bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Expression of Full-Length TrkA in the Prokaryotic Host Magnetospirillum magneticum AMB-1 by Using a Magnetosome Display System
Authors: Honda Toru , Yasuda Takayuki , Tanaka Tsuyoshi , Hagiwara Koji , Arai Tohru , Yoshino Tomoko
Published: December 2014

Tropomyosin receptor kinase A (TrkA), a receptor tyrosine kinase, is known to be associated with various diseases. Thus, TrkA has become a major drug-screening target for these diseases. Despite the fact that the production of recombinant proteins by prokaryotic hosts has advantages, such as fast growth and ease of genetic engineering, the efficient production of functional receptor tyrosine kinase by prokaryotic hosts remains a major experimental challenge. Here, we report the functional expression of full-length TrkA on magnetosomes in
Magnetospirillum magneticum
AMB-1 by using a magnetosome display system. TrkA was fused with the magnetosome-localized protein Mms13 and expressed on magnetosome surfaces. Recombinant TrkA showed both nerve growth factor (NGF)-binding and autophosphorylation activities. TrkA expressed on magnetosomes has the potential to be used, not only for further functional analysis of TrkA, but also for ligand screening.
Hierarchy in Pentose Sugar Metabolism in Clostridium acetobutylicum
Authors: Aristilde Ludmilla , Lewis Ian A. , Park Junyoung O. , Rabinowitz Joshua D.
Published: December 2014

Bacterial metabolism of polysaccharides from plant detritus into acids and solvents is an essential component of the terrestrial carbon cycle. Understanding the underlying metabolic pathways can also contribute to improved production of biofuels. Using a metabolomics approach involving liquid chromatography-mass spectrometry, we investigated the metabolism of mixtures of the cellulosic hexose sugar (glucose) and hemicellulosic pentose sugars (xylose and arabinose) in the anaerobic soil bacterium
Clostridium acetobutylicum
. Simultaneous feeding of stable isotope-labeled glucose and unlabeled xylose or arabinose revealed that, as expected, glucose was preferentially used as the carbon source. Assimilated pentose sugars accumulated in pentose phosphate pathway (PPP) intermediates with minimal flux into glycolysis. Simultaneous feeding of xylose and arabinose revealed an unexpected hierarchy among the pentose sugars, with arabinose utilized preferentially over xylose. The phosphoketolase pathway (PKP) provides an alternative route of pentose catabolism in
C. acetobutylicum
that directly converts xylulose-5-phosphate into acetyl-phosphate and glyceraldehyde-3-phosphate, bypassing most of the PPP. When feeding the mixture of pentose sugars, the labeling patterns of lower glycolytic intermediates indicated more flux through the PKP than through the PPP and upper glycolysis, and this was confirmed by quantitative flux modeling. Consistent with direct acetyl-phosphate production from the PKP, growth on the pentose mixture resulted in enhanced acetate excretion. Taken collectively, these findings reveal two hierarchies in clostridial pentose metabolism: xylose is subordinate to arabinose, and the PPP is used less than the PKP.
XoxF-Type Methanol Dehydrogenase from the Anaerobic Methanotroph “Candidatus Methylomirabilis oxyfera”
Authors: Wu Ming L. , Wessels Hans J. C. T. , Pol Arjan , Op den Camp Huub J. M. , Jetten Mike S. M. , van Niftrik Laura , Keltjens Jan T.
Published: December 2014

“
Candidatus
Methylomirabilis oxyfera” is a newly discovered anaerobic methanotroph that, surprisingly, oxidizes methane through an aerobic methane oxidation pathway. The second step in this aerobic pathway is the oxidation of methanol. In Gram-negative bacteria, the reaction is catalyzed by pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH). The genome of “
Ca
. Methylomirabilis oxyfera” putatively encodes three different MDHs that are localized in one large gene cluster: one so-called MxaFI-type MDH and two XoxF-type MDHs (XoxF1 and XoxF2). MxaFI MDHs represent the canonical enzymes, which are composed of two PQQ-containing large (α) subunits (MxaF) and two small (β) subunits (MxaI). XoxF MDHs are novel, ecologically widespread, but poorly investigated types of MDHs that can be phylogenetically divided into at least five different clades. The XoxF MDHs described thus far are homodimeric proteins containing a large subunit only. Here, we purified a heterotetrameric MDH from “
Ca
. Methylomirabilis oxyfera” that consisted of two XoxF and two MxaI subunits. The enzyme was localized in the periplasm of “
Ca
. Methylomirabilis oxyfera” cells and catalyzed methanol oxidation with appreciable specific activity and affinity (
V
max
of 10 μmol min
−1
mg
−1
protein,

