bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of
Mycoplasma
Proteins Carrying an Affinity Tag in
M. pneumoniae
Allows Rapid Purification and Circumvents Problems Related to the Aberrant Genetic Code
Authors: Schmidl Sebastian R. , Hames Claudine , Stülke Jörg
Published: October 2007

In
Mycoplasma pneumoniae
and several other mollicutes, the UGA opal codon specifies tryptophan rather than a translation stop. This often makes it difficult to express
Mycoplasma
proteins in heterologous hosts. In this work, we demonstrate that mollicute proteins can be fused to an affinity tag and be expressed directly in
M. pneumoniae
. The protein can then be purified by affinity chromatography and be used for biochemical or any other desired analysis.
Identification of a Malate Chemoreceptor in
Pseudomonas aeruginosa
by Screening for Chemotaxis Defects in an Energy Taxis-Deficient Mutant
Authors: Alvarez-Ortega Carolina , Harwood Caroline S.
Published: October 2007

We found that a robust energy taxis response mediated by the Aer receptor can sometimes mask chemotaxis mediated by other methyl-accepting chemotaxis proteins (MCPs) in
Pseudomonas aeruginosa
. We identified PA2652 as a chemoreceptor for malate by screening
aer mcp
double mutants by using swarm plate assays.
PaTrx1 and PaTrx3, Two Cytosolic Thioredoxins of the Filamentous Ascomycete
Podospora anserina
Involved in Sexual Development and Cell Degeneration
Authors: Malagnac Fabienne , Klapholz Benjamin , Silar Philippe
Published: October 2007

In various organisms, thioredoxins are known to be involved in the reduction of protein disulfide bonds and in protecting the cell from oxidative stress. Genes encoding thioredoxins were found by searching the complete genome sequence of the filamentous ascomycete
Podospora anserina
. Among them, PaTrx1, PaTrx2, and PaTrx3 are predicted to be canonical cytosolic proteins without additional domains. Targeted disruption of Pa
Trx1
, Pa
Trx2
, and Pa
Trx3
shows that PaTrx1 is the major thioredoxin involved in sulfur metabolism. Deletions have no effect on peroxide resistance; however, data show that either PaTrx1 or PaTrx3 is necessary for sexual reproduction and for the development of the crippled growth cell degeneration (CG), processes that also required the PaMpk1 mitogen-activated protein kinase (MAPK) pathway. Since Pa
Trx1
Pa
Trx3
mutants show not an enhancement but rather an impairment in CG, it seems unlikely that PaTrx1 and PaTrx3 thioredoxins participate in the inhibition of this MAPK pathway. Altogether, these results underscore a role for thioredoxins in fungal development.
A Rho3 Homolog Is Essential for Appressorium Development and Pathogenicity of
Magnaporthe grisea
Authors: Zheng Wu , Chen Jisheng , Liu Wende , Zheng Shiqin , Zhou Jie , Lu Guodong , Wang Zonghua
Published: October 2007

The small GTPase Rho3 is conserved in fungi and plays a key role in the control of cell polarity and exocytosis in yeast. In this report, we show that a Rho3 homolog, MgRho3, is dispensable for polarized hyphal growth in the rice blast fungus
Magnaporthe grisea
. However, MgRho3 is required for plant infection. Appressoria formed by the Mg
rho3
deletion mutants are morphologically abnormal and defective in plant penetration. Conidia of the Mg
rho3
deletion mutants are narrower than those of the wild-type strain and delayed in germination. Transformants expressing a dominant negative Mg
rho3
allele exhibit similar phenotypes as the Mg
rho3
deletion mutant, while transformants expressing a constitutively active allele of Mg
Rho3
can produce normal conidia but remain defective in appressorium formation and plant infection. In contrast, overexpression of wild-type Mg
Rho3
increases the infectivity of
M. grisea
. Our results reveal a new role for the conserved Rho3 as a critical regulator of developmental processes and pathogenicity of
M. grisea
.
Formation of Atroviridin by
Hypocrea atroviridis
Is Conidiation Associated and Positively Regulated by Blue Light and the G Protein GNA3
Authors: Komon-Zelazowska Monika , Neuhof Torsten , Dieckmann Ralf , von Döhren Hans , Herrera-Estrella Alfredo , Kubicek Christian P. , Druzhinina Irina S.
Published: October 2007

Species of the mycoparasitic fungal genus
Hypocrea/Trichoderma
are prominent producers of peptaibols, a class of small linear peptides of fungal origin. Some of these peptaibols have been shown to act synergistically with cell-wall-degrading enzymes in the inhibition of the growth of other fungi in vitro and in vivo. Here we present the structure of the
Hypocrea atroviridis
peptaibol synthetase gene (
pbs1
), deduced from the genome sequence of
H. atroviridis
. It consists of 19 typical peptide synthetase modules with the required additional modifying domains at the N and C termini. Phylogenetic and similarity analyses of the individual amino acid-activating modules is consistent with its ability to synthesize atroviridins. Matrix-assisted laser desorption ionization-time of flight mass spectrometry of surface-grown cultures of
H. atroviridis
showed that no peptaibols were formed during vegetative growth, but a microheterogenous mixture of atroviridins accumulated when the colonies started to sporulate. This correlation between sporulation and atroviridin formation was shown to be independent of the pathway inducing sporulation (i.e., light, mechanical injury and carbon starvation, respectively). Atroviridin formation was dependent on the function of the two blue light regulators, BLR1 and BLR2, under some but not all conditions of sporulation and was repressed in a
pkr1
(regulatory subunit of protein kinase A) antisense strain with constitutively active protein kinase A. Conversely, however, loss of function of the Gα-protein GNA3, which is a negative regulator of sporulation and leads to a hypersporulating phenotype, fully impairs atroviridin formation. Our data show that formation of atroviridin by
H. atroviridis
occurs in a sporulation-associated manner but is uncoupled from it at the stage of GNA3.
Endoplasmic Reticulum α-Glycosidases of
Candida albicans
Are Required for N Glycosylation, Cell Wall Integrity, and Normal Host-Fungus Interaction
Authors: Mora-Montes Héctor M. , Bates Steven , Netea Mihai G. , Díaz-Jiménez Diana F. , López-Romero Everardo , Zinker Samuel , Ponce-Noyola Patricia , Kullberg Bart Jan , Brown Alistair J. P. , Odds Frank C. , Flores-Carreón Arturo , Gow Neil A. R.
Published: October 2007

