bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Ndt80p in Sterol Metabolism Regulation and Azole Resistance in
Candida albicans
Authors: Sellam Adnane , Tebbji Faïza , Nantel André
Published: June 2009

The Ndt80p transcription factor modulates azole tolerance in
Candida albicans
by controlling the expression of the gene for the drug efflux pump Cdr1p. To date, the contribution of this transcriptional modulator to drug tolerance is not yet well understood. Here, we investigate the role of Ndt80p in mediating fluconazole tolerance by determining its genome-wide occupancy using chromatin immunoprecipitation coupled to high-density tiling arrays. Ndt80p was found to bind a large number of gene promoters with diverse biological functions. Gene ontology analysis of these Ndt80p targets revealed a significant enrichment in gene products related to the cell wall, carbohydrate metabolism, stress responses, hyphal development, multidrug transport, and the cell cycle. Ndt80p was found on the promoters of ergosterol biosynthesis genes, including on the azole target Erg11p. Additionally, expression profiling was used to identify fluconazole-responsive genes that require Ndt80p for their proper expression. We found that Ndt80p is crucial for the expression of numerous fluconazole-responsive genes, especially genes involved in ergosterol metabolism. Therefore, by combining genome-wide location and transcriptional profiling, we have characterized the Ndt80p fluconazole-dependent regulon and demonstrated the key role of this global transcriptional regulator in modulating sterol metabolism and drug resistance in
C. albicans
.
Functional Genomics of Adhesion, Invasion, and Mycelial Formation in
Schizosaccharomyces pombe
Authors: Dodgson James , Avula Hema , Hoe Kwang-Lae , Kim Dong-Uk , Park Han-Oh , Hayles Jacqueline , Armstrong John
Published: June 2009

Investigation into the switch between single-celled and filamentous forms of fungi may provide insights into cell polarity, differentiation, and fungal pathogenicity. At the molecular level, much of this investigation has fallen on two closely related budding yeasts,
Candida albicans
and
Saccharomyces cerevisiae
. Recently, the much more distant fission yeast
Schizosaccharomyces pombe
was shown to form invasive filaments after nitrogen limitation (E. Amoah-Buahin, N. Bone, and J. Armstrong, Eukaryot. Cell 4:1287-1297, 2005) and this genetically tractable organism provides an alternative system for the study of dimorphic growth. Here we describe a second mode of mycelial formation of
S. pombe
, on rich media. Screening of an
S. pombe
haploid deletion library identified 12 genes required for mycelial development which encode potential transcription factors, orthologues of
S. cerevisiae
Sec14p and Tlg2p, and the formin For3, among others. These were further grouped into two phenotypic classes representing different stages of the process. We show that galactose-dependent cell adhesion and actin assembly are both required for mycelial formation and mutants lacking a range of genes controlling cell polarity all produce mycelia but with radically altered morphology.
Special Sm Core Complex Functions in Assembly of the U2 Small Nuclear Ribonucleoprotein of
Trypanosoma brucei
Authors: Preußer Christian , Palfi Zsofia , Bindereif Albrecht
Published: June 2009

The processing of polycistronic pre-mRNAs in trypanosomes requires the spliceosomal small ribonucleoprotein complexes (snRNPs) U1, U2, U4/U6, U5, and SL, each of which contains a core of seven Sm proteins. Recently we reported the first evidence for a core variation in spliceosomal snRNPs; specifically, in the trypanosome U2 snRNP, two of the canonical Sm proteins, SmB and SmD3, are replaced by two U2-specific Sm proteins, Sm15K and Sm16.5K. Here we identify the U2-specific, nuclear-localized U2B″ protein from
Trypanosoma brucei
. U2B″ interacts with a second U2 snRNP protein, U2-40K (U2A′), which in turn contacts the U2-specific Sm16.5K/15K subcomplex. Together they form a high-affinity, U2-specific binding complex. This trypanosome-specific assembly differs from the mammalian system and provides a functional role for the Sm core variation found in the trypanosomal U2 snRNP.
Adaptations in the Glucose Metabolism of Procyclic
Trypanosoma brucei
Isolates from Tsetse Flies and during Differentiation of Bloodstream Forms
Authors: van Grinsven Koen W. A. , Van Den Abbeele Jan , Van den Bossche Peter , van Hellemond Jaap J. , Tielens Aloysius G. M.
Published: June 2009

Procyclic forms of
Trypanosoma brucei
isolated from the midguts of infected tsetse flies, or freshly transformed from a strain that is close to field isolates, do not use a complete Krebs cycle. Furthermore, short stumpy bloodstream forms produce acetate and are apparently metabolically preadapted to adequate functioning in the tsetse fly.
β-Glucanase Eng2 Is Required for Ascus Wall Endolysis after Sporulation in the Fission Yeast
Schizosaccharomyces pombe
Authors: Encinar del Dedo Javier , Dueñas Encarnación , Arnáiz Yolanda , del Rey Francisco , Vázquez de Aldana Carlos R.
Published: June 2009