K
m

of 17 μM). PQQ was present as the prosthetic group, which has to be taken up from the environment since the known gene inventory required for the synthesis of this cofactor is lacking. The MDH from “
Ca
. Methylomirabilis oxyfera” is the first representative of type 1 XoxF proteins to be described.
Bacterial Communities Associated with Surfaces of Leafy Greens: Shift in Composition and Decrease in Richness over Time
Authors: Dees Merete Wiken , Lysøe Erik , Nordskog Berit , Brurberg May Bente
Published: December 2014

The phyllosphere is colonized by a wide variety of bacteria and fungi; it harbors epiphytes, as well as plant-pathogenic bacteria and even human pathogens. However, little is known about how the bacterial community composition on leafy greens develops over time. The bacterial community of the leafy-green phyllosphere obtained from two plantings of rocket salad (
Diplotaxis tenuifolia
) and three plantings of lettuce (
Lactuca sativa
) at two farms in Norway were profiled by an Illumina MiSeq-based approach. We found that the bacterial richness of the
L. sativa
samples was significantly greater shortly (3 weeks) after planting than at harvest (5 to 7 weeks after planting) for plantings 1 and 3 at both farms. For the second planting, the bacterial diversity remained consistent at the two sites. This suggests that the effect on bacterial colonization of leaves, at least in part must, be seasonally driven rather than driven solely by leaf maturity. The distribution of phyllosphere communities varied between
D. tenuifolia
and
L. sativa
at harvest. The variability between these species at the same location suggests that the leaf-dwelling bacteria are not only passive inhabitants but interact with the host, which shapes niches favoring the growth of particular taxa. This work contributes to our understanding of host plant-specific microbial community structures and shows how these communities change throughout plant development.
Rapid Method Using Two Microbial Enzymes for Detection of
l
-Abrine in Food as a Marker for the Toxic Protein Abrin
Authors: Dodge Anthony G. , Carrasquillo Kelvin , Rivera Luis , Xu Lei , Wackett Lawrence P. , Sadowsky Michael J.
Published: December 2014

Abrin is a toxic protein produced by the ornamental plant
Abrus precatorius
, and it is of concern as a biothreat agent. The small coextracting molecule
N
-methyl-
l
-tryptophan (
l
-abrine) is specific to members of the genus
Abrus
and thus can be used as a marker for the presence or ingestion of abrin. Current methods for the detection of abrin or
l
-abrine in foods and other matrices require complex sample preparation and expensive instrumentation. To develop a fast and portable method for the detection of
l
-abrine in beverages and foods, the
Escherichia coli
proteins
N
-methyltryptophan oxidase (MTOX) and tryptophanase were expressed and purified. The two enzymes jointly degraded
l
-abrine to products that included ammonia and indole, and colorimetric assays for the detection of those analytes in beverage and food samples were evaluated. An indole assay using a modified version of Ehrlich's/Kovac's reagent was more sensitive and less subject to negative interferences from components in the samples than the Berthelot ammonia assay. The two enzymes were added into food and beverage samples spiked with
l
-abrine, and indole was detected as a degradation product, with the visual lower detection limit being 2.5 to 10.0 μM (∼0.6 to 2.2 ppm)
l
-abrine in the samples tested. Results could be obtained in as little as 15 min. Sample preparation was limited to pH adjustment of some samples. Visual detection was found to be about as sensitive as detection with a spectrophotometer, especially in milk-based matrices.
Escherichia coli Enoyl-Acyl Carrier Protein Reductase (FabI) Supports Efficient Operation of a Functional Reversal of the β-Oxidation Cycle
Authors: Vick Jacob E. , Clomburg James M. , Blankschien Matthew D. , Chou Alexander , Kim Seohyoung , Gonzalez Ramon
Published: December 2014