The cell surface of
Candida albicans
is enriched in highly glycosylated mannoproteins that are involved in the interaction with the host tissues. N glycosylation is a posttranslational modification that is initiated in the endoplasmic reticulum (ER), where the Glc
3
Man
9
GlcNAc
2
N-glycan is processed by α-glucosidases I and II and α1,2-mannosidase to generate Man
8
GlcNAc
2
. This N-oligosaccharide is then elaborated in the Golgi to form N-glycans with highly branched outer chains rich in mannose. In
Saccharomyces cerevisiae, CWH41, ROT2
, and
MNS1
encode for α-glucosidase I, α-glucosidase II catalytic subunit, and α1,2-mannosidase, respectively. We disrupted the
C. albicans CWH41, ROT2
, and
MNS1
homologs to determine the importance of N-oligosaccharide processing on the N-glycan outer-chain elongation and the host-fungus interaction. Yeast cells of Ca
cwh41
Δ, Ca
rot2
Δ, and Ca
mns1
Δ null mutants tended to aggregate, displayed reduced growth rates, had a lower content of cell wall phosphomannan and other changes in cell wall composition, underglycosylated β-
N
-acetylhexosaminidase, and had a constitutively activated PKC-Mkc1 cell wall integrity pathway. They were also attenuated in virulence in a murine model of systemic infection and stimulated an altered pro- and anti-inflammatory cytokine profile from human monocytes. Therefore, N-oligosaccharide processing by ER glycosidases is required for cell wall integrity and for host-fungus interactions.
Burkholderia cenocepacia
C5424 Produces a Pigment with Antioxidant Properties Using a Homogentisate Intermediate
Authors: Keith Karen E. , Killip Lauren , He Panqing , Moran Graham R. , Valvano Miguel A.
Published: October 2007

Burkholderia cenocepacia
is a gram-negative opportunistic pathogen that belongs to the
Burkholderia cepacia
complex.
B. cenocepacia
can survive intracellularly within phagocytic cells, and some epidemic strains produce a brown melanin-like pigment that can scavenge free radicals, resulting in the attenuation of the host cell oxidative burst. In this work, we demonstrate that the brown pigment produced by
B. cenocepacia
C5424 is synthesized from a homogentisate (HGA) precursor. The disruption of BCAL0207 (
hppD
) by insertional inactivation resulted in loss of pigmentation. Steady-state kinetic analysis of the BCAL0207 gene product demonstrated that it has 4-hydroxyphenylpyruvic acid dioxygenase (HppD) activity. Pigmentation could be restored by complementation providing
hppD
in
trans
. The
hppD
mutant was resistant to paraquat challenge but sensitive to H
2
O
2
and to extracellularly generated superoxide anions. Infection experiments in RAW 264.7 murine macrophages showed that the nonpigmented bacteria colocalized in a dextran-positive vacuole, suggesting that they are being trafficked to the lysosome. In contrast, the wild-type strain did not localize with dextran. Colocalization of the nonpigmented strain with dextran was reduced in the presence of the NADPH oxidase inhibitor diphenyleneiodonium, and also the inducible nitric oxide inhibitor aminoguanidine. Together, these observations suggest that the brown pigment produced by
B. cenocepacia
C5424 is a pyomelanin synthesized from an HGA intermediate that is capable of protecting the organism from in vitro and in vivo sources of oxidative stress.
Molecular Basis of the Defective Heat Stress Response inMycobacterium leprae
Authors: Williams Diana L. , Pittman Tana L. , Deshotel Mike , Oby-Robinson Sandra , Smith Issar , Husson Robert
Published: October 2007

Mycobacterium leprae, a major human pathogen, grows poorly at 37°C. The basis for its inability to survive at elevated temperatures was investigated. We determined thatM. lepraelacks a protective heat shock response as a result of the lack of transcriptional induction of the alternative sigma factor genessigEandsigBand the major heat shock operons, HSP70 and HSP60, even though heat shock promoters and regulatory circuits for these genes appear to be intact.M. leprae sigEwas found to be capable of complementing the defective heat shock response of mycobacterialsigEknockout mutants only in the presence of a functional mycobacterialsigH, which orchestrates the mycobacterial heat shock response. Since thesigHofM. lepraeis a pseudogene, these data support the conclusion that a key aspect of the defective heat shock response inM. lepraeis the absence of a functionalsigH. In addition, 68% of the genes induced during heat shock inM. tuberculosiswere shown to be either absent from theM. lepraegenome or were present as pseudogenes. Among these is thehsp/acr2gene, whose product is essential forM. tuberculosissurvival during heat shock. Taken together, these results suggest that the reduced ability ofM. lepraeto survive at elevated temperatures results from the lack of a functional transcriptional response to heat shock and the absence of a full repertoire of heat stress response genes, includingsigH.
Direct Transcriptional Control of the Plasminogen Activator Gene of
Yersinia pestis
by the Cyclic AMP Receptor Protein
Authors: Kim Tae-Jong , Chauhan Sadhana , Motin Vladimir L. , Goh Ee-Been , Igo Michele M. , Young Glenn M.
Published: October 2007