Meiosis is the developmental program by which sexually reproducing diploid organisms generate haploid gametes. In yeast, meiosis is followed by spore morphogenesis. When
Schizosaccharomyces pombe
diploid cells undergo meiosis, they differentiate into asci containing four haploid ascospores that are highly resistant to environmental stress. The formation of the ascospore wall requires the activity of several enzymes involved in the biosynthesis and modification of its components, such as α- and β-glucan synthases. Once the spores are completely mature, the wall of the ascus undergoes an endolytic process that results in the release of ascospores from the ascus, allowing their dispersal into the environment. This process requires the activity of the endo-α-1,3-glucanase Agn2. Here, we focus on the characterization of the endo-β-1,3-glucanase Eng2, which is also required for ascospore release from the ascus. Although Eng2 is present during the mitotic cycle, the protein accumulates after meiosis II. The expression of
eng2
+
is required for the efficient release of ascospores, as shown by placing
eng2
+
under the control of a repressible promoter. Furthermore, a point mutation that destroys the catalytic activity of the protein results in a phenotype similar to that of the mutant strain. Finally, we demonstrate that exogenous addition of purified Eng2 releases the ascospores from asci generated by an
eng2
Δ mutant. We propose that Eng2 would act together with Agn2 to completely hydrolyze the ascus wall, thereby assisting in the release of ascospores in
S. pombe
.
Isolation and Characterization ofXenorhabdus nematophilaTransposon Insertion Mutants Defective in Lipase Activity against Tween
Authors: Richards Gregory R. , Vivas Eugenio I. , Andersen Aaron W. , Rivera-Santos Delmarie , Gilmore Sara , Suen Garret , Goodrich-Blair Heidi
Published: June 2009

We identifiedXenorhabdus nematophilatransposon mutants with defects in lipase activity. One of the mutations, inyigL, a conserved gene of unknown function, resulted in attenuated virulence againstManduca sextainsects. We discuss possible connections between lipase production, YigL, and specific metabolic pathways.
Phylogenetic Ubiquity and Shuffling of the Bacterial RecBCD and AddAB Recombination Complexes
Authors: Cromie Gareth A.
Published: June 2009

RecBCD and AddAB are bacterial enzymes that share similar helicase and nuclease activities and initiate repair of DNA double-strand breaks by homologous recombination. Examination of the phylogenetic distribution of AddAB and RecBCD revealed that one or the other complex is present in most sequenced bacteria. In addition, horizontal gene transfer (HGT) events involving
addAB
and
recBCD
appear to be common, with the genes encoding one complex frequently replacing those encoding the other. HGT may also explain the unexpected identification of archaeal
addAB
genes. More than 85% of
addAB
and
recBCD
genes are clustered on the genome, suggesting operon structures. A few organisms, including the
Mycobacteria
, encode multiple copies of these complexes of either the same or mixed classes. The possibility that the enzymatic activities of the AddAB and RecBCD enzymes promote their horizontal transfer is discussed, and the distribution of AddAB/RecBCD is compared to that of the RecU/RuvC resolvases. Finally, it appears that two sequence motifs, the Walker A box involved in ATP binding and an iron-sulfur-cysteine cluster, are present only in subsets of AddB proteins, suggesting the existence of mechanistically distinct classes of AddB.
Optical Mapping Reveals a Large Genetic Inversion between Two Methicillin-Resistant
Staphylococcus aureus
Strains
Authors: Shukla Sanjay K. , Kislow Jennifer , Briska Adam , Henkhaus John , Dykes Colin
Published: June 2009

Staphylococcus aureus
is a highly versatile and evolving bacterium of great clinical importance.
S. aureus
can evolve by acquiring single nucleotide polymorphisms and mobile genetic elements and by recombination events. Identification and location of novel genomic elements in a bacterial genome are not straightforward, unless the whole genome is sequenced. Optical mapping is a new tool that creates a high-resolution, in situ ordered restriction map of a bacterial genome. These maps can be used to determine genomic organization and perform comparative genomics to identify genomic rearrangements, such as insertions, deletions, duplications, and inversions, compared to an in silico (virtual) restriction map of a known genome sequence. Using this technology, we report here the identification, approximate location, and characterization of a genetic inversion of ∼500 kb of a DNA element between the NRS387 (USA800) and FPR3757 (USA300) strains. The presence of the inversion and location of its junction sites were confirmed by site-specific PCR and sequencing. At both the left and right junction sites in NRS387, an IS
1181
element and a 73-bp sequence were identified as inverted repeats, which could explain the possible mechanism of the inversion event.
CtrA, a Global Response Regulator, Uses a Distinct Second Category of Weak DNA Binding Sites for Cell Cycle Transcription Control in
Caulobacter crescentus
Authors: Spencer William , Siam Rania , Ouimet Marie-Claude , Bastedo D. Patrick , Marczynski Gregory T.
Published: June 2009

CtrA controls cell cycle programs of chromosome replication and genetic transcription. Phosphorylated CtrA∼P exhibits high affinity (dissociation constant [

K
d

], <10 nM) for consensus TTAA-N7-TTAA binding sites with “typical” (N = 7) spacing. We show here that
ctrA
promoters P1 and P2 use low-affinity (

