coliB-derived ancestral strain used in the evolution experiment, a relationship that has not been described before. We further showed that this regulatory connection evolved over two different time scales, perhaps explaining why it was not observed before. On the longer time scale, we showed that this regulation ofompFby Fis is absent from the more widely studied K-12 strain and thus is specific to the B strain. On a shorter time scale, this regulatory linkage was lost during 20,000 generations of experimental evolution of the B strain. Finally, we mapped the Fis binding sites in theompFregulatory region, and we present a hypothetical model ofompFexpression that includes its other known regulators.
Comparison of the RealTime HIV-1, COBAS TaqMan 48 v1.0, Easy Q v1.2, and Versant v3.0 assays for Determination of HIV-1 Viral Loads in a Cohort of Canadian Patients with Diverse HIV Subtype Infections
Authors: Church Deirdre , Gregson Daniel , Lloyd Tracie , Klein Marina , Beckthold Brenda , Laupland Kevin , Gill M. John
Published: November 2010

HIV clinics in Canada provide care to an increasing number of patients born outside of Canada with HIV-1 non-B subtype infections. Because the Easy Q HIV-1 v1.2 assay (EQ; bioMérieux) failed to detect some non-B subtype infections, a multiassay HIV-1 viral load (VL) study was conducted with patients with diverse HIV subtype infections. Patients were enrolled from the Southern Alberta HIV Clinic (SAC), Calgary, Alberta, Canada (
n
= 349) and the McGill HIV Clinic (MHC), Montreal, Quebec, Canada (
n
= 20) and had four or five tubes of blood drawn for testing by EQ and three other commercial HIV VL assays: (i) the Versant 3.0 HIV-1 test, with the Versant 440 instrument (branched DNA [bDNA]; Siemens), (ii) the RealTime HIV-1 test, with the
m
2000rt instrument (
m
2000rt; Abbott Molecular Diagnostics), and (iii) the COBAS AmpliPrep TaqMan HIV-1 48 test (CAP-CTM; Roche Molecular Diagnostics). Blood was processed according to the individual manufacturer's requirements and stored frozen at −86°C. The HIV subtype was known for patients who had undergone HIV genotypic resistance testing (Virco, Belgium). Data analyses were done using standard statistical methods within Stata 9.0 (StataCorp, College Station, TX). A total of 371 samples were tested on 369 patients, of whom 291 (81%) had a Virco genotype result of B (195; 53%) or non-B (96; 26%) subtypes A to D and F to K, as well as circulating recombinant forms (CRFs) (i.e., CRF01_AE and CRF02_AG). Most (58/78; 74%) patients of unknown subtype were recent African emigrants who likely have non-subtype B infection. Overall bias was small in pairwise Bland-Altman plots, but the limits of agreement between assays were wide. Discordant viral load results occurred for 98 samples and were due to missing values, false negatives, and significant underquantification that varied by HIV subtype. Results were obtained for all 371 samples with
m
2000rt, but for only 357 (97%) with CAP-CTM, 338 (92%) with EQ, and 276 (75%) with bDNA due to errors/equipment failures. False-negative results (nondetection of viral RNA versus other assay results) occurred for all platforms, as follows: for
m
2000rt, 8 (2%) [B(4) and non-B(4) subtypes], CAP-CTM, 9 (2.5%) [B(6) and non-B(3) subtypes]; EQ, 20 (6%) [B(7) and non-B(13) subtypes]; bDNA, 5 (2%) [B(1) and C(4)]. EQ and bDNA had the highest rates of underquantification by ≥1.0 log
10
copies/ml, mainly for HIV non-B subtypes. Performance significantly varied between HIV VL platforms according to subtype. HIV viral diversity in the population being tested must be considered in selection of the viral load platform.
Multiple-Locus Variable-Number Tandem Repeat Analysis of
Neisseria gonorrhoeae
Authors: Heymans Raymond , Schouls Leo M. , van der Heide Han G. J. , Schim van der Loeff Maarten F. , Bruisten Sylvia M.
Published: November 2010