Horizontal gene transfer events followed by proper regulatory integration of a gene drive rapid evolution of bacterial pathogens. A key event in the evolution of the highly virulent plague bacterium
Yersinia pestis
was the acquisition of plasmid pPCP1, which carries the plasminogen activator gene,
pla
. This promoted the bubonic form of the disease by increasing bacterial dissemination from flea bite sites and incidentally enhanced replication in respiratory airways during pneumonic infection. We determined that expression of
pla
is controlled by the global regulator cyclic AMP (cAMP) receptor protein (Crp). This transcription factor is well conserved among distantly related bacteria, where it acts as a soluble receptor for the ubiquitous signaling molecule cAMP and controls a global network of metabolic and stress-protective genes. Crp has a similar physiological role in
Y. pestis
since loss of its function resulted in an inability to metabolize a variety of nonglucose substrates. Activation of
pla
expression requires a transcription activation element of the
pla
promoter that serves as a Crp binding site. Crp interaction with this site was demonstrated to occur only in the presence of cAMP. Alteration of the Crp binding site nucleotide sequence prevented in vitro formation of Crp-DNA complexes and inhibited in vivo expression of
pla
. The placement of
pla
under direct regulatory control of Crp highlights how highly adapted pathogens integrate laterally acquired genes to coordinate virulence factor expression with global gene networks to maintain homeostasis through the infectious life cycle.
Regulation of Multidrug Efflux Systems Involved in Multidrug and Metal Resistance of
Salmonella enterica
Serovar Typhimurium
Authors: Nishino Kunihiko , Nikaido Eiji , Yamaguchi Akihito
Published: October 2007

Multidrug-resistant strains of
Salmonella
are now encountered frequently, and the rates of multidrug resistance have increased considerably in recent years. Here, we report that the two-component regulatory system BaeSR increases multidrug and metal resistance in
Salmonella
through the induction of drug efflux systems. Screening of random fragments of genomic DNA for the ability to increase β-lactam resistance in
Salmonella enterica
led to the isolation of a plasmid containing
baeR
, which codes for the response regulator of BaeSR. When overexpressed,
baeR
significantly increased the resistance of the Δ
acrB
strain to oxacillin, cloxacillin, and nafcillin.
baeR
overexpression conferred resistance to novobiocin and deoxycholate, as well as to β-lactams in
Salmonella
. The increase in drug resistance caused by
baeR
overexpression was completely suppressed by deletion of the multifunctional outer membrane channel gene
tolC
. TolC interacts with different drug efflux systems. Among the nine drug efflux systems in
Salmonella
, quantitative real-time PCR analysis showed that BaeR induced the expression of
acrD
and
mdtABC
. Double deletion of these two genes completely suppressed BaeR-mediated multidrug resistance, whereas single deletion of either gene did not. The promoter regions of
acrD
and
mdtABC
harbor binding sites for the response regulator BaeR, which activates
acrD
and
mdtABC
transcription in response to indole, copper, and zinc. In addition to their role in multidrug resistance, we found that BaeSR, AcrD, and MdtABC contribute to copper and zinc resistance in
Salmonella
. Our results indicate that the BaeSR system increases multidrug and metal resistance in
Salmonella
by inducing the AcrD and MdtABC drug efflux systems. We found a previously uncharacterized physiological role for the AcrD and MdtABC multidrug efflux systems in metal resistance.
Conservation and Evolutionary Dynamics of the
agr
Cell-to-Cell Communication System across Firmicutes
Authors: Wuster Arthur , Babu M. Madan
Published: October 2007

We present evidence that the
agr
cell-to-cell communication system is present across firmicutes, including the human pathogen
Clostridium perfringens
. Although we find that the
agr
system is evolutionarily conserved and that the general functions which it regulates are similar in different species, the individual regulated genes are not the same. This suggests that the regulatory network controlled by
agr
is dynamic and evolves rapidly.
YhbO Protects Cells against Multiple Stresses
Authors: Abdallah Jad , Caldas Teresa , Kthiri Fatoum , Kern Renée , Richarme Gilbert
Published: October 2007

YhbO is a member of the DJ-1/ThiJ/Pfp1 superfamily, which includes chaperones, peptidases, and the Parkinson's disease protein DJ-1. AyhbO-disrupted mutant ofEscherichia coliis highly sensitive to oxidative, thermal, UV, and pH stresses, and the putative nucleophilic cysteine C104 of YhbO is required for stress resistance. These results suggest that YhbO affects a central process in stress management.
Cohabitation of Two DifferentlexARegulons inPseudomonas putida
Authors: Abella Marc , Campoy Susana , Erill Ivan , Rojo Fernando , Barbé Jordi
Published: October 2007