K
d

, >500 nM) CtrA binding sites with “atypical” (N ≠ 7) spacing. Footprints demonstrated that phosphorylated CtrA∼P does not exhibit increased affinity for “atypical” sites, as it does for sites in the replication origin. Instead, high levels of CtrA (>10 μM) accumulate, which can drive CtrA binding to “atypical” sites. In vivo cross-linking showed that when the stable CtrAΔ3 protein persists during the cell cycle, the “atypical” sites at
ctrA
and
motB
are persistently bound. Interestingly, the cell cycle timing of
ctrA
P1 and P2 transcription is not altered by persistent CtrAΔ3 binding. Therefore, operator DNA occupancy is not sufficient for regulation, and it is the cell cycle variation of CtrA∼P phosphorylation that provides the dominant “activation” signal. Protein dimerization is one potential means of “activation.” The glutathione
S
-transferase (GST) protein dimerizes, and fusion with CtrA (GST-CtrA) creates a stable dimer with enhanced affinity for TTAA motifs. Electrophoretic mobility shift assays with GST-CtrA revealed cooperative modes of binding that further distinguish the “atypical” sites. GST-CtrA also binds a single TTAA motif in
ctrA
P1 aided by DNA in the extended TTAACCAT motif. We discuss how “atypical” sites are a common yet distinct category of CtrA regulatory sites and new implications for the working and evolution of cell cycle control networks.
CsrRS Regulates Group BStreptococcusVirulence Gene Expression in Response to Environmental pH: a New Perspective on Vaccine Development
Authors: Santi Isabella , Grifantini Renata , Jiang Sheng-Mei , Brettoni Cecilia , Grandi Guido , Wessels Michael R. , Soriani Marco
Published: June 2009

To identify factors involved in the response of group B streptococci (GBS) to environmental pH, we performed a comparative global gene expression analysis of GBS at acidic and neutral pHs. We found that the transcription of 317 genes was increased at pH 5.5 relative to that at pH 7.0, while 61 genes were downregulated. The global response to acid stress included the differential expression of genes involved in transport, metabolism, stress response, and virulence. Known vaccine candidates, such as BibA and pilus components, were also regulated by pH. We observed that many of the genes involved in the GBS response to pH are known to be controlled by the CsrRS two-component system. Comparison of the regulon of wild-type strain 2603 V/R with that of acsrRSdeletion mutant strain revealed that the pH-dependent regulation of 90% of the downregulated genes and 59.3% of the up-regulated genes in strain 2603 V/R was CsrRS dependent and that many virulence factors were overexpressed at high pH. Beta-hemolysin regulation was abrogated by selective inactivation ofcsrS, suggesting the implication of the CsrS protein in pH sensing. These results imply that the translocation of GBS from the acidic milieu of the vagina to the neutral pH of the neonatal lung signals the up-regulation of GBS virulence factors and conversion from a colonizing to an invasive phenotype. In addition, the fact that increased exposure of BibA on the bacterial surface at pH 7.0 induced opsonophagocytic killing of GBS in immune serum highlights the importance of pH regulation in the protective efficacy of specific antibodies to surface-exposed GBS proteins.
Polyamines Are Not Required for Aerobic Growth of
Escherichia coli
: Preparation of a Strain with Deletions in All of the Genes for Polyamine Biosynthesis
Authors: Chattopadhyay Manas K. , Tabor Celia White , Tabor Herbert
Published: June 2009

A strain of
Escherichia coli
was constructed in which all of the genes involved in polyamine biosynthesis—
speA
(arginine decarboxylase),
speB
(agmatine ureohydrolase),
speC
(ornithine decarboxylase),
spe D
(adenosylmethionine decarboxylase),
speE
(spermidine synthase),
speF
(inducible ornithine decarboxylase),
cadA
(lysine decarboxylase), and
ldcC
(lysine decarboxylase)—had been deleted. Despite the complete absence of all of the polyamines, the strain grew indefinitely in air in amine-free medium, albeit at a slightly (ca. 40 to 50%) reduced growth rate. Even though this strain grew well in the absence of the amines in air, it was still sensitive to oxygen stress in the absence of added spermidine. In contrast to the ability to grow in air in the absence of polyamines, this strain, surprisingly, showed a requirement for polyamines for growth under strictly anaerobic conditions.
Enhancement of the Synthesis of RpoE and StpA by Polyamines at the Level of Translation in
Escherichia coli
under Heat Shock Conditions
Authors: Terui Yusuke , Higashi Kyohei , Tabei Yuzuru , Tomitori Hideyuki , Yamamoto Kaneyoshi , Ishihama Akira , Igarashi Kazuei , Kashiwagi Keiko
Published: June 2009

Proteins whose synthesis is enhanced by polyamines at the level of translation were identified with a polyamine-requiring mutant cultured in the presence of 0.1% glucose and 0.02% glutamate at 42°C. Polyamines had a greater effect on cell growth at 42°C than at 37°C. At 42°C, the synthesis of RpoE (σ
24
) and StpA, which are involved in the transcription of a number of heat shock response genes, was stimulated by polyamines at the level of translation. In the
rpoE
and
stpA
mRNAs, a Shine-Dalgarno (SD) sequence is located at 13 and 12 nucleotides, respectively, upstream of the initiation codon AUG. When the SD sequences were moved to the more common position 7 nucleotides upstream of the initiation codon AUG, the degree of polyamine stimulation was reduced, although the level of RpoE and StpA synthesis was markedly increased. The mechanism underlying polyamine stimulation of RpoE synthesis was then studied. Polyamine stimulation of RpoE synthesis was reduced by changing the bulged-out structure in the initiation site of
rpoE
mRNA, although the level of RpoE synthesis increased. A selective structural change of this bulged-out region induced by spermidine at 42°C was observed by circular dichroism. Polyamine stimulation of fMet-tRNA binding to ribosomes at 42°C also disappeared by changing the bulged-out structure in the initiation site of
rpoE
mRNA. The results suggest that polyamines enhance the synthesis of RpoE by changing the bulged-out structure in the initiation site of
rpoE
mRNA.
Role of the σD-Dependent Autolysins inBacillus subtilisPopulation Heterogeneity
Authors: Chen Rui , Guttenplan Sarah B. , Blair Kris M. , Kearns Daniel B.
Published: June 2009