The prevalence of
Neisseria gonorrhoeae
in the Netherlands has increased in recent years. A multiple-locus variable-number tandem repeat analysis (MLVA) was developed to assess the molecular epidemiology of
N
.
gonorrhoeae
and to elucidate transmission networks in high-risk groups in Amsterdam. The MLVA was evaluated using 5 variable-number tandem repeat (VNTR) loci with various degrees of polymorphism that were amplified in 2 multiplex PCRs and were then separated and sized on an automated sequencer. The assessed number of repeats was used to create MLVA profiles that consisted of strings of 5 integers. The stability of the VNTR loci was assessed using isolates obtained from multiple anatomical locations from the same patient (
n
= 118) and from patients and their sexual partners (
n
= 55). When isolates with a single locus variant were considered to belong to the same MLVA type, 87% of samples from multiple anatomical locations and 88% of samples from sexual partners shared an MLVA type. MLVA was ultimately performed on 880 isolates that were previously genotyped by restriction fragment length polymorphism (RFLP) analysis of the
por-opa
genes. Hierarchical cluster analysis of the MLVA profiles from 716 patient visits (one anatomical location per visit) classified 430 patient visits into 14 larger clusters (≥10 patient visits). In 7 clusters, 81% to 100% of isolates came from men who have sex with men (MSM); in 5 clusters, 79% to 100% of isolates came from heterosexuals; and 2 clusters contained isolates from fully mixed populations. Clusters also differed in characteristics such as ethnic background and coinfections. MLVA provided accurate identification of genetically related
N. gonorrhoeae
strains and revealed clusters of MSM and heterosexuals reflecting distinct transmission networks.
Comparison of Three Molecular Techniques for Typing
Pseudomonas aeruginosa
Isolates in Sputum Samples from Patients with Cystic Fibrosis
Authors: Kidd Timothy J. , Grimwood Keith , Ramsay Kay A. , Rainey Paul B. , Bell Scott C.
Published: November 2010

Monitoring the emergence and transmission of
Pseudomonas aeruginosa
strains among cystic fibrosis (CF) patients is important for infection control in CF centers internationally. A recently developed multilocus sequence typing (MLST) scheme is used for epidemiologic analyses of
P. aeruginosa
outbreaks; however, little is known about its suitability for isolates from CF patients compared with that of pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). As part of a prevalence study of
P. aeruginosa
strains in Australian CF clinics, we compared the discriminatory power and concordance of ERIC-PCR, PFGE, and MLST among 93 CF sputum and 11 control
P. aeruginosa
isolates. PFGE and MLST analyses were also performed on 30 paired isolates collected 85 to 354 days apart from 30 patients attending two CF centers separated by 3,600 kilometers in order to detect within-host evolution. Each of the three methods displayed high levels of concordance and discrimination; however, overall lower discrimination was seen with ERIC-PCR than with MLST and PFGE. Analysis of the 50 ERIC-PCR types yielded 54 PFGE types, which were related by ≤6 band differences, and 59 sequence types, which were classified into 7 BURST groups and 42 singletons. MLST also proved useful for detecting novel and known strains and for inferring relatedness among unique PFGE types. However, 47% of the paired isolates produced PFGE patterns that within 1 year differed by one to five bands, whereas with MLST all paired isolates remained identical. MLST thus represents a categorical analysis tool with resolving power similar to that of PFGE for typing
P. aeruginosa
. Its focus on highly conserved housekeeping genes is particularly suited for long-term clinical monitoring and detecting novel strains.
Granulomatous Amebic Encephalitis in a Child with Acute Lymphoblastic Leukemia Successfully Treated with Multimodal Antimicrobial Therapy and Hyperbaric Oxygen
Authors: Maritschnegg P. , Sovinz P. , Lackner H. , Benesch M. , Nebl A. , Schwinger W. , Walochnik J. , Urban C.
Published: November 2010