In contrast to the vast majority of the members of the domainBacteria, severalPseudomonasandXanthomonasspecies have twolexAgenes, whose products have been shown to recognize different LexA binding motifs, making them an interesting target for studying the interplay between cohabiting LexA regulons in a single species. Here we report an analysis of the genetic composition of the two LexA regulons ofPseudomonas putidaKT2440 performed with a genomic microarray. The data obtained indicate that one of the two LexA proteins (LexA1) seems to be in control of the conventionalEscherichia coli-like SOS response, while the other LexA protein (LexA2) regulates only its own transcriptional unit, which includes theimuA, imuB, anddnaE2genes, and a gene (PP_3901) from a residentP. putidaprophage. Furthermore, PP_3901 is also regulated by LexA1 and is required for DNA damage-mediated induction of severalP. putidaresident prophage genes. In silico searches suggested that this marked asymmetry in regulon contents also occurs in otherPseudomonasspecies with twolexAgenes, and the implications of this asymmetry in the evolution of the SOS network are discussed.
Functional Organization of the Autotransporter Adhesin Involved in Diffuse Adherence
Authors: Charbonneau Marie-Ève , Mourez Michael
Published: October 2007

TheEscherichia coliadhesin involved in diffuse adherence (AIDA-I) is a multifunctional autotransporter protein that mediates bacterial aggregation and biofilm formation, as well as adhesion and invasion of cultured epithelial cells. To elucidate the structure-function relationships of AIDA-I, we performed transposon-based linker scanning mutagenesis and constructed mutants with site-directed deletions. Twenty-nine different mutants with insertions that did not affect protein expression were obtained. Eleven mutants were deficient for one or two but not all of the functions associated with the expression of AIDA-I. Functional characterization of the transposon mutants and of an additional deletion mutant suggested that the N-terminal third of mature AIDA-I is involved in binding of this protein to cultured epithelial cells. The purified product of the putative domain could bind to cultured epithelial cells, confirming the importance of this region in adhesion. We also identified several different mutants in which invasion and adhesion were changed to different extents and two mutants in which autoaggregation and biofilm formation were also affected differently. These results suggest that although conceptually linked, adhesion and invasion, as well as autoaggregation and biofilm formation, are phenomena that may rely on distinct mechanisms when they are mediated by AIDA-I. This study sheds new light on the workings of a protein belonging to an emerging family of strikingly versatile virulence factors.
A Horizontal Gene Transfer Event Defines Two Distinct Groups within
Burkholderia pseudomallei
That Have Dissimilar Geographic Distributions
Authors: Tuanyok Apichai , Auerbach Raymond K. , Brettin Thomas S. , Bruce David C. , Munk A. Christine , Detter J. Chris , Pearson Talima , Hornstra Heidie , Sermswan Rasana W. , Wuthiekanun Vanaporn , Peacock Sharon J. , Currie Bart J. , Keim Paul , Wagner David M.
Published: October 2007

Burkholderia pseudomallei
is the etiologic agent of melioidosis. Many disease manifestations are associated with melioidosis, and the mechanisms causing this variation are unknown; genomic differences among strains offer one explanation. We compared the genome sequences of two strains of
B. pseudomallei
: the original reference strain K96243 from Thailand and strain MSHR305 from Australia. We identified a variable homologous region between the two strains. This region was previously identified in comparisons of the genome of
B. pseudomallei
strain K96243 with the genome of strain E264 from the closely related
B. thailandensis
. In that comparison, K96243 was shown to possess a horizontally acquired
Yersinia
-like fimbrial (YLF) gene cluster. Here, we show that the homologous genomic region in
B. pseudomallei
strain 305 is similar to that previously identified in
B. thailandensis
strain E264. We have named this region in
B. pseudomallei
strain 305 the
B. thailandensis
-like flagellum and chemotaxis (BTFC) gene cluster. We screened for these different genomic components across additional genome sequences and 571
B. pseudomallei
DNA extracts obtained from regions of endemicity. These alternate genomic states define two distinct groups within
B. pseudomallei
: all strains contained either the BTFC gene cluster (group BTFC) or the YLF gene cluster (group YLF). These two groups have distinct geographic distributions: group BTFC is dominant in Australia, and group YLF is dominant in Thailand and elsewhere. In addition, clinical isolates are more likely to belong to group YLF, whereas environmental isolates are more likely to belong to group BTFC. These groups should be further characterized in an animal model.
Characterization of MazF

Sa

, an Endoribonuclease from
Staphylococcus aureus
Authors: Fu Zhibiao , Donegan Niles P. , Memmi Guido , Cheung Ambrose L.
Published: October 2007

The
mazEF
homologs of
Staphylococcus aureus
, designated

mazEF
sa

, have been shown to cotranscribe with the
sigB
operon under stress conditions. In this study, we showed that MazEF

Sa

, as with their
Escherichia coli
counterparts, compose a toxin-antitoxin module wherein MazF

Sa

leads to rapid cell growth arrest and loss in viable CFU upon overexpression. MazF

Sa

is a novel sequence-specific endoribonuclease which cleaves mRNA to inhibit protein synthesis. Using
ctpA
mRNA as the model substrate both in vitro and in vivo, we demonstrated that MazF

Sa

cleaves single-strand RNA preferentially at the 5′ side of the first U or 3′ side of the second U residue within the consensus sequences VUUV′ (where V and V′ are A, C, or G and may or may not be identical). Binding studies confirmed that the antitoxin MazE

Sa

binds MazF

Sa

to form a complex to inhibit the endoribonuclease activity of MazF

Sa

. Contrary to the system in
E. coli
, exposure to selected antibiotics augmented

mazEF
sa

transcription, akin to what one would anticipate from the environmental stress response of the
sigB
system. These data indicate that the
mazEF
system of
S. aureus
differs from the gram-negative counterparts with respect to mRNA cleavage specificity and antibiotic stresses.
Single-Molecule Force Spectroscopy of Mycobacterial Adhesin-Adhesin Interactions
Authors: Verbelen Claire , Raze Dominique , Dewitte Frédérique , Locht Camille , Dufrêne Yves F.
Published: October 2007

