bluuebunny/crossref_metadata_2025 huggingface dataset:
Vehicle Handling Parameters Estimation in a Virtual Environment
Authors: Awan Muhammad Adeel , Hameed Amer
Published: May 2012

This article illustrates the use of a virtual vehicle, modelled in MSC Adams/Car, to study and analyse the technique that can be used to extract parameters for handling analysis and subsequently for control purposes. These parameters are usually not available for direct measurement and therefore need to be estimated from other vehicle states. However, presence of process and measurement noise limit the authenticity of these parameters. Kalman filtering algorithm is used as a virtual sensor for noise cancellation and estimation of non-measurable vehicle parameters. This technique uses mathematical model of the plant in conjunction with the system itself to predict the required states. In case of vehicle analysis an experimental vehicle equipped with various sensors is usually used to test and implement the filter; however not all the researchers have direct access to such resources. Therefore this paper describes the possibility of using a virtual vehicle for the purpose.
Fatigue Characterization of Plasma Spray Coated Composites
Authors: Haqu Ziaul , Ahmed Ikram , Talia George , Asmatulu Ramazan
Published: May 2012

In this work we study the fatigue behavior of plasma sprayed metallic coatings on graphite reinforced polymer matrix composites. In the absence of any standards for testing of coatings, the ASTM standard for composites was adopted. Stress-life curves for some of these coatings have been presented and discussed. Our initial results show potentials for fatigue resistance in spite of the materials being widely dissimilar. The coatings appear to be as good as (or superior to) the substrate in failure, i.e. in all the samples tested failure occurred in the substrate first.
Current Educational Issues in Mechanical Engineering Programs
Authors: Nazari G. , Hamidzadeh H. R.
Published: May 2012

It is envisaged that future developments in mechanical engineering point towards an ever increasing interaction among the various branches of the discipline and science, and, even more so between social demands and technology. In this paper a thorough consideration is given to the effects of various values for development and education of new mechanical engineers. Furthermore, the essential issues such as globalization of industry, commerce and the professions, increased community expectations, understanding of ethical conduct and responsibility toward society, and other challenges and opportunities for mechanical engineering education are presented. In addition, the need for effective professional self-regulation and activities such as accreditation and competency assessments are discussed for a viable mechanical engineering program.
Bactericidal Activity of ACH-702 against Nondividing and Biofilm Staphylococci
Authors: Podos Steven D. , Thanassi Jane A. , Leggio Melissa , Pucci Michael J.
Published: May 2012

Many bacterial infections involve slow or nondividing bacterial growth states and localized high cell densities. Antibiotics with demonstrated bactericidal activity rarely remain bactericidal at therapeutic concentrations under these conditions. The isothiazoloquinolone (ITQ) ACH-702 is a potent, bactericidal compound with activity against many antibiotic-resistant pathogens, including methicillin-resistant
Staphylococcus aureus
(MRSA). We evaluated its bactericidal activity under conditions where bacterial cells were not dividing and/or had slowed their growth. Against
S. aureus
cultures in stationary phase, ACH-702 showed concentration-dependent bactericidal activity and achieved a 3-log-unit reduction in viable cell counts within 6 h of treatment at ≥16× MIC values; in comparison, the bactericidal quinolone moxifloxacin and the additional comparator compounds vancomycin, linezolid, and rifampin at 16× to 32× MICs showed little or no bactericidal activity against stationary-phase cells. ACH-702 at 32× MIC retained bactericidal activity against stationary-phase
S. aureus
across a range of inoculum densities. ACH-702 did not kill cold-arrested cells yet remained bactericidal against cells arrested by protein synthesis inhibitors, suggesting that its bactericidal activity against nondividing cells requires active metabolism but not
de novo
protein synthesis. ACH-702 also showed a degree of bactericidal activity at 16× MIC against
S. epidermidis
biofilm cells that was superior to that of moxifloxacin, rifampin, and vancomycin. The bactericidal activity of ACH-702 against stationary-phase staphylococci and biofilms suggests potential clinical utility in infections containing cells in these physiological states.
The RAM Network in Pathogenic Fungi
Authors: Saputo Sarah , Chabrier-Rosello Yeissa , Luca Francis C. , Kumar Anuj , Krysan Damian J.
Published: April 2012

The
r
egulation of
A
ce2 and
m
orphogenesis (RAM) network is a protein kinase signaling pathway conserved among eukaryotes from yeasts to humans. Among fungi, the RAM network has been most extensively studied in the model yeast
Saccharomyces cerevisiae
and has been shown to regulate a range of cellular processes, including daughter cell-specific gene expression, cell cycle regulation, cell separation, mating, polarized growth, maintenance of cell wall integrity, and stress signaling. Increasing numbers of recent studies on the role of the RAM network in pathogenic fungal species have revealed that this network also plays an important role in the biology and pathogenesis of these organisms. In addition to providing a brief overview of the RAM network in
S. cerevisiae
, we summarize recent developments in the understanding of RAM network function in the human fungal pathogens
Candida albicans
,
Candida glabrata
,
Cryptococcus neoformans
,
Aspergillus fumigatus
, and
Pneumocystis
spp.
N
-Acetylglucosamine Induces White-to-Opaque Switching and Mating in Candida tropicalis, Providing New Insights into Adaptation and Fungal Sexual Evolution
Authors: Xie Jing , Du Han , Guan Guobo , Tong Yaojun , Kourkoumpetis Themistoklis K. , Zhang Lixin , Bai Feng-yan , Huang Guanghua
Published: April 2012