Acanthamoeba is the causative agent of granulomatous amebic encephalitis, a rare and usually fatal disease. We report a child with acute lymphoblastic leukemia who developed brain abscesses caused by
Acanthamoeba
during induction therapy. Multimodal antimicrobial chemotherapy and hyperbaric oxygen therapy resulted in complete resolution of symptoms and of pathology as seen by magnetic resonance imaging.
Rapid First- and Second-Line Drug Susceptibility Assay for
Mycobacterium tuberculosis
Isolates by Use of Quantitative PCR
Authors: Pholwat Suporn , Heysell Scott , Stroup Suzanne , Foongladda Suporn , Houpt Eric
Published: November 2010

The slow turnaround time for
Mycobacterium tuberculosis
drug susceptibility results is a barrier to care. We developed a rapid quantitative PCR (qPCR)-based phenotypic antimicrobial susceptibility test that utilizes amplification of the
M. tuberculosis
16S rRNA gene after 3 days of incubation with antituberculosis drugs. To decrease background from killed organisms, we used propidium monoazide (PMA), a DNA-binding dye that penetrates damaged bacterial cells and renders DNA unamplifiable.
M. tuberculosis
was cultured in broth media containing PMA with or without drugs for 3 days prior to DNA extraction and real-time PCR amplification. 16S rRNA qPCR exhibited a significant decrease in threshold cycle (

C
T

) time values (

C
T

control −

C
T

drug treated) with drug-susceptible strains compared with resistant strains. Susceptibility data were reported as Δ
CT
or as 2
Δ

CT

and with appropriate cutoffs yielded an accuracy of 89 to 100% on 38 susceptible, multidrug-resistant, and extensively drug-resistant strains compared with conventional agar proportion susceptibility results for isoniazid, rifampin, ethambutol, streptomycin, amikacin, kanamycin, capreomycin, ofloxacin, moxifloxacin, ethionamide,
para
-aminosalicylic acid, linezolid, and cycloserine and compared with Bactec MGIT results for pyrazinamide. This PMA-qPCR assay is useful as a rapid 3-day first- and second-line drug susceptibility test for
M. tuberculosis
.
Catheter-Associated
Nocardia higoensis
Bacteremia in a Child with Acute Lymphocytic Leukemia
Authors: Watson Michael E. , Estabrook Michele M. , Burnham Carey-Ann D.
Published: November 2010

A 23-month-old child with leukemia who was receiving chemotherapy developed fevers. Serial blood cultures from a central venous catheter and a peripheral venous site grew an organism identified by 16S rRNA gene sequencing and phenotypic analysis as
Nocardia higoensis
, an opportunistic organism isolated once previously from a pulmonary infection in Japan.
Early Events in Retrovirus XMRV Infection of the Wild-Derived MouseMus pahari
Authors: Sakuma Toshie , Tonne Jason M. , Squillace Karen A. , Ohmine Seiga , Thatava Tayaramma , Peng Kah-Whye , Barry Michael A. , Ikeda Yasuhiro
Published: November 2010

A novel gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV), has been identified in patients with prostate cancer and in patients with chronic fatigue syndromes. StandardMus musculuslaboratory mice lack a functional XPR1 receptor for XMRV and are therefore not a suitable model for the virus. In contrast, Gairdner's shrew-mice (Mus pahari) do express functional XPR1. To determine whetherMus paharicould serve as a model for XMRV, primaryMus paharifibroblasts and mice were infected with cell-free XMRV. Infection of cellsin vitroresulted in XMRV Gag expression and the production of XMRV virions. After intraperitoneal injection of XMRV intoMus paharimice, XMRV proviral DNA could be detected in spleen, blood, and brain. Intravenous administration of a green fluorescent protein (GFP) vector pseudotyped with XMRV produced GFP+CD4+T cells and CD19+B cells. Mice mounted adaptive immune responses against XMRV, as evidenced by the production of neutralizing and Env- and Gag-specific antibodies. Prominent G-to-A hypermutations were also found in viral genomes isolated from the spleen, suggesting intracellular restriction of XMRV infection by APOBEC3in vivo. These data demonstrate infection ofMus pahariby XMRV, potential cell tropism of the virus, and immunological and intracellular restriction of virus infectionin vivo. These data support the use ofMus paharias a model for XMRV pathogenesis and as a platform for vaccine and drug development against this potential human pathogen.
Cloning of the Epstein-Barr Virus-Related Rhesus Lymphocryptovirus as a Bacterial Artificial Chromosome: a Loss-of-Function Mutation of the rhBARF1 Immune Evasion Gene
Authors: Ohashi Makoto , Orlova Nina , Quink Carol , Wang Fred
Published: November 2010

