bluuebunny/crossref_metadata_2025_split huggingface dataset:
Plasmid Vectors for Proteomic Analyses in Giardia: Purification of Virulence Factors and Analysis of the Proteasome
Authors: Jerlström-Hultqvist Jon , Stadelmann Britta , Birkestedt Sandra , Hellman Ulf , Svärd Staffan G. 
Published: May 2012

ABSTRACT

In recent years, proteomics has come of age with the development of efficient tools for purification, identification, and characterization of gene products predicted by genome projects. The intestinal protozoan
Giardia intestinalis
can be transfected, but there is only a limited set of vectors available, and most of them are not user friendly. This work delineates the construction of a suite of cassette-based expression vectors for use in
Giardia
. Expression is provided by the strong constitutive ornithine carbamoyltransferase (OCT) promoter, and tagging is possible in both N- and C-terminal configurations. Taken together, the vectors are capable of providing protein localization and production of recombinant proteins, followed by efficient purification by a novel affinity tag combination, streptavidin binding peptide–glutathione
S
-transferase (SBP-GST). The option of removing the tags from purified proteins was provided by the inclusion of a PreScission protease site. The efficiency and feasibility of producing and purifying endogenous recombinant
Giardia
proteins with the developed vectors was demonstrated by the purification of active recombinant arginine deiminase (ADI) and OCT from stably transfected trophozoites. Moreover, we describe the tagging, purification by StrepTactin affinity chromatography, and compositional analysis by mass spectrometry of the
G. intestinalis
26S proteasome by employing the Strep II-FLAG–tandem affinity purification (SF-TAP) tag. This is the first report of efficient production and purification of recombinant proteins in and from
Giardia
, which will allow the study of specific parasite proteins and protein complexes.
Pseudomonas aeruginosa Thiol Peroxidase Protects against Hydrogen Peroxide Toxicity and Displays Atypical Patterns of Gene Regulation
Authors: Somprasong Nawarat , Jittawuttipoka Thichakorn , Duang-nkern Jintana , Romsang Adisak , Chaiyen Pimchai , Schweizer Herbert P. , Vattanaviboon Paiboon , Mongkolsuk Skorn 
Published: May 2012

ABSTRACT

The
Pseudomonas aeruginosa
PAO1 thiol peroxidase homolog (Tpx) belongs to a family of enzymes implicated in the removal of toxic peroxides. We have shown the expression of
tpx
to be highly inducible with redox cycling/superoxide generators and diamide and weakly inducible with organic hydroperoxides and hydrogen peroxide (H
2
O
2
). The PAO1
tpx
pattern is unlike the patterns for other peroxide-scavenging genes in
P. aeruginosa
. Analysis of the
tpx
promoter reveals the presence of a putative IscR binding site located near the promoter. The
tpx
expression profiles in PAO1 and the
iscR
mutant, together with results from gel mobility shift assays showing that purified IscR specifically binds the
tpx
promoter, support the role of IscR as a transcriptional repressor of
tpx
that also regulates the oxidant-inducible expression of the gene. Recombinant Tpx has been purified and biochemically characterized. The enzyme catalyzes thioredoxin-dependent peroxidation and can utilize organic hydroperoxides and H
2
O
2
as substrates. The Δ
tpx
mutant demonstrates differential sensitivity to H
2
O
2
only at moderate concentrations (0.5 mM) and not at high (20 mM) concentrations, suggesting a novel protective role of
tpx
against H
2
O
2
in
P. aeruginosa
. Altogether,
P. aeruginosa tpx
is a novel member of the IscR regulon and plays a primary role in protecting the bacteria from submillimolar concentrations of H
2
O
2
.
Surface-Layer (S-Layer) Proteins Sap and EA1 Govern the Binding of the S-Layer-Associated Protein BslO at the Cell Septa of Bacillus anthracis
Authors: Kern Valerie J. , Kern Justin W. , Theriot Julie A. , Schneewind Olaf , Missiakas Dominique 
Published: May 2012

ABSTRACT

The Gram-positive pathogen
Bacillus anthracis
contains 24 genes whose products harbor the structurally conserved surface-layer (S-layer) homology (SLH) domain. Proteins endowed with the SLH domain associate with the secondary cell wall polysaccharide (SCWP) following secretion. Two such proteins, Sap and EA1, have the unique ability to self-assemble into a paracrystalline layer on the surface of bacilli and form S layers. Other SLH domain proteins can also be found within the S layer and have been designated