Exponentially growing populations ofBacillus subtiliscontain two morphologically and functionally distinct cell types: motile individuals and nonmotile multicellular chains. Motility differentiation arises because RNA polymerase and the alternative sigma factor σDactivate expression of flagellin in a subpopulation of cells. Here we demonstrate that the peptidoglycan-remodeling autolysins under σDcontrol, LytC, LytD, and LytF, are expressed in the same subpopulation of cells that complete flagellar synthesis. Morphological heterogeneity is explained by the expression of LytF that is necessary and sufficient for cell separation. Moreover, LytC is required for motility but not at the level of cell separation or flagellum biosynthesis. Rather, LytC appears to be important for flagellar function, and motility was restored to aLytCmutant by mutation of eitherlonA, encoding the LonA protease, or a gene encoding a previously unannotated swarming motility inhibitor, SmiA. We conclude that heterogeneous activation of σD-dependent gene expression is sufficient to explain both the morphological heterogeneity and functional heterogeneity present in vegetativeB. subtilispopulations.
Site-Directed Mutagenesis Identifies a Molecular Switch Involved in Copper Sensing by the Histidine Kinase CinS in
Pseudomonas putida
KT2440
Authors: Quaranta Davide , McEvoy Megan M. , Rensing Christopher
Published: June 2009

In the presence of copper,
Pseudomonas putida
activates transcription of
cinAQ
via the two-component system CinS-CinR. The CinS-CinR TCS was responsive to 0.5 μM copper and was specifically activated only by copper and silver. Modeling studies of CinS identified a potential copper binding site containing H37 and H147. CinS mutants with H37R and H147R mutations had an almost 10-fold reduced copper-dependent induction of
cinAQ
compared to the wild type.
Effect of Perturbation of ATP Level on the Activity and Regulation of Nitrogenase in
Rhodospirillum rubrum
Authors: Zhang Yaoping , Pohlmann Edward L. , Roberts Gary P.
Published: June 2009

Nitrogenase activity in
Rhodospirillum rubrum
and in some other photosynthetic bacteria is regulated in part by the availability of light. This regulation is through a posttranslational modification system that is itself regulated by P
II
homologs in the cell. P
II
is one of the most broadly distributed regulatory proteins in nature and directly or indirectly senses nitrogen and carbon signals in the cell. However, its possible role in responding to light availability remains unclear. Because P
II
binds ATP, we tested the hypothesis that removal of light would affect P
II
by changing intracellular ATP levels, and this in turn would affect the regulation of nitrogenase activity. This in vivo test involved a variety of different methods for the measurement of ATP, as well as the deliberate perturbation of intracellular ATP levels by chemical and genetic means. To our surprise, we found fairly normal levels of nitrogenase activity and posttranslational regulation of nitrogenase even under conditions of drastically reduced ATP levels. This indicates that low ATP levels have no more than a modest impact on the P
II
-mediated regulation of NifA activity and on the posttranslational regulation of nitrogenase activity. The relatively high nitrogenase activity also shows that the ATP-dependent electron flux from dinitrogenase reductase to dinitrogenase is also surprisingly insensitive to a depleted ATP level. These in vivo results disprove the simple model of ATP as the key energy signal to P
II
under these conditions. We currently suppose that the ratio of ADP/ATP might be the relevant signal, as suggested by a number of recent in vitro analyses.
CodY Activates Transcription of a Small RNA inBacillus subtilis
Authors: Preis Heike , Eckart Rita A. , Gudipati Rajani K. , Heidrich Nadja , Brantl Sabine
Published: June 2009

Regulatory small RNAs (sRNAs) in bacterial genomes have become a focus of research over the past 8 years. Whereas more than 100 such sRNAs have been found inEscherichia coli, relatively little is known about sRNAs in gram-positive bacteria. Using a computational approach, we identified two sRNAs in intergenic regions of theBacillus subtilisgenome, SR1 and SR2 (renamed BsrF). Recently, we demonstrated that SR1 inhibits the translation initiation of the transcriptional activator AhrC. Here, we describe detection of BsrF, its expression profile, and its regulation by CodY. Furthermore, we mapped the secondary structure of BsrF. BsrF is expressed in complex and minimal media in all growth phases inB. subtilisand, with a similar expression profile, also inBacillus amyloliquefaciens. Neither overexpression nor deletion ofbsrFaffected the growth ofB. subtilis. BsrF was found to be long-lived in complex and minimal media. Analysis of 13 putative transcription factor binding sites upstream ofbsrFrevealed only an effect for CodY. Here, we showed by using Northern blotting,lacZreporter gene fusions, in vitro transcription, and DNase I footprinting that the transcription ofbsrFis activated by CodY in the presence of branched-chain amino acids and GTP. Furthermore, BsrF transcription was increased 1.5- to 2-fold by glucose in the presence of branched-chain amino acids, and this increase was independent of the known glucose-dependent regulators. BsrF is the second target for which transcriptional activation by CodY has been discovered.
Characterization of the
Synechocystis
Strain PCC 6803 Penicillin-Binding Proteins and Cytokinetic Proteins FtsQ and FtsW and Their Network of Interactions with ZipN
Authors: Marbouty Martial , Mazouni Khalil , Saguez Cyril , Cassier-Chauvat Corinne , Chauvat Franck
Published: June 2009