Rhesus macaques are naturally infected with a gammaherpesvirus which is in the same lymphocryptovirus (LCV) genus as and closely related to Epstein-Barr virus (EBV). The rhesus macaque LCV (rhLCV) contains a repertoire of genes identical to that of EBV, and experimental rhLCV infection of naive rhesus macaques accurately models acute and persistent EBV infection of humans. We cloned the LCL8664 rhLCV strain as a bacterial artificial chromosome to create recombinant rhLCV for investigation in this animal model system. A recombinant rhLCV (clone 16 rhLCV) carrying a mutation in the putative immune evasion gene rhBARF1 was created along with a rescued wild-type (rWT) rhLCV in which the rhBARF1 open reading frame (ORF) was repaired. The rWT rhLCV molecular clone demonstrated viral replication and B-cell immortalization properties comparable to those of the naturally derived LCL8664 rhLCV. Qualitatively, clone 16 rhLCV carrying a mutated rhBARF1 was competent for viral replication and B-cell immortalization, but quantitative assays showed that clone 16 rhLCV immortalized B cells less efficiently than LCL8664 and rWT rhLCV. Functional studies showed that rhBARF1 could block CSF-1 cytokine signaling as well as EBV BARF1, whereas the truncated rhBARF1 from clone 16 rhLCV was a loss-of-function mutant. These recombinant rhLCV can be used in the rhesus macaque animal model system to better understand how a putative viral immune evasion gene contributes to the pathogenesis of acute and persistent EBV infection. The development of a genetic system for making recombinant rhLCV constitutes a major advance in the study of EBV pathogenesis in the rhesus macaque animal model.
Minor Oral Lesions Facilitate Transmission of Chronic Wasting Disease
Authors: Denkers Nathaniel D. , Telling Glenn C. , Hoover Edward A.
Published: November 2010

While chronic wasting disease (CWD) prion transmission, entry, and trafficking remain incompletely elucidated, natural exposure of the oral and/or nasal mucous membranes seems certain. Cervids commonly sustain minor lesions on oral mucous membranes that could have an impact on susceptibility to prion infection. To explore this potential cofactor, we studied cohorts of cervid PrP transgenic mice with or without superficial abrasions on the lingual mucosa to determine whether minor oral mucosa lesions may enhance susceptibility to CWD infections. Results demonstrated that minor lingual abrasions substantially facilitate CWD transmission, revealing a cofactor that may be significant in cervids and perhaps other species.
Epitope Mapping of HIV-Specific CD8
+
T Cell Responses by Multiple Immunological Readouts Reveals Distinct Specificities Defined by Function
Authors: Richmond Meika , McKinnon Lyle R. , Kiazyk Sandra A. Koesters , Wachihi Charles , Kimani Makobu , Kimani Joshua , Plummer Francis A. , Ball T. Blake
Published: November 2010