We recently used a synthetic/bottom-up approach to establish the identity of the four enzymes composing an engineered functional reversal of the β-oxidation cycle for fuel and chemical production in
Escherichia coli
(J. M. Clomburg, J. E. Vick, M. D. Blankschien, M. Rodriguez-Moya, and R. Gonzalez, ACS Synth Biol 1:541–554, 2012,
http://dx.doi.org/10.1021/sb3000782
). While native enzymes that catalyze the first three steps of the pathway were identified, the identity of the native enzyme(s) acting as the
trans
-enoyl coenzyme A (CoA) reductase(s) remained unknown, limiting the amount of product that could be synthesized (e.g., 0.34 g/liter butyrate) and requiring the overexpression of a foreign enzyme (the
Euglena gracilis
trans
-enoyl-CoA reductase [
Eg
TER]) to achieve high titers (e.g., 3.4 g/liter butyrate). Here, we examine several native
E. coli
enzymes hypothesized to catalyze the reduction of enoyl-CoAs to acyl-CoAs. Our results indicate that FabI, the native enoyl-acyl carrier protein (enoyl-ACP) reductase (ENR) from type II fatty acid biosynthesis, possesses sufficient NADH-dependent TER activity to support the efficient operation of a β-oxidation reversal. Overexpression of FabI proved as effective as
Eg
TER for the production of butyrate and longer-chain carboxylic acids. Given the essential nature of
fabI
, we investigated whether bacterial ENRs from other families were able to complement a
fabI
deletion without promiscuous reduction of crotonyl-CoA. These characteristics from
Bacillus subtilis
FabL enabled Δ
fabI
complementation experiments that conclusively established that FabI encodes a native enoyl-CoA reductase activity that supports the β-oxidation reversal in
E. coli
.
Mechanisms of Methods for Hepatitis C Virus Inactivation
Authors: Pfaender Stephanie , Brinkmann Janine , Todt Daniel , Riebesehl Nina , Steinmann Joerg , Steinmann Jochen , Pietschmann Thomas , Steinmann Eike
Published: December 2014

Virus inactivation by chemical disinfectants is an important instrument for infection control in medical settings, but the mechanisms involved are poorly understood. In this study, we systematically investigated the effects of several antiviral treatments on hepatitis C virus (HCV) particles as model for enveloped viruses. Studies were performed with authentic cell culture-derived viruses, and the influence of chemical disinfectants, heat, and UV treatment on HCV was analyzed by the determination of infectious particles in a limiting-dilution assay, by quantitative reverse transcription-PCR, by core enzyme-linked immunosorbent assay, and by proteolytic protection assay. All different inactivation methods resulted in a loss of HCV infectivity by targeting different parts of the virus particle. Alcohols such as ethanol and 2-propanol did not affect the viral RNA genome integrity but disrupted the viral envelope membrane in a capsid protection assay. Heat and UV treatment of HCV particles resulted in direct damage of the viral genome since transfection of viral particle-associated RNA into permissive cells did not initiate RNA replication. In addition, heat incubation at 80°C disrupted the HCV envelope, rendering the viral capsid susceptible to proteolytic digest. This study demonstrated the molecular processes of viral inactivation of an enveloped virus and should facilitate the development of effective disinfection strategies in infection control not only against HCV but also against other enveloped viruses.
Diversity of Methane-Cycling Archaea in Hydrothermal Sediment Investigated by General and Group-Specific PCR Primers
Authors: Lever Mark A. , Teske Andreas P.
Published: December 2014

The zonation of anaerobic methane-cycling
Archaea
in hydrothermal sediment of Guaymas Basin was studied by general primer pairs (mcrI, ME1/ME2, mcrIRD) targeting the alpha subunit of methyl coenzyme M reductase gene (
mcrA
) and by new group-specific
mcrA
and 16S rRNA gene primer pairs. The mcrIRD primer pair outperformed the other general
mcrA
primer pairs in detection sensitivity and phylogenetic coverage. Methanotrophic ANME-1
Archaea
were the only group detected with group-specific primers only. The detection of 14
mcrA
lineages surpasses the diversity previously found in this location. Most phylotypes have high sequence similarities to hydrogenotrophs, methylotrophs, and anaerobic methanotrophs previously detected at Guaymas Basin or at hydrothermal vents, cold seeps, and oil reservoirs worldwide. Additionally, five
mcrA
phylotypes belonging to newly defined lineages are detected. Two of these belong to deeply branching new orders, while the others are new species or genera of
Methanopyraceae
and
Methermicoccaceae
. Downcore diversity decreases from all groups detected in the upper 6 cm (∼2 to 40°C, sulfate measurable to 4 cm) to only two groups below 6 cm (>40°C). Despite the presence of hyperthermophilic genera (
Methanopyrus
,
Methanocaldococcus
) in cooler surface strata, no genes were detected below 10 cm (≥60°C). While
mcrA
-based and 16S rRNA gene-based community compositions are generally congruent, the deeply branching
mcrA
cannot be assigned to specific 16S rRNA gene lineages. Our study indicates that even among well-studied metabolic groups and in previously characterized model environments, major evolutionary branches are overlooked. Detecting these groups by improved molecular biological methods is a crucial first step toward understanding their roles in nature.
Angioplasty for renovascular hypertension in 78 children
Authors: Kari Jameela A , Roebuck Derek J , McLaren Clare A , Davis Meryl , Dillon Michael J , Hamilton George , Shroff Rukshana , Marks Stephen D , Tullus Kjell
Published: December 2014