Because very little is known about cell division in noncylindrical bacteria and cyanobacteria, we investigated 10 putative cytokinetic proteins in the unicellular spherical cyanobacterium
Synechocystis
strain PCC 6803. Concerning the eight penicillin-binding proteins (PBPs), which define three classes, we found that
Synechocystis
can survive in the absence of one but not two PBPs of either class A or class C, whereas the unique class B PBP (also termed FtsI) is indispensable. Furthermore, we showed that all three classes of PBPs are required for normal cell size. Similarly, the putative FtsQ and FtsW proteins appeared to be required for viability and normal cell size. We also used a suitable bacterial two-hybrid system to characterize the interaction web among the eight PBPs, FtsQ, and FtsW, as well as ZipN, the crucial FtsZ partner that occurs only in cyanobacteria and plant chloroplasts. We showed that FtsI, FtsQ, and ZipN are self-interacting proteins and that both FtsI and FtsQ interact with class A PBPs, as well as with ZipN. Collectively, these findings indicate that ZipN, in interacting with FtsZ and both FtsI and FtQ, plays a similar role to the
Escherichia coli
FtsA protein, which is missing in cyanobacteria and chloroplasts.
Genome Sequence of the Emerging Pathogen
Helicobacter canadensis
Authors: Loman N. J. , Snyder L. A. S. , Linton J. D. , Langdon R. , Lawson A. J. , Weinstock G. M. , Wren B. W. , Pallen M. J.
Published: June 2009

We determined the genome sequence of the type strain of
Helicobacter canadensis
, an emerging human pathogen with diverse animal reservoirs. Potential virulence determinants carried by the genome include systems for N-linked glycosylation and capsular export. A protein-based phylogenetic analysis places
H. canadensis
close to
Wolinella succinogenes
.
Detection and Genotyping of Human Rotavirus VP4 and VP7 Genes by Reverse Transcriptase PCR and Reverse Hybridization
Authors: van Doorn Leen-Jan , Kleter Bernhard , Hoefnagel Evert , Stainier Isabelle , Poliszczak Annick , Colau Brigitte , Quint Wim
Published: June 2009

Rotavirus infections can be diagnosed in stool samples by serological and molecular methods. We developed a novel reverse transcriptase PCR (RT-PCR) method for the amplification of rotavirus RNA and a reverse hybridization assay on a strip to detect amplimers and identify the specific G and P genotypes present in human stool specimens. An additional aim was to permit specific identification of the rotavirus G1P[8] strain, used in the Rotarix vaccine. Novel broad-spectrum PCR primers were developed for both VP4 and VP7, permitting the amplification of a wide range of rotavirus genotypes. Primer sets comprise mixtures of defined primer sequences. For the identification of G and P genotypes, two reverse hybridization strip assays were developed. Both the VP4 and the VP7 strip contain universal probes for the detection of VP4 and VP7 sequences, irrespective of the G or P genotype. The VP4 strip contains type-specific probes for P[4], P[6], P[8], P[9], and P[10]. The VP7 strip contains type-specific probes for G1, G2, G3, G4, G5, G6, G8, and G9. In addition, probes to distinguish between wild-type G1 and G1 vaccine strain sequences were present. Testing by analysis of multiple reference strains confirmed that both RT-PCR methods allowed the detection of a broad spectrum of genotypes. RT-PCR for VP7 was more sensitive than RT-PCR for VP4, but all samples identified as positive for rotavirus antigen by an enzyme-linked immunosorbent assay (ELISA) were also positive for both VP4 and VP7. The high specificity of the reverse hybridization method was confirmed by sequence analysis as well as by type-specific PCR, and the vaccine strain could also be specifically identified. The reverse hybridization method permits accurate identification of mixed infections with different genotypes. Rotavirus genotypes for which no type-specific probes were present on the strip were adequately identified by the universal detection probes. The assay was formally validated by analyses of specificity, sensitivity, precision, accuracy, and robustness. In a panel of 149 ELISA-positive stool samples, comparison with conventional type-specific RT-PCR methods revealed the superiority of the novel method, mainly in cases of mixed rotavirus infections. This novel method permits highly accurate detection and identification of human rotavirus infections in stool samples. This validated assay could be useful for large-scale epidemiological and clinical trials.
Plasmid-Borne Virulence-Associated Genes Have a Conserved Organization in Virulent Strains of Avian Pathogenic
Escherichia coli
Authors: Tivendale Kelly A. , Allen Joanne L. , Browning Glenn F.
Published: June 2009

Avian pathogenic
Escherichia coli
(APEC) is an important respiratory pathogen of poultry. Various virulence factors are responsible for determining the pathogenicity of these strains, and it is commonly believed they are encoded on large plasmids the strains carry. This study examined a series of strains, the pathogenicity of which had previously been determined by aerosol exposure, for possession of large plasmids and found all isolates carried at least one large plasmid, regardless of the level of virulence. Virulence-associated genes carried on these plasmids were also examined, and it was shown that highly virulent strains carried at least four virulence-associated genes on their largest plasmid. Two of the virulence-associated genes were shown to be chromosomally located in a strain of intermediate virulence, while no virulence-associated genes were carried by the low-virulence strain. The organization of the virulence-associated genes was shown to be highly conserved among APEC isolates of high virulence, supporting the concept of a conserved portion of the putative virulence region that contributes to the pathogenicity of APEC strains.
Use of a New Single Multiplex PCR-Based Assay for Direct Simultaneous Characterization of Six
Neisseria meningitidis
Serogroups
Authors: Fraisier Christophe , Stor Richard , Tenebray Bernard , Sanson Yannick , Nicolas Pierre
Published: June 2009