The heparin-binding hemagglutinin (HBHA) is one of the few virulence factors identified for
Mycobacterium tuberculosis
. It is a surface-associated adhesin that expresses a number of different activities, including mycobacterial adhesion to nonphagocytic cells and microbial aggregation. Previous evidence indicated that HBHA is likely to form homodimers or homopolymers via a predicted coiled-coil region located within the N-terminal portion of the molecule. Here, we used single-molecule atomic-force microscopy to measure individual homophilic HBHA-HBHA interaction forces. Force curves recorded between tips and supports derivatized with HBHA proteins exposing their N-terminal domains showed a bimodal distribution of binding forces reflecting the formation of dimers or multimers. Moreover, the binding peaks showed elongation forces that were consistent with the unfolding of α-helical coiled-coil structures. By contrast, force curves obtained for proteins exposing their lysine-rich C-terminal domains showed a broader distribution of binding events, suggesting that they originate primarily from intermolecular electrostatic bridges between cationic and anionic residues rather than from specific coiled-coil interactions. Notably, similar homophilic HBHA-HBHA interactions were demonstrated on live mycobacteria producing HBHA, while they were not observed on an HBHA-deficient mutant. Together with the fact that HBHA mediates bacterial aggregation, these observations suggest that the single homophilic HBHA interactions measured here reflect the formation of multimers that may promote mycobacterial aggregation.
Regulation of
d
-Xylose Metabolism in
Caulobacter crescentus
by a LacI-Type Repressor
Authors: Stephens Craig , Christen Beat , Watanabe Kelly , Fuchs Thomas , Jenal Urs
Published: October 2007

In the oligotrophic freshwater bacterium
Caulobacter crescentus
,
d
-xylose induces expression of over 50 genes, including the
xyl
operon, which encodes key enzymes for xylose metabolism. The promoter (P

xylX

) controlling expression of the
xyl
operon is widely used as a tool for inducible heterologous gene expression in
C. crescentus
. We show here that P

xylX

and at least one other promoter in the xylose regulon (P

xylE

) are controlled by the CC3065 (
xylR
) gene product, a LacI-type repressor. Electrophoretic gel mobility shift assays showed that operator binding by XylR is greatly reduced in the presence of
d
-xylose. The data support the hypothesis that there is a simple regulatory mechanism in which XylR obstructs xylose-inducible promoters in the absence of the sugar; the repressor is induced to release DNA upon binding
d
-xylose, thereby freeing the promoter for productive interaction with RNA polymerase. XylR also has an effect on glucose metabolism, as
xylR
mutants exhibit reduced expression of the Entner-Doudoroff operon and their ability to utilize glucose as a sole carbon and energy source is compromised.
Isolation of Cold-Sensitive
yidC
Mutants Provides Insights into the Substrate Profile of the YidC Insertase and the Importance of Transmembrane 3 in YidC Function
Authors: Yuan Jijun , Phillips Gregory J. , Dalbey Ross E.
Published: October 2007

YidC, a 60-kDa integral membrane protein, plays an important role in membrane protein insertion in bacteria. YidC can function together with the SecYEG machinery or operate independently as a membrane protein insertase. In this paper, we describe two new
yidC
mutants that lead to a cold-sensitive phenotype in bacterial cell growth. Both alleles impart a cold-sensitive phenotype and result from point mutations localized to the third transmembrane (TM3) segment of YidC, indicating that this region is crucial for YidC function. We found that the
yidC
(
C423R
) mutant confers a weak phenotype on membrane protein insertion while a
yidC
(
P431L
) mutant leads to a stronger phenotype. In both cases, the affected substrates include the Pf3 coat protein and ATP synthase F
1
F
o
subunit c (F
o
C), while CyoA (the quinol binding subunit of the cytochrome
bo3
quinol oxidase complex) and wild-type procoat are slightly affected or not affected in either cold-sensitive mutant. To determine if the different substrates require various levels of YidC activity for membrane insertion, we performed studies where YidC was depleted using an arabinose-dependent expression system. We found that −3M-PC-Lep (a construct with three negatively charged residues inserted into the middle of the procoat-Lep [PC-Lep] protein) and Pf3 P2 (a construct with the Lep P2 domain added at the C terminus of Pf3 coat) required the highest amount of YidC and that CyoA-N-P2 (a construct with the amino-terminal part of CyoA fused to the Lep P2 soluble domain) and PC-Lep required the least, while F
o
C required moderate YidC levels. Although the cold-sensitive mutations can preferentially affect one substrate over another, our results indicate that different substrates require different levels of YidC activity for membrane insertion. Finally, we obtained several intragenic suppressors that overcame the cold sensitivity of the C423R mutation. One pair of mutations suggests an interaction between TM2 and TM3 of YidC. The studies reveal the critical regions of the YidC protein and provide insight into the substrate profile of the YidC insertase.
The role of a recombinant hybrid protein based ELISA for the serodiagnosis ofOnchocerca volvulus
Authors: Andrews J A , Bligh W J , Chiodini P L , Bradley J E , Nde P N , Lucius R
Published: October 2007

Aims:

Onchocerca volvulusinfection is traditionally diagnosed by examination of skin snips for the presence of microfilariae. A disadvantage of this method is the low sensitivity particularly with light or prepatent infection. Serodiagnosis using recombinant-antigen-based assays may provide a more sensitive diagnostic test. An ELISA based on a recombinant antigen OvH3 has previously been validated using sera from endemic areas. This study investigated the role of this ELISA-based assay for use in the serodiagnosis of onchocerciasis in non-endemic areas.