Pathogenic fungi are capable of switching between different phenotypes, each of which has a different biological advantage. In the most prevalent human fungal pathogen,
Candida albicans
, phenotypic transitions not only improve its adaptation to a continuously changing host microenvironment but also regulate sexual mating. In this report, we show that
Candida tropicalis
, another important human opportunistic pathogen, undergoes reversible and heritable phenotypic switching, referred to as the “white-opaque” transition. Here we show that
N
-acetylglucosamine (GlcNAc), an inducer of white-to-opaque switching in
C. albicans
, promotes opaque-cell formation and mating and also inhibits filamentation in a number of natural
C. tropicalis
strains. Our results suggest that host chemical signals may facilitate this phenotypic switching and mating of
C. tropicalis
, which had been previously thought to reproduce asexually. Overexpression of the
C. tropicalis WOR1
gene in
C. albicans
induces opaque-cell formation. Additionally, an intermediate phase between white and opaque was observed in
C. tropicalis
, indicating that the switching could be tristable.
Function of Cryptococcus neoformans KAR7 (
SEC66
) in Karyogamy during Unisexual and Opposite-Sex Mating
Authors: Lee Soo Chan , Heitman Joseph
Published: April 2012

The human basidiomycetous fungal pathogen
Cryptococcus neoformans
serves as a model fungus to study sexual development and produces infectious propagules, basidiospores, via the sexual cycle. Karyogamy is the process of nuclear fusion and an essential step to complete mating. Therefore, regulation of nuclear fusion is central to understanding sexual development of
C. neoformans
. However, our knowledge of karyogamy genes was limited. In this study, using a BLAST search with the
Saccharomyces cerevisiae KAR
genes, we identified five
C. neoformans
karyogamy gene orthologs:
CnKAR2
,
CnKAR3
,
CnKAR4
,
CnKAR7
(or
CnSEC66
), and
CnKAR8
. There are no apparent orthologs of the
S. cerevisiae
genes
ScKAR1
,
ScKAR5
, and
ScKar9
in
C. neoformans
. Karyogamy involves the congression of two nuclei followed by nuclear membrane fusion, which results in diploidization. ScKar7 (or ScSec66) is known to be involved in nuclear membrane fusion. In
C. neoformans
,
kar7
mutants display significant defects in hyphal growth and basidiospore chain formation during both
a
-α opposite and α-α unisexual reproduction. Fluorescent nuclear imaging revealed that during
kar7
×
kar7
bilateral mutant matings, the nuclei congress but fail to fuse in the basidia. These results demonstrate that the
KAR7
gene plays an integral role in both opposite-sex and unisexual mating, indicating that proper control of nuclear dynamics is important.
CnKAR2
was found to be essential for viability, and its function in mating is not known. No apparent phenotypes were observed during mating of
kar3
,
kar4
, or
kar8
mutants, suggesting that the role of these genes may be dispensable for
C. neoformans
mating, which demonstrates a different evolutionary trajectory for the
KAR
genes in
C. neoformans
compared to those in
S. cerevisiae
.
Lysine Acetylation Is Widespread on Proteins of Diverse Function and Localization in the Protozoan Parasite Toxoplasma gondii
Authors: Jeffers Victoria , Sullivan William J.
Published: April 2012

While histone proteins are the founding members of lysine acetylation substrates, it is now clear that hundreds of other proteins can be acetylated in multiple compartments of the cell. Our knowledge of the scope of this modification throughout the kingdom of life is beginning to emerge, as proteome-wide lysine acetylation has been documented in prokaryotes,
Arabidopsis thaliana
,
Drosophila melanogaster
, and human cells. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify parasite peptides enriched by immunopurification with acetyl-lysine antibody, we produced the first proteome-wide analysis of acetylation for a protozoan organism, the opportunistic apicomplexan parasite
Toxoplasma gondii
. The results show that lysine acetylation is abundant in the actively proliferating tachyzoite form of the parasite, which causes acute toxoplasmosis. Our approach successfully identified known acetylation marks on
Toxoplasma
histones and α-tubulin and detected over 400 novel acetylation sites on a wide variety of additional proteins, including those with roles in transcription, translation, metabolism, and stress responses. Importantly, an extensive set of parasite-specific proteins, including those found in organelles unique to
Apicomplexa
, is acetylated in the parasite. Our data provide a wealth of new information that improves our understanding of the evolution of this vital regulatory modification while potentially revealing novel therapeutic avenues. We conclude from this study that lysine acetylation was prevalent in the early stages of eukaryotic cell evolution and occurs on proteins involved in a remarkably diverse array of cellular functions, including those that are specific to parasites.
Divergent Targets of Candida albicans Biofilm Regulator Bcr1
In Vitro
and
In Vivo
Authors: Fanning Saranna , Xu Wenjie , Solis Norma , Woolford Carol A. , Filler Scott G. , Mitchell Aaron P.
Published: April 2012