B

acillus
S
-
l
ayer-associated protein (BSLs). While both S-layer proteins and BSLs bind the same SCWP, their deposition on the cell surface is not random. For example, BslO is targeted to septal peptidoglycan zones, where it catalyzes the separation of daughter cells. Here we show that an insertional lesion in the
sap
structural gene results in elongated chains of bacilli, as observed with a
bslO
mutant. The chain length of the
sap
mutant can be reduced by the addition of purified BslO in the culture medium. This complementation in
trans
can be explained by an increased deposition of BslO onto the surface of
sap
mutant bacilli that extends beyond chain septa. Using fluorescence microscopy, we observed that the Sap S layer does not overlap the EA1 S layer and slowly yields to the EA1 S layer in a growth-phase-dependent manner. Although present all over bacilli, Sap S-layer patches are not observed at septa. Thus, we propose that the dynamic Sap/EA1 S-layer coverage of the envelope restricts the deposition of BslO to the SCWP at septal rings.
Delineation of the Caffeine C-8 Oxidation Pathway in Pseudomonas sp. Strain CBB1 via Characterization of a New Trimethyluric Acid Monooxygenase and Genes Involved in Trimethyluric Acid Metabolism
Authors: Mohanty Sujit Kumar , Yu Chi-Li , Das Shuvendu , Louie Tai Man , Gakhar Lokesh , Subramanian Mani 
Published: May 2012

ABSTRACT

The molecular basis of the ability of bacteria to live on caffeine via the C-8 oxidation pathway is unknown. The first step of this pathway, caffeine to trimethyluric acid (TMU), has been attributed to poorly characterized caffeine oxidases and a novel quinone-dependent caffeine dehydrogenase. Here, we report the detailed characterization of the second enzyme, a novel NADH-dependent trimethyluric acid monooxygenase (TmuM), a flavoprotein that catalyzes the conversion of TMU to 1,3,7-trimethyl-5-hydroxyisourate (TM-HIU). This product spontaneously decomposes to racemic 3,6,8-trimethylallantoin (TMA). TmuM prefers trimethyluric acids and, to a lesser extent, dimethyluric acids as substrates, but it exhibits no activity on uric acid. Homology models of TmuM against uric acid oxidase HpxO (which catalyzes uric acid to 5-hydroxyisourate) reveal a much bigger and hydrophobic cavity to accommodate the larger substrates. Genes involved in the caffeine C-8 oxidation pathway are located in a 25.2-kb genomic DNA fragment of CBB1, including
cdhABC
(coding for caffeine dehydrogenase) and
tmuM
(coding for TmuM). Comparison of this gene cluster to the uric acid-metabolizing gene cluster and pathway of
Klebsiella pneumoniae
revealed two major open reading frames coding for the conversion of TM-HIU to
S
-(+)-trimethylallantoin [
S
-(+)-TMA]. The first one, designated
tmuH
, codes for a putative TM-HIU hydrolase, which catalyzes the conversion of TM-HIU to 3,6,8-trimethyl-2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (TM-OHCU). The second one, designated
tmuD
, codes for a putative TM-OHCU decarboxylase which catalyzes the conversion of TM-OHCU to
S
-(+)-TMA. Based on a combination of enzymology and gene-analysis, a new degradative pathway for caffeine has been proposed via TMU, TM-HIU, TM-OHCU to
S
-(+)-TMA.
Novel Role for RNase PH in the Degradation of Structured RNA
Authors: Jain Chaitanya 
Published: May 2012

ABSTRACTEscherichia colicontains multiple 3′ to 5′ RNases, of which two, RNase PH and polynucleotide phosphorylase (PNPase), use inorganic phosphate as a nucleophile to catalyze RNA cleavage. It is known that an absence of these two enzymes causes growth defects, but the basis for these defects has remained undefined. To further an understanding of the function of these enzymes, the degradation pattern of different cellular RNAs was analyzed. It was observed that an absence of both enzymes results in the appearance of novel mRNA degradation fragments. Such fragments were also observed in strains containing mutations in RNase R and PNPase, enzymes whose collective absence is known to cause an accumulation of structured RNA fragments. Additional experiments indicated that the growth defects of strains containing RNase R and PNPase mutations were exacerbated upon RNase PH removal. Taken together, these observations suggested that RNase PH could play a role in structured RNA degradation. Biochemical experiments with RNase PH demonstrated that this enzyme digests through RNA duplexes of moderate stability. In addition, mapping and sequence analysis of an mRNA degradation fragment that accumulates in the absence of the phosphorolytic enzymes revealed the presence of an extended stem-loop motif at the 3′ end. Overall, these results indicate that RNase PH plays a novel role in the degradation of structured RNAs and provides a potential explanation for the growth defects caused by an absence of the phosphorolytic RNases.
Identification of Ata, a Multifunctional Trimeric Autotransporter of Acinetobacter baumannii
Authors: Bentancor Leticia V. , Camacho-Peiro Ana , Bozkurt-Guzel Cagla , Pier Gerald B. , Maira-Litrán Tomás 
Published: May 2012