Objectives

To evaluate the outcome of percutaneous transluminal angioplasty (PTA) in children with renovascular hypertension (RVH) treated at a single centre over 29 years.

Methods

A retrospective study of the medical charts of all children with RVH who underwent PTA between 1984 and 2012. The primary outcome measurement was blood pressure (BP) achieved after the procedure. The BP before the procedure was compared with that at last available follow-up, 6 (range 0.6–16) years after the initial procedure.

Results

Seventy-eight children with median (range) age of 6.5 (0.5–17) years were studied. Twenty-three (29.5%) had an underlying syndrome, 35 (44.9%) children had bilateral renal artery stenosis (RAS), 18 (23%) intrarenal disease and 11(14%) showed bilateral RAS and intrarenal disease. Twenty (25.6%) children had mid-aortic syndrome and 14 (17.9%) cerebrovascular disease. One hundred and fourteen PTA procedures were carried out including 31 stent insertions. Following PTA, BP was improved in 49 (62.8%) children and of those 18 (23.1%) were cured. Children with involvement of only the main renal arteries showed improved BP control in 79.9% of the children with cure in 39.5%. BP was intentionally maintained above the 95th centile for age and height in four children with coexistent cerebrovascular disease. No change in BP was seen in 18 children despite observed technical success of the PTA, and in seven children due to technical failure of the procedure.

Conclusions

PTA provided a clinical benefit in 62.8% of children with RVH.
Improving vitamin A and D intake among Inuit and Inuvialuit in Arctic Canada: evidence from the Healthy Foods North study
Authors: Pakseresht Mohammadreza , Kolahdooz Fariba , Gittelsohn Joel , Roache Cindy , Corriveau André , Sharma Sangita
Published: December 2014

Background

People in Arctic Canada are undergoing a nutritional transition and increased prevalence of chronic disease. The Healthy Foods North diet and physical activity intervention was developed in 2007–2008 while working with populations in six communities in Nunavut and the Northwest Territories, Canada.

Methods

Four communities received the 1-year intervention (eg, conducting workshops, cooking classes and walking clubs) and two communities served as controls. Among the 263 adult evaluation participants, food frequency questionnaires were used to assess dietary intake at baseline and postintervention. Changes in mean nutrient intakes, nutrient density and dietary adequacy from baseline to postintervention were determined. The intervention impact on nutrient intakes was assessed through multivariate linear regression analysis.

Results

Post-intervention assessment showed a reductions in total fat, saturated, monounsaturated and polyunsaturated fatty acids, and increases in iron intake, only in the intervention group. More than a 4%-increase in the percentage of adherence to vitamins A and D recommendations was observed in the intervention group. After adjusting the regression models, respondents in the intervention communities significantly reduced their energy intake and increased their vitamins A and D intake.

Conclusions

The Healthy Foods North is an effective programme to improve dietary quality among populations of the Canadian Arctic. Long-term interventions are expected to be important factors in the prevention of diet-related chronic diseases in these communities.
Assessment and comparison of left ventricular shear in normal and situs inversus totalis hearts by means of magnetic resonance tagging
Authors: Rossi Alessandro C. , Pluijmert Marieke , Bovendeerd Peter H. M. , Kroon Wilco , Arts Theo , Delhaas Tammo
Published: December 2014