Methods:

The ELISA-based assay was tested on sera from untreated patients with known onchocerciasis and on untreated and treated patients with definite or probable onchocerciasis identified from a hospital diagnostic database. The assay was also tested on sera from patients with other helminthic infections to determine the sensitivity and specificity of this assay in a tertiary referral laboratory dealing with sera from a variety of patients.

Results:

The sensitivity and specificity of the OvH3 assay were 93.2% and 93.5%, respectively, when tested on non-endemic patients with clinical diagnosis of onchocerciasis.

Conclusions:

This study demonstrates the potential role of the assay as a sensitive and specific test for use in the serodiagnosis of onchocerciasis in a reference laboratory dealing with sera from patients in non-endemic setting.
A study of the prevalence of vitamin K deficiency in patients with cancer referred to a hospital palliative care team and its association with abnormal haemostasis
Authors: Harrington D J , Western H , Seton-Jones C , Rangarajan S , Beynon T , Shearer M J
Published: October 2007

Background:

Many patients with advanced cancer are malnourished. Anorexia is common, as is the use of chemotherapy, which may cause nausea and poor appetite. Ten per cent of these patients experience haemorrhagic events.

Aim:

Since vitamin K deficiency (VKD) causes bleeding, to establish the prevalence of VKD in patients with advanced cancer receiving palliative care.

Methods:

Serum concentrations of vitamin K1and undercarboxylated factor II (PIVKA-II) were determined in 46 (17 male/29 female) inpatients aged 26–85 (mean 58) years. INR and liver function tests (bilirubin, ALT, GGT and ALP) were also performed.

Results:

Vitamin K1was below the lower limit of the reference range (0.33 nmol/l) in 22% of patients. 78% of patients had some degree of functional VKD indicated by raised (>0.2 AU/ml) PIVKA-II. Six patients (13%) had a prolonged INR, all of whom had raised PIVKA-II and GGT; 4 also had vitamin K1<0.33 nmol/l. Three patients (6.5%) had clinically significant VKD characterised by INR >1.5, PIVKA-II >10 AU/ml, and undetectable vitamin K1.

Conclusions:

Patients with advanced cancer are prone to VKD which, while usually subclinical, may develop to a clinically relevant prolongation of the INR. Serum measurements of vitamin K1and PIVKA-II can be used to detect VKD and monitor vitamin K status before an increased risk of bleeding develops.
Distribution of neural cell adhesion molecule (NCAM/CD56) in gastrointestinal stromal tumours and their intra-abdominal mesenchymal mimics
Authors: Agaimy A , Wünsch P H
Published: October 2007

Background:

The distribution and reactivity pattern of neural cell adhesion molecule (NCAM/CD56) in gastrointestinal stromal tumours (GISTs) and their mesenchymal mimics have not been investigated in the KIT era.

Methods:

275 histologically and immunohistochemically well characterised primary and metastatic intra-abdominal mesenchymal lesions were analysed by conventional immunohistochemistry, with emphasis on GIST and GI smooth muscle neoplasms.

Results:

CD56 expression was seen in 18/21 (86%), 4/5 (80%), 26/34 (76%), and 32/168 (19%) of primary GI leiomyomas, schwannomas, leiomyosarcomas, and GISTs, respectively. Reactivity in GISTs was mostly focal. Of 6% strongly staining GISTs, 71% were either malignant clinically or assigned a high risk prognostic group. CD56 expression in GISTs varied greatly with histological type (seen in 50% and 7% of epithelioid and spindled GISTs, respectively) and anatomical site (in 33%, 10%, 1%, and 0% of rectal, gastric, small intestinal and oesophageal GISTs, respectively). A variable, but inconsistent expression was seen in miscellaneous lesions including dedifferentiated liposarcoma, abdominopelvic PEComa, myo/fibroblastic sarcoma and malignant fibrous histiocytoma. Mesenteric fibromatoses, angiosarcoma/Kaposi sarcoma, reactive tumefactive fibrogenic lesions and 12/13 primary anorectal and oesophageal melanomas were negative.

Conclusion:

Results confirmed the ubiquity and non-specificity of CD56 as a neurogenic marker. Except for a subset of epithelioid gastric and high-grade rectal GISTs, CD56 expression is rare in GISTs, contrasting with true leiomyomatous and neurogenic neoplasms. CD56 plays a limited role in the differential diagnosis of GIST. Its potential role as a marker of adverse outcome in GISTs remains to be further investigated.
Effects of PCBs, p,p′-DDT, p,p′-DDE, HCB and β-HCH on thyroid function in preschool children
Authors: Álvarez-Pedrerol M , Ribas-Fitó N , Torrent M , Carrizo D , Grimalt J O , Sunyer J
Published: October 2007

Objective:

Several studies have shown that some organochlorine compounds (OCs) can interfere with the thyroid system. As thyroid hormones (THs) are essential for normal brain development, it is important to study the association between THs and OCs during pregnancy and childhood. We have evaluated the relationship between thyroid function and OCs in preschool children.

Methods:

Children from a general population birth cohort in Menorca (n = 259), Spain were assessed at the age of 4 years. Concentrations of THs (free T4 and total T3), thyrotropin (TSH) and a range of OCs were measured in peripheral blood.

Results:

Blood levels of dichlorodiphenyl trichloroethane (p,p′-DDT), β-hexachlorocylcohexane (β-HCH), polychlorinated biphenyls (congeners PCB-138, PCB-153 and PCB-118) were related to lower total T3 levels (p<0.05). In addition, free T4 was inversely associated with PCB-118, while no relationship was found between TSH and any of the measured OCs.