The limited success of HIV vaccine candidates to date highlights our need to better characterize protective cell-mediated immunity (CMI). While HIV-specific CD8
+
T cell responses have been defined largely by measuring gamma interferon (IFN-γ), these responses are not always protective, and it is unclear whether the same epitopes would predominate if other functional parameters were examined. Here, we assessed the epitope specificity of HIV-specific CD8
+
T cell responses by multiparametric flow cytometry, measuring five CD8
+
T cell functions (IFN-γ, macrophage inflammatory protein 1β [MIP-1β], tumor necrosis factor alpha [TNF-α], interleukin-2 [IL-2], and proliferative capacity) in 24 chronically HIV-infected individuals. Sixty-nine epitope-specific responses to 50 epitopes within p24 were measured. Surprisingly, most epitope-specific responses were IFN-γ negative (50/69 responses). Many responses had polyfunctional (33%) and proliferative (19%) components. An inverse association between IL-2 and proliferation responses was also observed, contrary to what was described previously. We confirm that long-term nonprogressors (LTNP) have more polyfunctional responses and also have higher-magnitude and broader p24-specific proliferation and higher levels of IL-2 and TNF-α production than do progressing controls. Together, these data suggest that the specificity of CD8
+
T cell responses differs depending on the immunological readout, with a 3.5-fold increase in breadth detected by including multiple parameters. Furthermore, the identification of epitopes that elicit polyfunctional responses reinforces the need for the comprehensive evaluation of HIV vaccine candidates, and these epitopes may represent novel targets for CMI-based vaccines.
Mapping the Landscape of Host-Pathogen Coevolution: HLA Class I Binding and Its Relationship with Evolutionary Conservation in Human and Viral Proteins
Authors: Hertz Tomer , Nolan David , James Ian , John Mina , Gaudieri Silvana , Phillips Elizabeth , Huang Jim C. , Riadi Gonzalo , Mallal Simon , Jojic Nebojsa
Published: November 2010

The high diversity of HLA binding preferences has been driven by the sequence diversity of short segments of relevant pathogenic proteins presented by HLA molecules to the immune system. To identify possible commonalities in HLA binding preferences, we quantify these using a novel measure termed “targeting efficiency,” which captures the correlation between HLA-peptide binding affinities and the conservation of the targeted proteomic regions. Analysis of targeting efficiencies for 95 HLA class I alleles over thousands of human proteins and 52 human viruses indicates that HLA molecules preferentially target conserved regions in these proteomes, although the arboviralFlaviviridaeare a notable exception where nonconserved regions are preferentially targeted by most alleles. HLA-A alleles and several HLA-B alleles that have maintained close sequence identity with chimpanzee homologues target conserved human proteins and DNA viruses such asHerpesviridaeandAdenoviridaemost efficiently, while all HLA-B alleles studied efficiently target RNA viruses. These patterns of host and pathogen specialization are both consistent with coevolutionary selection and functionally relevant in specific cases; for example, preferential HLA targeting of conserved proteomic regions is associated with improved outcomes in HIV infection and with protection against dengue hemorrhagic fever. Efficiency analysis provides a novel perspective on the coevolutionary relationship between HLA class I molecular diversity, self-derived peptides that shape T-cell immunity through ontogeny, and the broad range of viruses that subsequently engage with the adaptive immune response.
Confirmation of Putative HIV-1 Group P in Cameroon
Authors: Vallari Ana , Holzmayer Vera , Harris Barbara , Yamaguchi Julie , Ngansop Charlotte , Makamche Florence , Mbanya Dora , Kaptué Lazare , Ndembi Nicaise , Gürtler Lutz , Devare Sushil , Brennan Catherine A.
Published: November 2010

We report the second human immunodeficiency virus (HIV) belonging to the new HIV type 1 (HIV-1) group P lineage that is closely related to the simian immunodeficiency virus found in gorillas. This virus was identified in an HIV-seropositive male hospital patient in Cameroon, confirming that the group P virus is circulating in humans. Results from screening 1,736 HIV-seropositive specimens collected in Cameroon indicate that HIV-1 group P infections are rare, accounting for only 0.06% of HIV infections. Despite its rarity, group P shows evidence of adaptation to humans.
Rat liver microsomal metabolism of o-aminophenol and N-(2-methoxyphenyl)hydroxylamine, two metabolites of the environmental pollutant and carcinogen o-anisidine in humans
Authors: Naiman Karel , Hodek Petr , Liberda Jiří , Schmeiser Heinz H. , Frei Eva , Stiborová Marie
Published: November 2010