Situs inversus totalis (SIT) is characterized by complete mirroring of gross cardiac anatomy and position combined with an incompletely mirrored myofiber arrangement, being normal at the apex but inverted at the base of the left ventricle (LV). This study relates myocardial structure to mechanical function by analyzing and comparing myocardial deformation patterns of normal and SIT subjects, focusing especially on circumferential-radial shear. In nine control and nine SIT normotensive human subjects, myocardial deformation was assessed from magnetic resonance tagging (MRT) image sequences of five LV short-axis slices. During ejection, no significant difference in either circumferential shortening (εcc) or its axial gradient (Δεcc) is found between corresponding LV levels in control and SIT hearts. Circumferential-radial shear (εcr) has a clear linear trend from apex-to-base in controls, while in SIT it hovers close to zero at all levels. Torsion as well as axial change in εcr (Δεcr) is as in controls in apical sections of SIT hearts but deviates significantly towards the base, changing sign close to the LV equator. Interindividual variability in torsion and Δεcr values is higher in SIT than in controls. Apex-to-base trends of torsion and Δεcr in SIT, changing sign near the LV equator, further substantiate a structural transition in myofiber arrangement close to the LV equator itself. Invariance of εcc and Δεcc patterns between controls and SIT subjects shows that normal LV pump function is achieved in SIT despite partial mirroring of myocardial structure leading to torsional and shear patterns that are far from normality.
Single-wire pressure and flow velocity measurement for quantifying microvascular dysfunction in patients with coronary vasospastic angina
Authors: Yamanaga Kenshi , Tsujita Kenichi , Komura Naohiro , Kaikita Koichi , Sakamoto Kenji , Miyazaki Takashi , Saito Michiyo , Ishii Masanobu , Tabata Noriaki , Akasaka Tomonori , Sato Koji , Horio Eiji , Arima Yuichiro , Kojima Sunao , Tayama Shinji , Nakamura Sunao , Hokimoto Seiji , Ogawa Hisao
Published: December 2014

Endothelial and vascular smooth muscle dysfunction of epicardial coronary arteries play a pivotal role in the pathogenesis of vasospastic angina (VSA). However, coronary microvascular (MV) function in patients with VSA is not fully understood. In the present study, subjects without coronary obstruction were divided into two groups according to the acetylcholine provocation test: VSA group ( n = 29) and non-VSA group ( n = 21). Hyperemic MV resistance (hMR) was measured using a dual-sensor (Doppler velocity and pressure)-equipped guidewire, and guidewire-derived hemodynamic parameters were compared. There were no between-group differences in clinical demographics, including potential factors affecting MV function (e.g., diabetes). Although coronary flow velocity reserve was similar between the two groups [2.4 ± 1.0 (VSA group) vs. 2.4 ± 0.9 (non-VSA group); P = 0.8], coronary vessel resistance and hMR were significantly elevated in the VSA group compared with the non-VSA group (2.6 ± 3.1 vs. 1.2 ± 0.8, P = 0.04; 1.9 ± 0.6 vs. 1.6 ± 0.5, P = 0.03, respectively). Coronary vasospasm, older age, E/e', and estimated glomerular filtration rate were significantly associated with MV dysfunction [defined as ≥ median value of hMR (1.6)] in univariate analysis. Coronary vasospasm most strongly predicted higher hMR in multivariate logistic regression analysis (odds ratio, 4.61; 95% confidence interval, 0.98–21.60; P = 0.053). In conclusion, coronary MV resistance is impaired in patients with VSA compared with non-VSA patients, whereas coronary flow velocity reserve is maintained at normal levels in both groups. In vivo assessment of hMR might be a promising index of coronary MV dysfunction in patients with VSA.
Diving and exercise: The interaction of trigeminal receptors and muscle metaboreceptors on muscle sympathetic nerve activity in humans
Authors: Fisher James P. , Fernandes Igor A. , Barbosa Thales C. , Prodel Eliza , Coote John H. , Nóbrega Antonio Claudio L. , Vianna Lauro C.
Published: December 2014