ABSTRACT

Acinetobacter baumannii
has recently emerged as a highly troublesome nosocomial pathogen, especially in patients in intensive care units and in those undergoing mechanical ventilation. We have identified a surface protein adhesin of
A. baumannii
, designated the

A
cinetobacter

t
rimeric
a
utotransporter (Ata), that contains all of the typical features of trimeric autotransporters (TA), including a long signal peptide followed by an N-terminal, surface-exposed passenger domain and a C-terminal domain encoding 4 β-strands. To demonstrate that Ata encoded a TA, we created a fusion protein in which we replaced the entire passenger domain of Ata with the epitope tag V5, which can be tracked with specific monoclonal antibodies, and demonstrated that the C-terminal 101 amino acids of Ata were capable of exporting the heterologous V5 tag to the surface of
A. baumannii
in a trimeric form. We found that Ata played a role in biofilm formation and bound to various extracellular matrix/basal membrane (ECM/BM) components, including collagen types I, III, IV, and V and laminin. Moreover, Ata mediated the adhesion of whole
A. baumannii
cells to immobilized collagen type IV and played a role in the survival of
A. baumannii
in a lethal model of systemic infection in immunocompetent mice. Taken together, these results reveal that Ata is a TA of
A. baumannii
involved in virulence, including biofilm formation, binding to ECM/BM proteins, mediating the adhesion of
A. baumannii
cells to collagen type IV, and contributing to the survival of
A. baumannii
in a mouse model of lethal infection.
Pulse oximetry screening for congenital heart defects in newborn infants: an evaluation of acceptability to mothers
Authors: Powell Rachael , Pattison Helen M , Bhoyar Abhay , Furmston Alexandra T , Middleton Lee J , Daniels Jane P , Ewer Andrew K 
Published: May 2012

BackgroundIntroducing neonatal screening procedures may not be readily accepted by parents and may increase anxiety. The acceptability of pulse oximetry screening to parents has not been previously reported.ObjectiveTo assess maternal acceptability of pulse oximetry screening for congenital heart defects and to identify factors predictive of participation in screening.Design and settingA questionnaire was completed by a cross-sectional sample of mothers whose babies were recruited into the PulseOx Study which investigated the test accuracy of pulse oximetry screening.ParticipantsA total of 119 mothers of babies with false-positive (FP) results, 15 with true-positive and 679 with true-negative results following screening.Main outcome measuresQuestionnaires included measures of satisfaction with screening, anxiety, depression and perceptions of test results.ResultsParticipants were predominantly satisfied with screening. The anxiety of mothers given FP results was not significantly higher than that of mothers given true-negative results (median score 32.7 vs 30.0, p=0.09). White British/Irish mothers were more likely to participate in screening, with a decline rate of 5%; other ethnic groups were more likely to decline with the largest increase in declining being for Black African mothers (21%, OR 4.6, 95% CI 3.8 to 5.5). White British mothers were also less anxious (p<0.001) and more satisfied (p<0.001) than those of other ethnicitiesConclusionsPulse oximetry screening was acceptable to mothers and FP results were not found to increase anxiety. Factors leading to differences in participation and satisfaction across ethnic groups need to be identified so that staff can support parents appropriately.
Proline Oxidase Promotes Tumor Cell Survival in Hypoxic Tumor Microenvironments
Authors: Liu Wei , Glunde Kristine , Bhujwalla Zaver M. , Raman Venu , Sharma Anit , Phang James M. 
Published: May 2012