Candida albicans
is a causative agent of oropharyngeal candidiasis (OPC), a biofilm-like infection of the oral mucosa. Biofilm formation depends upon the
C. albicans
transcription factor Bcr1, and previous studies indicate that Bcr1 is required for OPC in a mouse model of infection. Here we have used a nanoString gene expression measurement platform to elucidate the role of Bcr1 in OPC-related gene expression. We chose for assays a panel of 134 genes that represent a range of morphogenetic and cell cycle functions as well as environmental and stress response pathways. We assayed gene expression in whole infected tongue samples. The results sketch a portrait of
C. albicans
gene expression in which numerous stress response pathways are activated during OPC. This one set of experiments identifies 64 new genes with significantly altered RNA levels during OPC, thus increasing substantially the number of known genes in this expression class. The
bcr1
Δ/Δ mutant had a much more limited gene expression defect during OPC infection than previously reported for
in vitro
growth conditions. Among major functional Bcr1 targets, we observed that
ALS3
was Bcr1 dependent
in vivo
while
HWP1
was not. We used null mutants and complemented strains to verify that Bcr1 and Hwp1 are required for OPC infection in this model. The role of Als3 is transient and mild, though significant. Our findings suggest that the versatility of
C. albicans
as a pathogen may reflect its ability to persist in the face of multiple stresses and underscore that transcriptional circuitry during infection may be distinct from that detailed during
in vitro
growth.
Titan Cells Confer Protection from Phagocytosis in Cryptococcus neoformans Infections
Authors: Okagaki Laura H. , Nielsen Kirsten
Published: April 2012

The human fungal pathogenCryptococcus neoformansproduces an enlarged “titan” cell morphology when exposed to the host pulmonary environment. Titan cells exhibit traits that promote survival in the host. Previous studies showed that titan cells are not phagocytosed and that increased titan cell production in the lungs results in reduced phagocytosis of cryptococcal cells by host immune cells. Here, the effect of titan cell production on host-pathogen interactions during early stages of pulmonary cryptococcosis was explored. The relationship between titan cell production and phagocytosis was found to be nonlinear; moderate increases in titan cell production resulted in profound decreases in phagocytosis, with significant differences occurring within the first 24 h of the infection. Not only were titan cells themselves protected from phagocytosis, but titan cell formation also conferred protection from phagocytosis to normal-size cryptococcal cells. Large particles introduced into the lungs were not phagocytosed, suggesting the large size of titan cells protects against phagocytosis. The presence of large particles was unable to protect smaller particles from phagocytosis, revealing that titan cell size alone is not sufficient to provide the observed cross-protection of normal-size cryptococcal cells. These data suggest that titan cells play a critical role in establishment of the pulmonary infection by promoting the survival of the entire population of cryptococcal cells.
Functional Analysis of the Single Est1/Ebs1 Homologue in Kluyveromyces lactis Reveals Roles in both Telomere Maintenance and Rapamycin Resistance
Authors: Hsu Min , Yu Eun Young , Sprušanský Ondrej , McEachern Michael J. , Lue Neal F.
Published: April 2012

Est1 and Ebs1 in
Saccharomyces cerevisiae
are paralogous proteins that arose through whole-genome duplication and that serve distinct functions in telomere maintenance and translational regulation. Here we present our functional analysis of the sole Est1/Ebs1 homologue in the related budding yeast
Kluyveromyces lactis
(named
Kl
Est1). We show that similar to other Est1s,
Kl
Est1 is required for normal telomere maintenance
in vivo
and full telomerase primer extension activity
in vitro
.
Kl
Est1 also associates with telomerase RNA (Ter1) and an active telomerase complex in cell extracts. Both the telomere maintenance and the Ter1 association functions of
Kl
Est1 require its N-terminal domain but not its C terminus. Analysis of clusters of point mutations revealed residues in both the N-terminal TPR subdomain and the downstream helical subdomain (DSH) that are important for telomere maintenance and Ter1 association. A UV cross-linking assay was used to establish a direct physical interaction between
Kl
Est1 and a putative stem-loop in Ter1, which also requires both the TPR and DSH subdomains. Moreover, similar to
S. cerevisiae
Ebs1 (
Sc
Ebs1) (but not
Sc
Est1),
Kl
Est1 confers rapamycin sensitivity and may be involved in nonsense-mediated decay. Interestingly, unlike telomere regulation, this apparently separate function of
Kl
Est1 requires its C-terminal domain. Our findings provide insights on the mechanisms and evolution of Est1/Ebs1 homologues in budding yeast and present an attractive model system for analyzing members of this multifunctional protein family.
P65 Truncation Impacts P30 Dynamics during Mycoplasma pneumoniae Gliding
Authors: Hasselbring Benjamin M. , Sheppard Edward S. , Krause Duncan C.
Published: April 2012

The cell wall-less prokaryote
Mycoplasma pneumoniae
is a major cause of community-acquired bronchitis and pneumonia in humans. Colonization is mediated largely by a differentiated terminal organelle, which is also the leading end in gliding motility. Cytadherence-associated proteins P30 and P65 appear to traffic concurrently to the distal end of developing terminal organelles. Here, truncation of P65 due to transposon insertion in the corresponding gene resulted in lower gliding velocity, reduced cytadherence, and decreased steady-state levels of several terminal organelle proteins, including P30. Utilizing fluorescent protein fusions, we followed terminal organelle development over time. New P30 foci appeared at nascent terminal organelles in P65 mutants, as in the wild type. However, with forward cell motility, P30 in the P65 mutants appeared to drag toward the trailing cell pole, where it was released, yielding a fluorescent trail to which truncated P65 colocalized. In contrast, P30 was only rarely observed at the trailing end of gliding wild-type cells. Complementation with the recombinant wild-type P65 allele by transposon delivery restored P65 levels and stabilized P30 localization to the terminal organelle.
The Divergent AmoC
3
Subunit of Ammonia Monooxygenase Functions as Part of a Stress Response System in Nitrosomonas europaea
Authors: Berube Paul M. , Stahl David A.
Published: April 2012