We developed a new
Neisseria meningitidis
multiplex PCR to determine six serogroups, including X-specific primers, and to allow direct W135/Y discrimination. This assay offers a simple and low-cost method for serogrouping
N
.
meningitidis
from cerebrospinal fluid that could be useful in Africa.
The Ubiquitin-Specific Peptidase USP15 Regulates Human Papillomavirus Type 16 E6 Protein Stability
Authors: Vos Robin M. , Altreuter Jennifer , White Elizabeth A. , Howley Peter M.
Published: June 2009

Proteomic identification of human papillomavirus type 16 (HPV16) E6-interacting proteins revealed several proteins involved in ubiquitin-mediated proteolysis. In addition to the well-characterized E6AP ubiquitin-protein ligase, a second HECT domain protein (HERC2) and a deubiquitylating enzyme (USP15) were identified by tandem affinity purification of HPV16 E6-associated proteins. This study focuses on the functional consequences of the interaction of E6 with USP15. Overexpression of USP15 resulted in increased levels of the E6 protein, and the small interfering RNA-mediated knockdown of USP15 decreased E6 protein levels. These results implicate USP15 directly in the regulation of E6 protein stability and suggest that ubiquitylated E6 could be a substrate for USP15 ubiquitin peptidase activity. It remains possible that E6 could affect the activity of USP15 on specific cellular substrates, a hypothesis that can be tested as more is learned about the substrates and pathways controlled by USP15.
Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype
Authors: Nedellec R. , Coetzer M. , Shimizu N. , Hoshino H. , Polonis V. R. , Morris L. , Mårtensson U. E. A. , Binley J. , Overbaugh J. , Mosier D. E.
Published: June 2009

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.
Subtype-Specific Differences in the Human Immunodeficiency Virus Type 1 Reverse Transcriptase Connection Subdomain of CRF01_AE Are Associated with Higher Levels of Resistance to 3′-Azido-3′-Deoxythymidine
Authors: Delviks-Frankenberry Krista A. , Nikolenko Galina N. , Maldarelli Frank , Hase Saiki , Takebe Yutaka , Pathak Vinay K.
Published: June 2009

We previously shown that mutations in the connection (CN) subdomain of human immunodeficiency virus type 1 (HIV-1) subtype B reverse transcriptase (RT) increase 3′-azido-3′-deoxythymidine (AZT) resistance in the context of thymidine analog mutations (TAMs) by affecting the balance between polymerization and RNase H activity. To determine whether this balance affects drug resistance in other HIV-1 subtypes, recombinant subtype CRF01_AE was analyzed. Interestingly, CRF01_AE containing TAMs exhibited 64-fold higher AZT resistance relative to wild-type B, whereas AZT resistance of subtype B containing the same TAMs was 13-fold higher, which in turn correlated with higher levels of AZT-monophosphate (AZTMP) excision on both RNA and DNA templates. The high level of AZT resistance exhibited by CRF01_AE was primarily associated with the T400 residue in wild-type subtype AE CN subdomain. An A400T substitution in subtype B enhanced AZT resistance, increased AZTMP excision on both RNA and DNA templates, and reduced RNase H cleavage. Replacing the T400 residue in CRF01_AE with alanine restored AZT sensitivity and reduced AZTMP excision on both RNA and DNA templates, suggesting that the T400 residue increases AZT resistance in CRF01_AE at least in part by directly increasing the efficiency of AZTMP excision. These results show for the first time that CRF01_AE exhibits higher levels of AZT resistance in the presence of TAMs and that this resistance is primarily associated with T400. Our results also show that mixing the RT polymerase, CN, and RNase H domains from different subtypes can underestimate AZT resistance levels, and they emphasize the need to develop subtype-specific genotypic and phenotypic assays to provide more accurate estimates of clinical drug resistance.
Measurement of Human Immunodeficiency Virus Type 1 Preintegration Transcription by Using Rev-Dependent Rev-CEM Cells Reveals a Sizable Transcribing DNA Population Comparable to That from Proviral Templates
Authors: Iyer Subashini R. , Yu Dongyang , Biancotto Angélique , Margolis Leonid B. , Wu Yuntao
Published: June 2009