Abstract
Proline is a readily released stress substrate that can be metabolized by proline oxidase (POX) to generate either reactive oxygen species (ROS) to induce apoptosis or autophagy or ATP during times of nutrient stress. However, the contribution of proline metabolism to tumorigenesis in hypoxic microenvironments has not been explored. In this study, we investigated the different functions of POX under hypoxia and glucose depletion. We found that hypoxia induced POX expression in cancer cells in vitro and that POX upregulation colocalized with hypoxic tissues in vivo. In addition, the combination of hypoxia and low glucose showed additive effects on POX expression. Similar to conditions of low glucose, hypoxia-mediated POX induction was dependent on AMP-activated protein kinase activation but was independent of HIF-1α and HIF-2α. Under low-glucose and combined low-glucose and hypoxic conditions, proline catabolized by POX was used preferentially for ATP production, whereas under hypoxia, POX mediated autophagic signaling for survival by generating ROS. Although the specific mechanism was different for hypoxia and glucose deprivation, POX consistently contributed to tumor cell survival under these conditions. Together, our findings offer new insights into the metabolic reprogramming of tumor cells present within a hostile microenvironment and suggest that proline metabolism is a potential target for cancer therapeutics. Cancer Res; 72(14); 3677–86. ©2012 AACR.
Brachyury, a Driver of the Epithelial–Mesenchymal Transition, Is Overexpressed in Human Lung Tumors: An Opportunity for Novel Interventions against Lung Cancer
Authors: Roselli Mario , Fernando Romaine I. , Guadagni Fiorella , Spila Antonella , Alessandroni Jhessica , Palmirotta Raffaele , Costarelli Leopoldo , Litzinger Mary , Hamilton Duane , Huang Bruce , Tucker Joanne , Tsang Kwong-Yok , Schlom Jeffrey , Palena Claudia 
Published: May 2012

Abstract
Purpose: The epithelial–mesenchymal transition (EMT) is emerging as a critical factor for the progression and metastasis of carcinomas, as well as drug resistance. The T-box transcription factor Brachyury has been recently characterized as a driver of EMT in human carcinoma cells. The purpose of this study was to characterize Brachyury as a potential target for lung cancer therapy.
Experimental Design: The expression of Brachyury was evaluated by PCR and by immunohistochemistry in human lung tumors and adult normal tissues. Brachyury gene copy number and promoter methylation status were analyzed in tumor tissues with various levels of Brachyury expression. Lung carcinoma cells' susceptibility to T-cell lysis and EGF receptor (EGFR) kinase inhibition were also evaluated relative to the levels of Brachyury.
Results: Our results showed Brachyury protein expression in 41% of primary lung carcinomas, including 48% of adenocarcinomas and 25% of squamous cell carcinomas. With the exception of normal testis and some thyroid tissues, the majority of normal tissues evaluated in this study were negative for the expression of Brachyury protein. Brachyury-specific T cells could lyse Brachyury-positive tumors and the level of Brachyury corresponded to resistance of tumor cells to EGFR kinase inhibition.
Conclusion: We hypothesize that the elimination of Brachyury-positive tumor cells may be able to prevent and/or diminish tumor dissemination and the establishment of metastases. The ability of Brachyury-specific T-cell lines to lyse Brachyury-positive tumor cells, in vitro, supports the development of Brachyury-based immunotherapeutic approaches for the treatment of lung cancer. Clin Cancer Res; 18(14); 3868–79. ©2012 AACR.
The Outgrowth of Micrometastases Is Enabled by the Formation of Filopodium-like Protrusions
Authors: Shibue Tsukasa , Brooks Mary W. , Inan M. Fatih , Reinhardt Ferenc , Weinberg Robert A. 
Published: May 2012

Abstract
Disseminated cancer cells that have extravasated into the tissue parenchyma must interact productively with its extracellular matrix components to survive, proliferate, and form macroscopic metastases. The biochemical and cell biologic mechanisms enabling this interaction remain poorly understood. We find that the formation of elongated integrin β1–containing adhesion plaques by cancer cells that have extravasated into the lung parenchyma enables the proliferation of these cells via activation of focal adhesion kinase. These plaques originate in and appear only after the formation of filopodium-like protrusions (FLP) that harbor integrin β1 along their shafts. The cytoskeleton-regulating proteins Rif and mDia2 contribute critically to the formation of these protrusions and thereby enable the proliferation of extravasated cancer cells. Hence, the formation of FLPs represents a critical rate-limiting step for the subsequent development of macroscopic metastases.
Significance: Although the mechanisms of metastatic dissemination have begun to be uncovered, those involved in the establishment of extravasated cancer cells in foreign tissue microenvironments remained largely obscure. We have studied the behavior of recently extravasated cancer cells in the lungs and identified a series of cell biologic processes involving the formation of filopodium-like protrusions and the subsequent development of elongated, mature adhesion plaques, which contribute critically to the rapid proliferation of the micrometastatic cells and thus are prerequisites to the eventual lung colonization by these cells. Cancer Discov; 2(8); 706–21. ©2012 AACR.
This article is highlighted in the In This Issue feature, p. 653.
β-Adrenergic Receptor Stimulation Transactivates Protease-Activated Receptor 1 via Matrix Metalloproteinase 13 in Cardiac Cells
Authors: Jaffré Fabrice , Friedman Alan E. , Hu Zhaoyang , Mackman Nigel , Blaxall Burns C. 
Published: May 2012

Background—
Chronic β-adrenergic receptor (β-AR) overstimulation, a hallmark of heart failure, is associated with increased cardiac expression of matrix metalloproteinases (MMPs). MMP-1 has been shown to cleave and activate the protease-activated receptor 1 (PAR1) in noncardiac cells. In the present study, we hypothesized that β-AR stimulation would result in MMP-dependent PAR1 transactivation in cardiac cells.