Conclusions:

This study suggests that even at background levels of exposure, OCs may affect the thyroid system, particularly total T3 levels.
An occupational health intervention programme for workers at high risk for sickness absence. Cost effectiveness analysis based on a randomised controlled trial
Authors: Taimela S , Justén S , Aronen P , Sintonen H , Läärä E , Malmivaara A , Tiekso J , Aro T
Published: October 2007

Objectives:

To determine whether, from a healthcare perspective, a specific occupational health intervention is cost effective in reducing sickness absence when compared with usual care in occupational health in workers with high risk of sickness absence.

Methods:

Economic evaluation alongside a randomised controlled trial. 418 workers with high risk of sickness absence from one corporation were randomised to intervention (n = 209) or to usual care (n = 209). The subjects in the intervention group were invited to occupational health service for a consultation. The intervention included, if appropriate, a referral to specialist treatment. Register data of sickness absence were available for 384 subjects and questionnaire data on healthcare costs from 272 subjects. Missing direct total cost data were imputed using a two-part regression model. Primary outcome measures were sickness absence days and direct healthcare costs up to 12 months after randomisation. Cost effectiveness (CE) was expressed as an incremental CE ratio, CE plane and CE acceptability curve with both available direct total cost data and missing total cost data imputed.

Results:

After one year, the mean of sickness absence was 30 days in the usual care group (n = 192) and 11 days less (95% CI 1 to 20 days) in the intervention group (n = 192). Among the employees with available cost data, the mean days of sickness absence were 22 and 24, and the mean total cost €974 and €1049 in the intervention group (n = 134) and in the usual care group (n = 138), respectively. The intervention turned out to be dominant—both cost saving and more effective than usual occupational health care. The saving was €43 per sickness absence day avoided with available direct total cost data, and €17 with missing total cost data imputed.

Conclusions:

One year follow-up data show that occupational health intervention for workers with high risk of sickness absence is a cost effective use of healthcare resources.
A normative study of levels of uranium in the urine of British Forces personnel
Authors: Miller B G , Colvin A P , Hutchison P A , Tait H , Dempsey S , Lewis D , Soutar C A
Published: October 2007

Objectives:

The UK Ministry of Defence (MoD) controls a biological monitoring programme that includes testing for uranium in personnel who served in the conflict in Iraq in 2003. To help interpret the results, the MoD commissioned this study to quantify a normative reference distribution of urinary uranium concentrations in military personnel who had not served in that conflict.

Methods:

The study selected and visited various military establishments to recruit a representative mix of ranks, genders and occupational groups (combat, support and auxiliary). A standardised protocol and recruitment questionnaire were used. The 125 ml spot urine samples collected were analysed for uranium and creatinine concentrations and (where possible) for uranium isotope238U/235U ratio.

Results:

Samples from 732 eligible subjects were analysed. Adjusted uranium concentrations ranged up to 556 ng.g−1creatinine, somewhat higher than reference values quoted for the USA but much lower than recorded in granite areas such as Finland. Isotope ratio measurements were available for 125 samples (17) with the highest concentrations; these all had a natural isotope signature and no evidence of depleted uranium (DU). On average, urinary uranium concentrations were somewhat lower in officers than in other ranks; they differed also across the services, the Navy being lowest and the Army highest. The levels give no concern regarding health risks in the personnel studied.

Conclusion:

Since even the highest values were from natural sources, we assume the differences represent differences in ingestion of natural uranium. Definition of a reference distribution or normal values will depend on the subpopulation of interest.
Geochemical constraints on the provenance and depositional setting of sedimentary rocks from the islands of Chios, Inousses and Psara, Aegean Sea, Greece: implications for the evolution of Palaeotethys
Authors: Meinhold Guido , Kostopoulos Dimitrios , Reischmann Thomas
Published: October 2007

The provenance and depositional setting of Late Palaeozoic and Early Mesozoic clastic sediments from the eastern Aegean archipelago are examined here for the first time using whole-rock geochemistry and composition of detrital chrome spinel. Major- and trace-element data for Late Palaeozoic and Permo-Triassic clastic sediments from the Lower and Upper Units of Chios are compatible with an acidic to intermediate source, minor input of (ultra)mafic detritus and recycling of older sedimentary components. Chondrite-normalized REE profiles are uniform with light REE enrichments (La
N
/Yb
N
c
. 7.7), negative Eu anomalies (Eu/Eu*
c
. 0.67) and flat heavy REE patterns (Gd
N
/Yb
N
c
. 1.5), indicating an upper continental crustal source and/or young differentiated arc material. Detrital chrome spinel from the clastic sediments of Chios has Cr-number (Cr/(Cr + Al)) values between 0.29 and 0.89 and Mg-number (Mg/(Mg + Fe
2+
)) values between 0.24 and 0.70, suggesting a probably mixed (ultra)mafic source involving ridge peridotites (mid-ocean ridge type), fore-arc peridotites and island-arc basalts. The metasediments from the islands of Inousses and Psara have similar whole-rock chemical signatures to those of Chios, although no evidence was found for an (ultra)mafic source. We conclude that both the Late Palaeozoic sediments from the Lower and Upper Units of Chios and the metasediments from Inousses and Psara were deposited in a continental island-arc setting until at least Late Permian times, probably at a single Palaeotethyan margin. They are interpreted to be allochthonous, tectonically transported to their present position by Late Mesozoic to Cenozoic orogenic processes.
The geological history of Geysir, Iceland: a tephrochronological approach to the dating of sinter
Authors: Jones Brian , Renaut Robin W. , Torfason Helgi , Owen R.Bernhart
Published: October 2007