The ammonia monooxygenase of chemolithotrophic ammonia-oxidizing bacteria (AOB) catalyzes the first step in ammonia oxidation by converting ammonia to hydroxylamine. The monooxygenase of
Nitrosomonas europaea
is encoded by two nearly identical operon copies (

amoCAB
1

,
2

). Several AOB, including
N. europaea
, also possess a divergent monocistronic copy of
amoC
(

amoC
3

) of unknown function. Previous work suggested a possible functional role for

amoC
3

as part of the σ
E
stress response regulon during the recovery of
N. europaea
from extended ammonia starvation, thus indicating its importance during the exit of cells from starvation. We here used global transcription analysis to show that expression of

amoC
3

is part of a general poststarvation cellular response system in
N. europaea
. We also found that

amoC
3

is required for an efficient response to some stress conditions, as deleting this gene impaired growth at elevated temperatures and recovery following starvation under high oxygen tensions. Deletion of the σ
32
global stress response regulator demonstrated that the heat shock regulon plays a significant role in mediating the recovery of
N. europaea
from starvation. These findings provide the first described phenotype associated with the divergent AmoC
3
subunit which appears to function as a stress-responsive subunit capable of maintaining ammonia oxidation activity under stress conditions. While this study was limited to starvation and heat shock, it is possible that the AmoC
3
subunit may be responsive to other membrane stressors (e.g., solvent or osmotic shocks) that are prevalent in the environments of AOB.
Clostridium difficile MazF Toxin Exhibits Selective, Not Global, mRNA Cleavage
Authors: Rothenbacher Francesca P. , Suzuki Motoo , Hurley Jennifer M. , Montville Thomas J. , Kirn Thomas J. , Ouyang Ming , Woychik Nancy A.
Published: April 2012

Clostridium difficile
is an important, emerging nosocomial pathogen. The transition from harmless colonization to disease is typically preceded by antimicrobial therapy, which alters the balance of the intestinal flora, enabling
C. difficile
to proliferate in the colon. One of the most perplexing aspects of the
C. difficile
infectious cycle is its ability to survive antimicrobial therapy and transition from inert colonization to active infection. Toxin-antitoxin (TA) systems have been implicated in facilitating persistence after antibiotic treatment. We identified only one TA system in
C. difficile
strain 630 (epidemic type X), designated MazE-cd and MazF-cd, a counterpart of the well-characterized
Escherichia coli
MazEF TA system. This
E. coli
MazF toxin cleaves mRNA at ACA sequences, leading to global mRNA degradation, growth arrest, and death. Likewise, MazF-cd expression in
E. coli
or
Clostridium perfringens
resulted in growth arrest. Primer extension analysis revealed that MazF-cd cleaved RNA at the five-base consensus sequence UACAU, suggesting that the mRNAs susceptible to cleavage comprise a subset of total mRNAs. In agreement, we observed differential cleavage of several mRNAs by MazF-cd
in vivo
, revealing a direct correlation between the number of cleavage recognition sites within a given transcript and its susceptibility to degradation by MazF-cd. Interestingly, upon detailed statistical analyses of the
C. difficile
transcriptome, the major
C. difficile
virulence factor toxin B (TcdB) and CwpV, a cell wall protein involved in aggregation, were predicted to be significantly resistant to MazF-cd cleavage.
Effects of the ERES Pathogenicity Region Regulator Ralp3 on Streptococcus pyogenes Serotype M49 Virulence Factor Expression
Authors: Siemens Nikolai , Fiedler Tomas , Normann Jana , Klein Johannes , Münch Richard , Patenge Nadja , Kreikemeyer Bernd
Published: April 2012

Streptococcus pyogenes
(group A streptococcus [GAS]) is a highly virulent Gram-positive bacterium. For successful infection, GAS expresses many virulence factors, which are clustered together with transcriptional regulators in distinct genomic regions. Ralp3 is a central regulator of the ERES region. In this study, we investigated the role of Ralp3 in GAS M49 pathogenesis. The inactivation of Ralp3 resulted in reduced attachment to and internalization into human keratinocytes. The Δ
ralp3
mutant failed to survive in human blood and serum, and the hyaluronic acid capsule was slightly decreased. In addition, the mutant showed a lower binding capacity to human plasminogen, and the SpeB activity was significantly decreased. Complementation of the Δ
ralp3
mutant restored the wild-type phenotype. The transcriptome and quantitative reverse transcription-PCR analysis of the serotype M49 GAS strain and its isogenic Δ
ralp3
mutant identified 16 genes as upregulated, and 43 genes were found to be downregulated. Among the downregulated genes, there were open reading frames encoding proteins involved in metabolism (e.g., both
lac
operons and the
fru
operon), genes encoding lantibiotics (e.g., the putative salivaricin operon), and ORFs encoding virulence factors (such as the whole Mga core regulon and further genes under Mga control). In summary, the ERES region regulator Ralp3 is an important serotype-specific transcriptional regulator for virulence and metabolic control.
EbfC (YbaB) Is a New Type of Bacterial Nucleoid-Associated Protein and a Global Regulator of Gene Expression in the Lyme Disease Spirochete
Authors: Jutras Brandon L. , Bowman Amy , Brissette Catherine A. , Adams Claire A. , Verma Ashutosh , Chenail Alicia M. , Stevenson Brian
Published: April 2012