Swimming involves muscular activity and submersion, creating a conflict of autonomic reflexes elicited by the trigeminal receptors and skeletal muscle afferents. We sought to determine the autonomic cardiovascular responses to separate and concurrent stimulation of the trigeminal cutaneous receptors and metabolically sensitive skeletal muscle afferents (muscle metaboreflex). In eight healthy men (30 ± 2 yr) muscle sympathetic nerve activity (MSNA; microneurography), mean arterial pressure (MAP; Finometer), femoral artery blood flow (duplex Doppler ultrasonography), and femoral vascular conductance (femoral artery blood flow/MAP) were assessed during the following three experimental conditions: 1) facial cooling (trigeminal nerve stimulation), 2) postexercise ischemia (PEI; muscle metaboreflex activation) following isometric handgrip, and 3) trigeminal nerve stimulation with concurrent PEI. Trigeminal nerve stimulation produced significant increases in MSNA total activity (Δ347 ± 167%) and MAP (Δ21 ± 5%) and a reduction in femoral artery vascular conductance (Δ−17 ± 9%). PEI also evoked significant increases in MSNA total activity (Δ234 ± 83%) and MAP (Δ36 ± 4%) and a slight nonsignificant reduction in femoral artery vascular conductance (Δ−9 ± 12%). Trigeminal nerve stimulation with concurrent PEI evoked changes in MSNA total activity (Δ341 ± 96%), MAP (Δ39 ± 4%), and femoral artery vascular conductance (Δ−20 ± 9%) that were similar to those evoked by either separate trigeminal nerve stimulation or separate PEI. Thus, excitatory inputs from the trigeminal nerve and metabolically sensitive skeletal muscle afferents do not summate algebraically in eliciting a MSNA and cardiovascular response but rather exhibit synaptic occlusion, suggesting a high degree of convergent inputs on output neurons.
Impaired oxidative metabolism and calcium mishandling underlie cardiac dysfunction in a rat model of post-acute isoproterenol-induced cardiomyopathy
Authors: Willis B. Cicero , Salazar-Cantú Ayleen , Silva-Platas Christian , Fernández-Sada Evaristo , Villegas César A. , Rios-Argaiz Eduardo , González-Serrano Pilar , Sánchez Luis A. , Guerrero-Beltrán Carlos E. , García Noemí , Torre-Amione Guillermo , García-Rivas Gerardo J. , Altamirano Julio
Published: December 2014

Stress-induced cardiomyopathy, triggered by acute catecholamine discharge, is a syndrome characterized by transient, apical ballooning linked to acute heart failure and ventricular arrhythmias. Rats receiving an acute isoproterenol (ISO) overdose (OV) suffer cardiac apex ischemia-reperfusion damage and arrhythmia, and then undergo cardiac remodeling and dysfunction. Nevertheless, the subcellular mechanisms underlying cardiac dysfunction after acute damage subsides are not thoroughly understood. To address this question, Wistar rats received a single ISO injection (67 mg/kg). We found in vivo moderate systolic and diastolic dysfunction at 2 wk post-ISO-OV; however, systolic dysfunction recovered after 4 wk, while diastolic dysfunction worsened. At 2 wk post-ISO-OV, cardiac function was assessed ex vivo, while mitochondrial oxidative metabolism and stress were assessed in vitro, and Ca2+handling in ventricular myocytes. These were complemented with sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), phospholamban (PLB), and RyR2 expression studies. Ex vivo, basal mechanical performance index (MPI) and oxygen consumption rate (MVO2) were unchanged. Nevertheless, upon increase of metabolic demand, by β-adrenergic stimulation (1–100 nM ISO), the MPI versus MVO2relation decreased and shifted to the right, suggesting MPI and mitochondrial energy production uncoupling. Mitochondria showed decreased oxidative metabolism, membrane fragility, and enhanced oxidative stress. Myocytes presented systolic and diastolic Ca2+mishandling, and blunted response to ISO (100 nM), and all these without apparent changes in SERCA, PLB, or RyR2 expression. We suggest that post-ISO-OV mitochondrial dysfunction may underlie decreased cardiac contractility, mainly by depletion of ATP needed for myofilaments and Ca2+transport by SERCA, while exacerbated oxidative stress may enhance diastolic RyR2 activity.
Elevated peripheral blood mononuclear cell-derived superoxide production in healthy young black men
Authors: Deo Shekhar H. , Holwerda Seth W. , Keller David M. , Fadel Paul J.
Published: December 2014