Preintegration transcription is an early process in human immunodeficiency virus type 1 infection and has been suggested to occur at a low level. The templates have also been suggested to represent a small population of nonintegrated viral DNA, particularly the two-long-terminal-repeat (2-LTR) circles. However, these determinations were made by either using PCR amplification of viral transcripts in bulk cell populations or utilizing the LTR-driving reporter cells that measure the synthesis of Tat. The intrinsic leakiness of LTR often makes the measurement of low-level viral transcription inaccurate. Since preintegration transcription also generates Rev, to eliminate the nonspecificity associated with the use of LTR alone we have developed a novel Rev-dependent indicator cell, Rev-CEM, to measure preintegration transcription based on the amount of Rev generated. In this report, using Rev-CEM cells, we demonstrate that preintegration transcription occurs on a much larger scale than expected. The transcribing population derived from nonintegrated viral DNA was comparable (at approximately 70%) to that derived from provirus in a productive viral replication cycle. Nevertheless, each nonintegrated viral DNA template exhibited a significant reduction in the level of transcriptional activity in the absence of integration. We also performed flow cytometry sorting of infected cells to identify viral templates. Surprisingly, our results suggest that the majority of 2-LTR circles are not active in directing transcription. It is likely that the nonintegrated templates are from the predominant DNA species, such as the full-length, linear DNA. Our results also suggest that a nonintegrating lentiviral vector can be as effective as an integrating vector in directing gene expression in nondividing cells, with the proper choice of an internal promoter.
Dexamethasone Treatment of Calves Latently Infected with Bovine Herpesvirus 1 Leads to Activation of the bICP0 Early Promoter, in Part by the Cellular Transcription Factor C/EBP-Alpha
Authors: Workman Aspen , Perez Sandra , Doster Alan , Jones Clinton
Published: June 2009

Sensory neurons within trigeminal ganglia (TG) are the primary site for bovine herpesvirus 1 (BHV-1) latency. During latency, viral gene expression is restricted to the latency-related (LR) gene and the open reading frame ORF-E. We previously constructed an LR mutant virus that expresses LR RNA but not any of the known LR proteins. In contrast to calves latently infected with wild-type (wt) BHV-1 or the LR rescued virus, the LR mutant virus does not reactivate from latency following dexamethasone (DEX) treatment. In this study, we demonstrated that bICP0, but not bICP4, transcripts were consistently detected in TG of calves infected with the LR mutant or LR rescued virus following DEX treatment. Calves latently infected with the LR rescued virus but not the LR mutant virus expressed late transcripts, which correlated with shedding of infectious virus following DEX treatment. The bICP4 and bICP0 genes share a common immediate-early promoter, suggesting that this promoter was not consistently activated during DEX-induced reactivation from latency. The bICP0 gene also contains a novel early promoter that was activated by DEX in mouse neuroblastoma cells. Expression of a cellular transcription factor, C/EBP-alpha, was stimulated by DEX, and C/EBP-alpha expression was necessary for DEX induction of bICP0 early promoter activity. C/EBP-alpha directly interacted with bICP0 early promoter sequences that were necessary for
trans
activation by C/EBP-alpha. In summary, DEX treatment of latently infected calves induced cellular factors that stimulated bICP0 early promoter activity. Activation of bICP0 early promoter activity does not necessarily lead to late gene expression and virus shedding.
Tactile acuity and lumbopelvic motor control in patients with back pain and healthy controls
Authors: Luomajoki H , Moseley G L
Published: June 2009

Background

Voluntary lumbopelvic control is compromised in patients with back pain. Loss of proprioceptive acuity is one contributor to decreased control. Several reasons for decreased proprioceptive acuity have been proposed, but the integrity of cortical body maps has been overlooked. We investigated whether tactile acuity, a clear clinical signature of primary sensory cortex organisation, relates to lumbopelvic control in people with back pain.

Methods

Forty-five patients with back pain and 45 age- and sex-matched healthy controls participated in this cross-sectional study. Tactile acuity at the back was assessed using two-point discrimination (TPD) threshold in vertical and horizontal directions. Voluntary motor control was assessed using an established battery of clinical tests.

Results

Patients performed worse on the voluntary lumbopelvic tasks than healthy controls did (p<0.001). TPD threshold was larger in patients (mean (SD)=61 (13) mm) than in healthy controls (44 (10) mm). Moreover, larger TPD threshold was positively related to worse performance on the voluntary lumbopelvic tasks (Pearson's r=0.49; p<0.001).

Discussion

Tactile acuity, a clear clinical signature of primary sensory cortex organisation, relates to voluntary lumbopelvic control. This relationship raises the possibility that the former contributes to the latter, in which case training tactile acuity may aid recovery and assist in achieving normal motor performance after back injury.
Traffic particles and occurrence of acute myocardial infarction: a case–control analysis
Authors: Tonne C , Yanosky J , Gryparis A , Melly S , Mittleman M , Goldberg R , von Klot S , Schwartz J
Published: June 2009

Objectives:

We modelled exposure to traffic particles using a latent variable approach and investigated whether long-term exposure to traffic particles is associated with an increase in the occurrence of acute myocardial infarction (AMI) using data from a population-based coronary disease registry.

Methods:

Cases of individually validated AMI were identified between 1995 and 2003 as part of the Worcester Heart Attack Study. Population controls were selected from Massachusetts, USA, resident lists. NO2and PM2.5filter absorbance were measured at 36 locations throughout the study area. The air pollution data were used to estimate exposure to traffic particles using a semiparametric latent variable regression model. Conditional logistic models were used to estimate the association between exposure to traffic particles and occurrence of AMI.

Results:

Modelled exposure to traffic particles was highest near the city of Worcester. Cases of AMI were more exposed to traffic and traffic particles compared to controls. An interquartile range increase in modelled traffic particles was associated with a 10% (95% CI 4% to 16%) increase in the odds of AMI. Accounting for spatial dependence at the census tract, but not block group, scale substantially attenuated this association.