Nearly every known species ofEubacteriaencodes a homolog of theBorrelia burgdorferiEbfC DNA-binding protein. We now demonstrate that fluorescently tagged EbfC associates withB. burgdorferinucleoidsin vivoand that chromatin immunoprecipitation (ChIP) of wild-type EbfC showed it to bindin vivoto sites throughout the genome, two hallmarks of nucleoid-associated proteins. Comparative RNA sequencing (RNA-Seq) of a mutantB. burgdorferistrain that overexpresses EbfC indicated that approximately 4.5% of borrelial genes are significantly impacted by EbfC. TheebfCgene was highly expressed in rapidly growing bacteria, butebfCmRNA was undetectable in stationary phase. Combined with previous data showing that EbfC induces bends in DNA, these results demonstrate that EbfC is a nucleoid-associated protein and lead to the hypothesis thatB. burgdorferiutilizes cellular fluctuations in EbfC levels to globally control transcription of numerous genes. The ubiquity of EbfC proteins inEubacteriasuggests that these results apply to a wide range of pathogens and other bacteria.
Rrp2, a Prokaryotic Enhancer-Like Binding Protein, Is Essential for Viability of Borrelia burgdorferi
Authors: Groshong Ashley M. , Gibbons Nora E. , Yang X. Frank , Blevins Jon S.
Published: April 2012

The Lyme disease spirochete,
Borrelia burgdorferi
, exists in two diverse niches (i.e., an arthropod tick vector and mammalian host) during its enzootic life cycle. To effectively adapt to these unique environments, the bacterium alters the expression of numerous genes, including several major outer surface (lipo)proteins that are required for infection and transmission. An enhancer-binding protein (EBP), known as Rrp2, is one identified activator of the RpoN/RpoS alternative sigma factor cascade. Because initial efforts to generate an
rrp2
deletion strain were unsuccessful, the role of Rrp2 in the activation of the RpoN/RpoS pathway was first defined using a strain of
B. burgdorferi
carrying an
rrp2
point mutant that was defective in its ability to activate RpoN-dependent transcription. The fact that subsequent attempts to disrupt
rrp2
have also been unsuccessful has led investigators to hypothesize that Rrp2 has other undefined functions which are essential for
B. burgdorferi
survival and independent of its EBP function. We used a
lac
-based inducible expression system to generate a conditional
rrp2
mutant in virulent
B. burgdorferi
. In this strain, an isopropyl-β-
d
-thiogalactopyranoside-inducible copy of the
rrp2
gene is expressed in
trans
from a borrelial shuttle vector. We found that the chromosomal copy of
rrp2
could be inactivated only when
rrp2
was induced, and the maintenance of
rrp2
expression was required for the growth of the mutants. In addition, the overexpression of
rrp2
is detrimental to
B. burgdorferi
growth in a manner that is independent of the RpoN/RpoS pathway. These studies provide the first direct evidence that
rrp2
is an essential gene in
B. burgdorferi
.
In Vivo
and
In Vitro
Analyses of Regulation of the Pheromone-Responsive
prgQ
Promoter by the PrgX Pheromone Receptor Protein
Authors: Caserta Enrico , Haemig Heather A. H. , Manias Dawn A. , Tomsic Jerneja , Grundy Frank J. , Henkin Tina M. , Dunny Gary M.
Published: April 2012

Expression of conjugative transfer and virulence functions of the
Enterococcus faecalis
antibiotic resistance plasmid pCF10 is regulated by the interaction of the pheromone receptor protein PrgX with two DNA binding operator sites (XBS1 and XBS2) upstream from the transcription start site of the
prgQ
operon (encoding the pCF10 transfer machinery) and by posttranscriptional mechanisms. Occupancy of both binding sites by PrgX dimers results in repression of the
prgQ
promoter. Structural and genetic studies suggest that the peptide pheromone cCF10 functions by binding to PrgX and altering its oligomerization state, resulting in reduced occupancy of XBSs and increased
prgQ
transcription. The DNA binding activity of PrgX has additional indirect regulatory effects on
prgQ
transcript levels related to the position of the convergently transcribed
prgX
operon. This has complicated interpretation of previous analyses of the control of
prgQ
expression by PrgX. We report here the results of
in vivo
and
in vitro
experiments examining the direct effects of PrgX on transcription from the
prgQ
promoter, as well as quantitative correlation between the concentrations of XBSs, PrgX protein, and
prgQ
promoter activity
in vivo
. The results of electrophoretic mobility shift assays and quantitative analysis of
prgQ
transcription
in vitro
and
in vivo
support the predicted roles of the PrgX DNA binding sites in
prgQ
transcription regulation. The results also suggest the existence of other factors that impede PrgX repression or enhance its antagonism by cCF10
in vivo
.
Escherichia coli DNA Polymerase IV (Pol IV), but Not Pol II, Dynamically Switches with a Stalled Pol III* Replicase
Authors: Heltzel Justin M. H. , Maul Robert W. , Wolff David W. , Sutton Mark D.
Published: April 2012

The
dnaN159
allele encodes a temperature-sensitive mutant form of the β sliding clamp (β159). SOS-induced levels of DNA polymerase IV (Pol IV) confer UV sensitivity upon the
dnaN159
strain, while levels of Pol IV ∼4-fold higher than those induced by the SOS response severely impede its growth. Here, we used mutations in Pol IV that disrupted specific interactions with the β clamp to test our hypothesis that these phenotypes were the result of Pol IV gaining inappropriate access to the replication fork via a Pol III*-Pol IV switch relying on both the rim and cleft of the clamp. Our results clearly demonstrate that Pol IV relied on both the clamp rim and cleft interactions for these phenotypes. In contrast to the case for Pol IV, elevated levels of the other Pols, including Pol II, which was expressed at levels ∼8-fold higher than the normal SOS-induced levels, failed to impede growth of the
dnaN159
strain. These findings suggest that the mechanism used by Pol IV to switch with Pol III* is distinct from those used by the other Pols. Results of experiments utilizing purified components to reconstitute the Pol III*-Pol II switch
in vitro
indicated that Pol II switched equally well with both a stalled and an actively replicating Pol III* in a manner that was independent of the rim contact required by Pol IV. These results provide compelling support for the Pol III*-Pol IV two-step switch model and demonstrate important mechanistic differences in how Pol IV and Pol II switch with Pol III*.
The Entner-Doudoroff Pathway Is Obligatory for Gluconate Utilization and Contributes to the Pathogenicity of Vibrio cholerae
Authors: Patra Tapas , Koley Hemanta , Ramamurthy Thandavarayan , Ghose Asoke C. , Nandy Ranjan K.
Published: April 2012