Several studies have demonstrated that blacks exhibit elevations in systemic oxidative stress. However, the source(s) and mechanism(s) contributing to the elevation in oxidative stress remain unclear. Given that peripheral blood mononuclear cells (PBMCs) can be a major source of NADPH oxidase-derived superoxide production, we tested the hypothesis that young black men demonstrate greater superoxide production and NADPH oxidase expression in PBMCs compared with whites. PBMCs were freshly isolated from whole blood in young normotensive black ( n = 18) and white ( n = 16) men. Intracellular superoxide production in PBMCs was measured using dihydroethidium fluorescence, protein expression of NADPH oxidase subunits, gp91 phox (membranous) and p47 phox (cytosolic) in PBMCs were assessed using Western blot analysis, and plasma protein carbonyls were measured as a marker of systemic oxidative stress. Black men showed elevated intracellular superoxide production (4.3 ± 0.5 vs. 2.0 ± 0.6 relative fluorescence units; black men vs. white men, P < 0.05), increased protein expression for gp91 phox and p47 phox (e.g., p47 phox: 1.1 ± 0.2, black men vs. 0.4 ± 0.1, white men, P < 0.05) in PBMCs and higher circulating protein carbonyl levels (22 ± 4 vs. 14 ± 2 nmol/ml; black men vs. white men, P < 0.05). Interestingly, a positive family history of hypertension in black men did not further enhance PBMC-derived intracellular superoxide production or NADPH oxidase subunit protein expression. These findings indicate that black men exhibit greater resting PBMC-derived superoxide production and an upregulation of the NADPH oxidase pathway with a possible contribution to increases in systemic oxidative stress.
IRS2Copy Number Gain,KRASandBRAFMutation Status as Predictive Biomarkers for Response to the IGF-1R/IR Inhibitor BMS-754807 in Colorectal Cancer Cell Lines
Authors: Huang Fei , Chang Han , Greer Ann , Hillerman Stephen , Reeves Karen A. , Hurlburt Warren , Cogswell John , Patel Dharmesh , Qi Zhenhao , Fairchild Craig , Ryseck Rolf-Peter , Wong Tai W. , Finckenstein Friedrich G. , Jackson Jeffrey , Carboni Joan M.
Published: December 2014

Insulin-like growth factor receptor 1 (IGF-1R)–targeting therapies are currently at an important crossroad given the low clinical response rates seen in unselected patients. Predictive biomarkers for patient selection are critical for improving clinical benefit. Coupling in vitro sensitivity testing of BMS-754807, a dual IGF-1R/IR inhibitor, with genomic interrogations in 60 human colorectal cancer cell lines, we identified biomarkers correlated with response to BMS-754807. The results showed that cell lines with BRAFV600E or KRASG13D mutation were resistant, whereas cell lines with wild-type of both KRAS and BRAF were particularly sensitive to BMS-754807 if they have either higher RNA expression levels of IR-A or lower levels of IGFBP6. In addition, the cell lines with KRAS mutations, those with either insulin receptor substrate 2 (IRS2) copy number gain (CNG) or higher IGF-1R expression levels, were more sensitive to the drug. Furthermore, cell lines with IRS2 CNG had higher levels of ligand-stimulated activation of IGF-1R and AKT, suggesting that these cell lines with IGF-IR signaling pathways more actively coupled to AKT signaling are more responsive to IGF-1R/IR inhibition. IRS2 siRNA knockdown reduced IRS2 protein expression levels and decreased sensitivity to BMS-754807, providing evidence for the functional involvement of IRS2 in mediating the drug response. The prevalence of IRS2 CNG in colorectal cancer tumors as measured by qPCR-CNV is approximately 35%. In summary, we identified IRS2 CNG, IGF-1R, IR-A, and IGFBP6 RNA expression levels, and KRAS and BRAF mutational status as candidate predictive biomarkers for response to BMS-754807. This work proposed clinical development opportunities for BMS-754807 in colorectal cancer with patient selection to improve clinical benefit. Mol Cancer Ther; 14(2); 620–30. ©2014 AACR.
PD-L1 Expression Correlates with Tumor-Infiltrating Lymphocytes and Response to Neoadjuvant Chemotherapy in Breast Cancer
Authors: Wimberly Hallie , Brown Jason R. , Schalper Kurt , Haack Herbert , Silver Matthew R. , Nixon Christian , Bossuyt Veerle , Pusztai Lajos , Lannin Donald R. , Rimm David L.
Published: December 2014