The vertical succession through the discharge apron of Geysir, up to 3.5 m thick, is formed of five lithological units, A–E. Units A, B, C, and E are formed of siliceous sinters whereas Unit D is a brown ash bed. Tephra grains in units C, D, and E came from Katla, Hekla, and Veiðivötn. Katla tephra in Unit D has been largely altered to palagonite. Integration of tephrochronological ages, historical records, and geological and geomorphological relationships shows that the Geysir succession developed in four main phases. During Phase I,
c
. 10 000–4000 years ago, laminated sinters formed from hot spring waters. Phase II, initiated
c
. 3300 years ago, saw the termination of sinter formation and blanketing of the area with tephra from Katla and Hekla. Phase III,
c
. 3000–900 years ago, represented by the boundary between units D and E, was characterized by weathering and little sinter deposition. Phase IV was initiated with the (re)birth of Geysir 800–900 years ago. Sinter deposited from the discharged waters has, however, been largely confined to the southern part of the discharge apron. Only limited opal-A precipitation has occurred since 1916 because of a decline in Geysir's eruptions.
Temporal floorplanning using the three-dimensional transitive closure subGraph
Authors: Yuh Ping-Hung , Yang Chia-Lin , Chang Yao-Wen
Published: October 2007

Improving logic capacity by time-sharing, dynamically reconfigurable Field Gate Programmable Arrays (FPGAs) are employed to handle designs of high complexity and functionality. In this paper, we use a novel graph-based topological floorplan representation, named
3D-subTCG
(3-Dimensional Transitive Closure subGraph), to deal with the 3-dimensional (temporal) floorplanning/placement problem, arising from dynamically reconfigurable FPGAs. The 3D-subTCG uses three transitive closure graphs to model the temporal and spatial relations between modules. We derive the feasibility conditions for the precedence constraints induced by the execution of the dynamically reconfigurable FPGAs. Because the geometric relationship is transparent to the 3D-subTCG and its induced operations (i.e., we can directly detect the relationship between any two tasks from the representation), we can easily detect any violation of the temporal precedence constraints on 3D-subTCG. We also derive important properties of the 3D-subTCG to reduce the solution space and shorten the running time for 3D (temporal) foorplanning/placement. Experimental results show that our 3D-subTCG-based algorithm is very effective and efficient.
A practical dynamic single assignment transformation
Authors: Vanbroekhoven Peter , Janssens Gerda , Bruynooghe Maurice , Catthoor Francky
Published: October 2007

This paper presents a novel method to construct a dynamic single assignment (DSA) form of array intensive, pointer free C programs. A program in DSA form does not perform any destructive update of scalars and array elements; that is, each element is written at most once. As DSA makes the dependencies between variable references explicit, it facilitates complex analyses and optimizations of programs. Existing transformations into DSA perform a complex data flow analysis with exponential analysis time, and they work only for a limited class of input programs. Our method removes irregularities from the data flow by adding copy assignments to the program, so that it can use simple data flow analyses. The presented DSA transformation scales very well with growing program sizes and overcomes a number of important limitations of existing methods. We have implemented the method and it is being used in the context of memory optimization and verification of those optimizations. Experiments show that in practice, the method scales well indeed, and that added copy operations can be removed in case they are unwanted.
Methodology for operation shuffling and L0 cluster generation for low energy heterogeneous VLIW processors
Authors: Kobayashi Yuki , Jayapala Murali , Raghavan Praveen , Catthoor Francky , Imai Masaharu
Published: October 2007

Clustering L0 buffers is effective for energy reduction in the instruction memory hierarchy of embedded VLIW processors. However, the efficiency of the clustering depends on the schedule of the target application. Especially in heterogeneous or data clustered VLIW processors, determining energy efficient scheduling is more constraining.

This article proposes a realistic technique supported by a tool flow to explore operation shuffling for improving generation of L0 clusters. The tool flow explores assignment of operations for each cycle and generates various schedules. This approach makes it possible to reduce energy consumption for various processor architectures. However, the computational complexity is large because of the huge exploration space. Therefore, some heuristics are also developed, which reduce the size of the exploration space while the solution quality remains reasonable. Furthermore, we also propose a technique to support VLIW processors with multiple data clusters, which is essential to apply the methodology to real world processors.

The experimental results indicate potential gains of up to 27.6% in energy in L0 buffers, through operation shuffling for heterogeneous processor architectures as well as a homogeneous architecture. Furthermore, the proposed heuristics drastically reduce the exploration search space by about 90%, while the results are comparable to full search, with average differences of less than 1%. The experimental results indicate that energy efficiency can be improved in most of the media benchmarks by the proposed methodology, where the average gain is around 10% in comparison with generating clusters without operation shuffling.
Techniques for the synthesis of reversible Toffoli networks
Authors: Maslov D. , Dueck G. W. , Miller D. M.
Published: October 2007

We present certain new techniques for the synthesis of reversible networks of Toffoli gates, as well as improvements to previous methods. Gate count and technology oriented cost metrics are used. Two new synthesis procedures employing Reed-Muller spectra are introduced and shown to complement earlier synthesis approaches. The previously proposed template simplification method is enhanced through the introduction of a faster and more efficient template application algorithm, an updated classification of the templates, and the addition of new templates of sizes 7 and 9. A resynthesis approach is introduced wherein a sequence of gates is chosen from a network, and the reversible specification it realizes is resynthesized as an independent problem in hopes of reducing the network cost. Empirical results are presented to show that the methods are efficient in terms of the realization of reversible benchmark specifications.