Conclusions:

These results provide some support for an association between long-term exposure to traffic particles and risk of AMI. The results were sensitive to the scale selected for the analysis of spatial dependence, an issue that requires further investigation. The latent variable model captured variation in exposure, although on a relatively large spatial scale.
COUNTING d-DIMENSIONAL POLYCUBES AND NONRECTANGULAR PLANAR POLYOMINOES
Authors: ALEKSANDROWICZ GADI , BAREQUET GILL
Published: June 2009

A planar polyomino of size n is an edge-connected set of n squares on a rectangular two-dimensional lattice. Similarly, a d-dimensional polycube (for d ≥ 2) of size n is a connected set of n hypercubes on an orthogonal d-dimensional lattice, where two hypercubes are neighbors if they share a (d - 1)-dimensional face. There are also two-dimensional polyominoes that lie on a triangular or hexagonal lattice. In this paper we describe a generalization of Redelmeier's algorithm for counting two-dimensional rectangular polyominoes, which counts all the above types of polyominoes. For example, our program computed the number of distinct three-dimensional polycubes of size 18. To the best of our knowledge, this is the first tabulation of this value.
Accretionary orogens through Earth history
Authors: Cawood Peter A. , Kröner Alfred , Collins William J. , Kusky Timothy M. , Mooney Walter D. , Windley Brian F.
Published: June 2009

Accretionary orogens form at intraoceanic and continental margin convergent plate boundaries. They include the supra-subduction zone forearc, magmatic arc and back-arc components. Accretionary orogens can be grouped into retreating and advancing types, based on their kinematic framework and resulting geological character. Retreating orogens (e.g. modern western Pacific) are undergoing long-term extension in response to the site of subduction of the lower plate retreating with respect to the overriding plate and are characterized by back-arc basins. Advancing orogens (e.g. Andes) develop in an environment in which the overriding plate is advancing towards the downgoing plate, resulting in the development of foreland fold and thrust belts and crustal thickening. Cratonization of accretionary orogens occurs during continuing plate convergence and requires transient coupling across the plate boundary with strain concentrated in zones of mechanical and thermal weakening such as the magmatic arc and back-arc region. Potential driving mechanisms for coupling include accretion of buoyant lithosphere (terrane accretion), flat-slab subduction, and rapid absolute upper plate motion overriding the downgoing plate. Accretionary orogens have been active throughout Earth history, extending back until at least 3.2 Ga, and potentially earlier, and provide an important constraint on the initiation of horizontal motion of lithospheric plates on Earth. They have been responsible for major growth of the continental lithosphere through the addition of juvenile magmatic products but are also major sites of consumption and reworking of continental crust through time, through sediment subduction and subduction erosion. It is probable that the rates of crustal growth and destruction are roughly equal, implying that net growth since the Archaean is effectively zero.
Metamorphic patterns in orogenic systems and the geological record
Authors: Brown Michael
Published: June 2009

Regional metamorphism occurs in plate boundary zones. Accretionary orogenic systems form at subduction boundaries in the absence of continent collision, whereas collisional orogenic systems form where ocean basins close and subduction steps back and flips (arc collisions), simply steps back and continues with the same polarity (block and terrane collisions) or ultimately ceases (continental collisions). As a result, collisional orogenic systems may be superimposed on accretionary orogenic systems. Metamorphism associated with orogenesis provides a mineral record that may be inverted to yield apparent thermal gradients for different metamorphic belts, which in turn may be used to infer tectonic setting. Potentially, peak mineral assemblages are robust recorders of metamorphic
P
and
T
, particularly at high
P
–
T
conditions, because prograde dehydration and melting with melt loss produce nominally anhydrous mineral assemblages that are difficult to retrogress or overprint without fluid influx. Currently on Earth, lower thermal gradients are associated with subduction (and early stages of collision) whereas higher thermal gradients are characteristic of back-arcs and orogenic hinterlands. This duality of thermal regimes is the hallmark of asymmetric or one-sided subduction and plate tectonics on modern Earth, and a duality of metamorphic belts will be the characteristic imprint of asymmetric or one-sided subduction in the geological record. Accretionary orogenic systems may exhibit retreating trench–advancing trench cycles, associated with high (>750 °C GPa
−1
) thermal gradient type of metamorphism, or advancing trench–retreating trench cycles, associated with low (<350 °C GPa
−1
) to intermediate (350–750 °C GPa
−1
) thermal gradient types of metamorphism. Whether the subducting boundary advances or retreats determines the mode of evolution. Accretionary orogenic systems may involve accretion of allochthonous and/or para-autochthonous elements to continental margins at subduction boundaries. Paired metamorphic belts,
sensu
Miyashiro, comprising a low thermal gradient metamorphic belt outboard and a high thermal gradient metamorphic belt inboard, are characteristic and may record orogen-parallel terrane migration and juxtaposition by accretion of contemporary belts of contrasting type. A wider definition of ‘paired’ metamorphism is proposed to incorporate all types of dual metamorphic belts. An additional feature is ridge subduction, which may be reflected in the pattern of high d
T
/d
P
metamorphism and associated magmatism. Apparent thermal gradients derived from inversion of age-constrained metamorphic
P
–
T
data are used to identify tectonic settings of ancient metamorphism, to evaluate the age distribution of metamorphism in the rock record from the Neoarchaean Era to the Cenozoic Era, and to consider how this relates to the supercontinent cycle and the process of terrane export and accretion. In addition, I speculate about metamorphism and tectonics before the Mesoarchaean Era.