Methods and Results—

β-AR stimulation of neonatal rat ventricular myocytes (NRVMs) or cardiac fibroblasts with isoproterenol transduced with an alkaline phosphatase–tagged PAR1 elicited a significant increase in alkaline phosphatase–PAR1 cleavage. This isoproterenol-dependent cleavage was significantly reduced by the broad-spectrum MMP inhibitor GM6001. Importantly, specific MMP-13 inhibitors also decreased alkaline phosphatase–PAR1 cleavage in isoproterenol-stimulated NRVMs, as well as in NRVMs stimulated with conditioned medium from isoproterenol-stimulated cardiac fibroblasts. Moreover, we found that recombinant MMP-13 stimulation cleaved alkaline phosphatase–PAR1 in NRVMs at DPRS
42
↓
43
FLLRN. This also led to the activation of the ERK1/2 pathway through Gαq in NRVMs and via the Gαq/ErbB receptor pathways in cardiac fibroblasts. MMP-13 elicited similar levels of ERK1/2 activation but lower levels of generation of inositol phosphates in comparison to thrombin. Finally, we demonstrated that either PAR1 genetic ablation or pharmacological inhibition of MMP-13 prevented isoproterenol-dependent cardiac dysfunction in mice.

Conclusions—
In this study, we demonstrate that β-AR stimulation leads to MMP-13 transactivation of PAR1 in both cardiac fibroblasts and cardiomyocytes and that this likely contributes to pathological activation of Gαq and ErbB receptor–dependent pathways in the heart. We propose that this mechanism may underlie the development of β-AR overstimulation–dependent cardiac dysfunction.
Using Multisensory Cues to Facilitate Air Traffic Management
Authors: Ngo Mary K. , Pierce Russell S. , Spence Charles 
Published: May 2012

Objective: In the present study, we sought to investigate whether auditory and tactile cuing could be used to facilitate a complex, real-world air traffic management scenario. Background: Auditory and tactile cuing provides an effective means of improving both the speed and accuracy of participants’ performance in a variety of laboratory-based visual target detection and identification tasks. Method: A low-fidelity air traffic simulation task was used in which participants monitored and controlled aircraft. The participants had to ensure that the aircraft landed or exited at the correct altitude, speed, and direction and that they maintained a safe separation from all other aircraft and boundaries. The performance measures recorded included en route time, handoff delay, and conflict resolution delay (the performance measure of interest). In a baseline condition, the aircraft in conflict was highlighted in red (visual cue), and in the experimental conditions, this standard visual cue was accompanied by a simultaneously presented auditory, vibrotactile, or audiotactile cue. Results: Participants responded significantly more rapidly, but no less accurately, to conflicts when presented with an additional auditory or audiotactile cue than with either a vibrotactile or visual cue alone. Conclusion: Auditory and audiotactile cues have the potential for improving operator performance by reducing the time it takes to detect and respond to potential visual target events. Application: These results have important implications for the design and use of multisensory cues in air traffic management.
Evolution of cerebral perfusion techniques in type a aortic dissection surgery: a single center experience
Authors: Salah K , van Straten AHM , Soliman Hamad MA , ter Woorst JF , Tan MESH 
Published: May 2012

Background: The purpose of this study was to investigate the effect of using antegrade selective cerebral perfusion (ASCP) with moderate hypothermia on hospital mortality after surgery for acute type A aortic dissection (AAAD). Methods: Between January 1998 and December 2008, 142 consecutive patients were operated on for AAAD. Patients were divided into two subgroups: the cohort of patients operated on from January 1998 until December 2003 (without ASCP) (P1998-2003, n=64) and the cohort operated on from January 2004 until December 2008 (with ASCP)(P2004-2008, n=78). Results: The difference in hospital mortality was statistically significant (P1998-2003: 42.2%; P2004-2008: 14.1%, p<0.0005). Survival rates were 51.6±6.2% vs. 75.1±5.5% and 45.9±6.2% vs. 69.7±7.3% for one and four years, respectively (p=0.001). Multivariate logistic regression analysis revealed that ASCP was the only independent protective factor of hospital mortality (p=0.047). Conclusion: In patients operated on for AAAD, antegrade selective cerebral perfusion with moderate hypothermia is a significant factor in decreasing hospital mortality.