The Entner-Doudoroff (ED) pathway has recently been shown to play an important role in sugar catabolism for many organisms although very little information is available on the functionality of this pathway in
Vibrio cholerae
, the causative agent of cholera. In this study, activation of the genes
edd
and
eda
, encoding 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase, was used as a marker of a functional ED pathway in
V. cholerae
. Transcriptional activation analyses and gene silencing experiments with cells grown in sugar-supplemented M9 medium demonstrated that the ED pathway is functional in
V. cholerae
and is obligatory for gluconate catabolism. Importantly, selective activation of the ED pathway led to concurrent elevation of transcripts of prime virulence genes (
ctxA
and
tcpA
) and their regulator (
toxT
). Further, lowering of these transcript levels and cholera toxin production
in vitro
by an ED pathway-defective mutant (strain N16961 with a Δ
edd
mutation [Δ
edd
N16961
strain]) suggested the importance of this pathway in regulating
V. cholerae
virulence. The
in vivo
relevance of these data was established as the mutant failed to colonize in suckling mice intestine or to induce fluid accumulation in ligated rabbit ileal loops. Activation of the ED pathway in
V. cholerae
was shown to inhibit biofilm formation
in vitro
that could be reversed in the mutant. As further support for these results, comparative transcriptome analysis with cells grown in the presence of glucose or gluconate revealed that a functional ED pathway led to activation of a subset of previously reported
in vivo
expressed genes. All of these results suggest the importance of the ED pathway in
V. cholerae
pathogenesis.
MexT Functions as a Redox-Responsive Regulator Modulating Disulfide Stress Resistance in Pseudomonas aeruginosa
Authors: Fargier Emilie , Mac Aogáin Micheál , Mooij Marlies J. , Woods David F. , Morrissey John P. , Dobson Alan D. W. , Adams Claire , O'Gara Fergal
Published: April 2012

MexT is a global LysR transcriptional regulator known to modulate antibiotic resistance and virulence in
Pseudomonas aeruginosa
. In this study, a novel role for MexT in mediating intrinsic disulfide stress resistance was demonstrated, representing the first identified phenotype associated with inactivation of this regulator in wild-type cells. Disruption of
mexT
resulted in increased susceptibility to the disulfide stress elicitor diamide [diazenedicarboxylic acid bis(
N
,
N
,-di-methylamide)]. This compound is known to elicit a specific stress response via depletion of reduced glutathione and alteration of the cellular redox environment, implicating MexT in redox control. In support of this, MexT-regulated targets, including the MexEF-OprN multidrug efflux system, were induced by subinhibitory concentrations of diamide. A
mexF
insertion mutant also exhibited increased diamide susceptibility, implicating the MexEF-OprN efflux system in MexT-associated disulfide stress resistance. Purified MexT protein was observed to form an oligomeric complex in the presence of oxidized glutathione, with a calculated redox potential of −189 mV. This value far exceeds the thiol-disulfide redox potential of the bacterial cytoplasm, ensuring that MexT remains reduced under normal physiological conditions. MexT is activated by mutational disruption of the predicted quinone oxidoreductase encoded by
mexS
. Alterations in the cellular redox state were observed in a
mexS
mutant (PA14
nfxC
), supporting a model whereby the perception of MexS-associated redox signals by MexT leads to the induction of the MexEF-OprN efflux system, which, in turn, may mediate disulfide stress resistance via efflux of electrophilic compounds.
Genetic, Biochemical, and Molecular Characterization of the Polypeptide Transport-Associated Domain of Escherichia coli BamA
Authors: Workman Patricia , Heide Kristina , Giuliano Nicolas , Lee Nanhee , Mar James , Vuong Phu , Bennion Drew , Misra Rajeev
Published: April 2012

The BamA protein of
Escherichia coli
plays a central role in the assembly of β-barrel outer membrane proteins (OMPs). The C-terminal domain of BamA folds into an integral outer membrane β-barrel, and the N terminus forms a periplasmic polypeptide transport-associated (POTRA) domain for OMP reception and assembly. We show here that BamA misfolding, caused by the deletion of the R44 residue from the α2 helix of the POTRA 1 domain (ΔR44), can be overcome by the insertion of alanine 2 residues upstream or downstream from the ΔR44 site. This highlights the importance of the side chain orientation of the α2 helix residues for normal POTRA 1 activity. The ΔR44-mediated POTRA folding defect and its correction by the insertion of alanine were further demonstrated by using a construct expressing just the soluble POTRA domain. Besides misfolding, the expression of BamA
ΔR44
from a low-copy-number plasmid confers a severe drug hypersensitivity phenotype. A spontaneous drug-resistant revertant of BamA
ΔR44
was found to carry an A18S substitution in the α1 helix of POTRA 1. In the BamA
ΔR44, A18S
background, OMP biogenesis improved dramatically, and this correlated with improved BamA folding, BamA-SurA interactions, and LptD (lipopolysaccharide transporter) biogenesis. The presence of the A18S substitution in the wild-type BamA protein did not affect the activity of BamA. The discovery of the A18S substitution in the α1 helix of the POTRA 1 domain as a suppressor of the folding defect caused by ΔR44 underscores the importance of the helix 1 and 2 regions in BamA folding.
Mucin Promotes Rapid Surface Motility in Pseudomonas aeruginosa
Authors: Yeung Amy T. Y. , Parayno Alicia , Hancock Robert E. W.
Published: May 2012