o-Aminophenol and N-(2-methoxyphenyl)hydroxylamine are human metabolites of the industrial and environmental pollutant and bladder carcinogen 2-methoxyaniline (o-anisidine). The latter one is also a human metabolite of another pollutant and bladder carcinogen, 2-methoxynitrobenzene (o-nitroanisole). Here, we investigated the ability of rat hepatic micro- somes to metabolize these metabolites. N-(2-methoxyphenyl)hydroxylamine is metabolized by rat hepatic microsomes to o-aminophenol and predominantly o-anisidine, the parent carcinogen from which N-(2-methoxyphenyl)hydroxylamine is formed. In addition, two N-(2-methoxyphenyl)hydroxylamine metabolites, whose exact structures have not been identified as yet, were generated. On the contrary, no metabolites were found to be formed from o-aminophenol by rat hepatic microsomes. Whereas N-(2-methoxyphenyl)hydroxylamine is responsible for formation of three deoxyguanosine adducts in DNA, o-aminophenol seems to be a detoxication metabolite of N-(2-methoxyphenyl)hydroxylamine and/or a parental carcinogen, o-anisidine; no o-aminophenol-derived DNA adducts were found after its reaction with microsomal cytochromes P450 and peroxidases.
Ethical dilemmas in medical humanitarian practice: cases for reflection from Médecins Sans Frontières
Authors: Sheather Julian , Shah Tejshri
Published: November 2010

Médecins Sans Frontières (MSF) is an independent medical humanitarian organisation working in over 70 countries. It has provided medical assistance for over 35 years to populations vulnerable through conflict, disease and inadequate health systems. Medical ethics define the starting point of the relationship between medical staff and patients. The ethics of humanitarian interventions and of research in conflict settings are much debated. However, less is known about the ethical dilemmas faced by medical humanitarian staff in their daily work. Ethical dilemmas can be intensified in humanitarian contexts by insecure environments, lack of optimum care, language barriers, potentially heightened power discrepancies between care providers and patients, differing cultural values and perceptions of patients, communities and medical staff. Time constraints, stressful conditions and lack of familiarity with ethical frameworks can prevent reflection on these dilemmas, as can frustration that such reflection does not necessarily provide instant solutions. Lack of reflection, however, can be distressing for medical practitioners and can reduce the quality of care. Ethical reflection has a central role in MSF, and the organisation uses ethical frameworks to help with clinical and programmatic decisions as well as in deliberations over operational research. We illustrate and discuss some real ethical dilemmas facing MSF teams. Only by sharing and seeking guidance can MSF and similar actors make more thoughtful and appropriate decisions. Our aim in sharing these cases is to invite discussion and dialogue in the wider medical community working in crisis, conflict or with severe resource limitations.
The right to die in the minimally conscious state
Authors: Johnson L Syd M
Published: November 2010

The right to die has for decades been recognised for persons in a vegetative state, but there remains controversy about ending life-sustaining medical treatment for persons in the minimally conscious state (MCS). The controversy is rooted in assumptions about the moral significance of consciousness, and the value of life for patients who are conscious and not terminally ill. This paper evaluates these assumptions in light of evidence that generates concerns about quality of life in the MCS. It is argued that surrogates should be permitted to make decisions to withdraw life-sustaining medical treatment from patients in the MCS.
The moral primacy of the human being: a reply to Parker
Authors: Helgesson Gert , Eriksson Stefan
Published: November 2010

In a previous paper in theJournal of Medical Ethics, the authors argued that the research ethical principle stating that the individual shall have priority over science, found in many guidelines, is utterly unclear and because of this should be explicated or otherwise deleted. In a recent commentary, Parker argued that this leaves us defending a position that would allow totalitarian regimes to pursue glory at the expense of its citizens. The present response addresses this and similar accusations.