An important environmental factor that determines the mode of motility adopted by
Pseudomonas aeruginosa
is the viscosity of the medium, often provided by adjusting agar concentrations
in vitro
. However, the viscous gel-like property of the mucus layer that overlays epithelial surfaces is largely due to the glycoprotein mucin.
P. aeruginosa
is known to swim within 0.3% (wt/vol) agar and swarm on the surface at 0.5% (wt/vol) agar with amino acids as a weak nitrogen source. When physiological concentrations or as little as 0.05% (wt/vol) mucin was added to the swimming agar, in addition to swimming,
P. aeruginosa
was observed to undergo highly accelerated motility on the surface of the agar. The surface motility colonies in the presence of mucin appeared to be circular, with a bright green center surrounded by a thicker white edge. While intact flagella were required for the surface motility in the presence of mucin, type IV pili and rhamnolipid production were not. Replacement of mucin with other wetting agents indicated that the lubricant properties of mucin might contribute to the surface motility. Based on studies with mutants, the quorum-sensing systems (
las
and
rhl
) and the orphan autoinducer receptor QscR played important roles in this form of surface motility. Transcriptional analysis of cells taken from the motility zone revealed the upregulation of genes involved in virulence and resistance. Based on these results, we suggest that mucin may be promoting a new or highly modified form of surface motility, which we propose should be termed “surfing.”

IMPORTANCE
An important factor that dictates the mode of motility adopted by
P. aeruginosa
is the viscosity of the medium, often provided by adjusting agar concentrations
in vitro
. However, the gel-like properties of the mucous layers that overlay epithelial surfaces, such as those of the lung, a major site of
Pseudomonas
infection, are contributed mostly by the production of the glycoprotein mucin. In this study, we added mucin to swimming media and found that it promoted the ability of
P. aeruginosa
to exhibit rapid surface motility. These motility colonies appeared in a circular form, with a bright green center surrounded by a thicker white edge. Interestingly, bacterial cells at the thick edge appeared piled up and lacked flagella, while cells at the motility center had flagella. Our data from various genetic and phenotypic studies suggest that mucin may be promoting a modified form of swarming or a novel form of surface motility in
P. aeruginosa
.
Ten challenges in improving quality in healthcare: lessons from the Health Foundation's programme evaluations and relevant literature: Table 1
Authors: Dixon-Woods Mary , McNicol Sarah , Martin Graham
Published: April 2012

Background

Formal evaluations of programmes are an important source of learning about the challenges faced in improving quality in healthcare and how they can be addressed. The authors aimed to integrate lessons from evaluations of the Health Foundation's improvement programmes with relevant literature.

Methods

The authors analysed evaluation reports relating to five Health Foundation improvement programmes using a form of ‘best fit’ synthesis, where a pre-existing framework was used for initial coding and then updated in response to the emerging analysis. A rapid narrative review of relevant literature was also undertaken.

Results

The authors identified ten key challenges: convincing people that there is a problem that is relevant to them; convincing them that the solution chosen is the right one; getting data collection and monitoring systems right; excess ambitions and ‘projectness’; organisational cultures, capacities and contexts; tribalism and lack of staff engagement; leadership; incentivising participation and ‘hard edges’; securing sustainability; and risk of unintended consequences. The authors identified a range of tactics that may be used to respond to these challenges.

Discussion

Securing improvement may be hard and slow and faces many challenges. Formal evaluations assist in recognising the nature of these challenges and help in addressing them.
AggressiveHelicobacter pylori-negative peptic ulceration as the initial manifestation of Crohn's disease
Authors: Callaghan James , Brown Sarah , Battcock Tim , Parry Sally , Snook Jonathon
Published: May 2012

Peptic ulceration is a recognised feature of Crohn's disease, but the characteristics of this manifestation are rather poorly described. Furthermore, most reports in the literature relate to ulcer disease in cases of established Crohn's disease.

The authors report a series of four cases in which the diagnosis of Crohn's disease was preceded by peptic ulceration. Potential confounding factors were as far as possible excluded, implying a true association. Characteristics of the ulcer disease included (1) a multifocal distribution, (2)Helicobacter pylorinegativity and (3) an unusually aggressive clinical course despite proton pump inhibitor therapy, necessitating endoscopic or surgical intervention in three cases.

Crohn's-related peptic ulceration is a relatively common manifestation which may precede the diagnosis of Crohn's disease itself. Recognition of the underlying diagnosis may be hampered by non-specific histology, but is important in view of the aggressive course of the ulceration, which may respond to medical therapy for Crohn's disease.
Citric acid as a trifunctional organocatalyst for thiocyanation of aromatic and heteroaromatic compounds in aqueous media
Authors: Khazaei Ardeshir , Zolfigol Mohammad Ali , Mokhlesi Mohmmad , Pirveysian Mahtab
Published: May 2012

A green and simple procedure for the thiocynation of aromatic and heteroaromatic rings in the presence of a catalytic amount of citric acid in water are described. The reactions proceed in high yields, with short reaction times and under mild conditions.
Theoretical studies on clusters of carbonate with carbon dioxide, CO31–/2–(CO2)n, forn= 1–5 — Comparison of carbonate clusters with sulfate clusters
Authors: Grein Friedrich , Chevrier Daniel M.
Published: May 2012

Density functional theory (DFT) calculations were performed on the geometries and energies of CO31–/2–(CO2)nclusters with n = 1–5. For small clusters (n = 1 or 2), coupled cluster energies were obtained. Up to three CO2molecules are bound covalently to the dianion. Only weak electrostatic bonds were found in the monoanions. Calculated binding energies for the monoanions are in reasonable agreement with experimental values. The calculated adiabatic electron detachment energy for the dianion is –0.07 eV at n = 5, indicating that at least six CO2molecules will have to be added to CO32–before the dianionic cluster becomes, in the gas phase, more stable than the monoanionic one. In comparison, for sulfate – carbon dioxide clusters, stabilization occurs at n = 2. Carbonate clusters are compared with sulfate clusters for three solvent molecules: CO2, SO2, and H2O. Carbonate clusters have larger binding energies than sulfate clusters. For a given dianion, binding energies are largest for SO2and smallest for H2O. However, in all cases, stabilization of the carbonate dianion by clustering is more difficult to achieve than stabilization of the sulfate dianion.
Distinctive heat-shock response of bioleaching microorganismAcidithiobacillus ferrooxidansobserved using genome-wide microarray
Authors: Yin Huaqun , Tang Min , Zhou Zhijun , Fu Xian , Shen Li , Liang Yili , Li Qian , Liu Hongwei , Liu Xueduan
Published: May 2012

Temperature plays an important role in the heap bioleaching. The maldistribution of ventilation in the heap leads to local hyperthermia, which does exert a tremendous stress on bioleaching microbes. In this study, the genome-wide expression profiles of Acidithiobacillus ferrooxidans at 40 °C were detected using the microarray. The results showed that some classic proteases like Lon and small heat-shock proteins were not induced, and heat-inducible membrane proteins were suggested to be under the control of σE. Moreover, expression changes of energy metabolism are noteworthy, which is different from that in heterotrophic bacteria upon heat stress. The induced enzymes catalyzed the central carbon metabolism pathway that might mainly provide precursors of amino acids for protein synthesis. These results will deepen the understanding of the mechanisms of heat-shock response on autotrophic bacteria.
Production of natural folates by lactic acid bacteria starter cultures isolated from artisanal Argentinean yogurts
Authors: Laiño Jonathan Emiliano , LeBlanc Jean Guy , Savoy de Giori Graciela
Published: May 2012

Folate is a B-group vitamin that cannot be synthesized by humans and must be obtained exogenously. Although some species of lactic acid bacteria (LAB) can produce folates, little is known about the production of this vitamin by yogurt starter cultures. Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains were isolated from artisanal Argentinean yogurts and were grown in folate-free culture medium (FACM) and nonfat milk after which intracellular and extracellular folate production were evaluated. From the initial 92 isolated LAB strains, 4 L. delbrueckii subsp. bulgaricus and 32 S. thermophilus were able to grow in the absence of folate. Lactobacillus delbrueckii subsp. bulgaricus CRL 863 and S. thermophilus CRL 415 and CRL 803 produced the highest extracellular folate levels (from 22.3 to 135 µg/L) in FACM. In nonfat milk, these strains were able to increase the initial folate concentrations by almost 190%. This is the first report where native strains of L. delbrueckii subsp. bulgaricus were shown to produce natural folate. The LAB strains identified in this study could be used in developing novel fermented products bio-enriched in natural folates that could in turn be used as an alternative to fortification with the controversial synthetic chemical folic acid.
A COMPREHENSIVE DFT STUDY OF ZINC OXIDE IN DIFFERENT PHASES
Authors: UL HAQ BAKHTIAR , AFAQ A. , AHMED R. , NASEEM S.
Published: April 2012

A density functional study for structural and electronic properties of Zinc Oxide (ZnO), in wurtzite, rock salt and zinc-blende phases has been performed using full potential-linearized augmented plane wave/linearized augmented plane wave plus local ideal orbital (FP-LAPW/L(APW+lo) approach as realized in WIEN2k code. To approximate exchange correlation energy and corresponding potential, a special GGA parameterized by Wu–Cohen has been implemented. Our results of lattice constants, bulk moduli as well as for internal parameter with GGA-WC are found to be more reliable. This study reveals that value of internal parameter decreases with increasing volume whereas computed electronic band structure confirms the direct band gap behavior of ZnO in B4 and B3 phases while indirect band gap behavior in B1 phase. Moreover, two fold degeneracy at the maxima of valence band for B4 and B1 phases whereas three fold for B3 is observed. A detailed comparison with experimental and other first principles studies is also made.
TDDFT CALCULATION FOR OPTICAL ABSORPTION SPECTRA OF Na4 ISOMERS
Authors: HONG X. H. , WANG F. , DU C. H. , GOU B. C.
Published: May 2012

The optical absorption spectra have been calculated for two isomers of Na 4 based on a real-space, real-time implementation of time-dependent density functional theory (TDDFT). We find that our spectra of two Na 4 isomers are in very good agreement with experimental spectra in peak positions and peak intensities. Our results give a good explanation of the discrepancy between the two experimental spectra. Furthermore, we discuss the influence of different isomers on the optical absorption spectra.