Programmed death 1 ligand 1 (PD-L1) is an immune regulatory molecule that limits antitumor immune activity. Targeting of PD-L1 and other immune checkpoint proteins has shown therapeutic activity in various tumor types. The expression of PD-L1 and its correlation with response to neoadjuvant chemotherapy in breast cancer has not been studied extensively. Our goal was to assess PD-L1 expression in a cohort of breast cancer patients treated with neoadjuvant chemotherapy. Pretreatment biopsies from 105 patients with breast cancer from Yale New Haven Hospital that subsequently received neoadjuvant chemotherapy were assessed for PD-L1 protein expression by automated quantitative analysis with a rabbit monoclonal antibody (E1L3N) to the cytoplasmic domain of PD-L1. In addition, tumor-infiltrating lymphocytes (TIL) were assessed on hematoxylin and eosin slides. PD-L1 expression was observed in 30% of patients, and it was positively associated with hormone-receptor–negative and triple-negative status and high levels of TILs. Both TILs and PD-L1 measured in the epithelium or stroma predicted pathologic complete response (pCR) to neoadjuvant chemotherapy in univariate and multivariate analyses. However, because they are strongly associated, TILs and PD-L1 cannot both be included in a significant multivariate model. PD-L1 expression is prevalent in breast cancer, particularly hormone-receptor–negative and triple-negative patients, indicating a subset of patients that may benefit from immune therapy. Furthermore, PD-L1 and TILs correlate with pCR, and high PD-L1 predicts pCR in multivariate analysis. Cancer Immunol Res; 3(4); 326–32. ©2014 AACR.
Stereotactic Radiation Therapy Augments Antigen-Specific PD-1–Mediated Antitumor Immune Responses via Cross-Presentation of Tumor Antigen
Authors: Sharabi Andrew B. , Nirschl Christopher J. , Kochel Christina M. , Nirschl Thomas R. , Francica Brian J. , Velarde Esteban , Deweese Theodore L. , Drake Charles G.
Published: December 2014

The immune-modulating effects of radiotherapy (XRT) have gained considerable interest recently, and there have been multiple reports of synergy between XRT and immunotherapy. However, additional preclinical studies are needed to demonstrate the antigen-specific nature of radiation-induced immune responses and elucidate potential mechanisms of synergy with immunotherapy. Here, we demonstrate the ability of stereotactic XRT to induce endogenous antigen-specific immune responses when it is combined with anti–PD-1 checkpoint blockade immunotherapy. Using the small animal radiation research platform (SARRP), image-guided stereotactic XRT delivered to B16-OVA melanoma or 4T1-HA breast carcinoma tumors resulted in the development of antigen-specific T cell– and B cell–mediated immune responses. These immune-stimulating effects of XRT were significantly increased when XRT was combined with either anti–PD-1 therapy or regulatory T cell (Treg) depletion, resulting in improved local tumor control. Phenotypic analyses of antigen-specific CD8 T cells revealed that XRT increased the percentage of antigen-experienced T cells and effector memory T cells. Mechanistically, we found that XRT upregulates tumor-associated antigen–MHC complexes, enhances antigen cross-presentation in the draining lymph node, and increases T-cell infiltration into tumors. These findings demonstrate the ability of XRT to prime an endogenous antigen-specific immune response and provide an additional mechanistic rationale for combining radiation with PD-1 blockade in the clinic. Cancer Immunol Res; 3(4); 345–55. ©2014 AACR.
Proteasome-Mediated Reduction in Proapoptotic Molecule Bim Renders CD4
+
CD28
null
T Cells Resistant to Apoptosis in Acute Coronary Syndrome
Authors: Kovalcsik Edit , Antunes Ricardo F. , Baruah Paramita , Kaski Juan Carlos , Dumitriu Ingrid E.
Published: December 2014

Background—

The number of CD4
+
CD28
null
(CD28
null
) T cells, a unique subset of T lymphocytes with proinflammatory and cell-lytic phenotype, increases markedly in patients with acute coronary syndrome (ACS). ACS patients harboring high numbers of CD28
null
T cells have increased risk of recurrent severe acute coronary events and unfavorable prognosis. The mechanisms that govern the increase in CD28
null
T cells in ACS remain elusive. We investigated whether apoptosis pathways regulating T-cell homeostasis are perturbed in CD28
null
T cells in ACS.

Methods and Results—

We found that CD28
null
T cells in ACS were resistant to apoptosis induction via Fas-ligation or ceramide. This was attributable to a dramatic reduction in proapoptotic molecules Bim, Bax, and Fas in CD28
null
T cells, whereas antiapoptotic molecules Bcl-2 and Bcl-xL were similar in CD28
null
and CD28
+
T cells. We also show that Bim is phosphorylated in CD28
null
T cells and degraded by the proteasome. Moreover, we demonstrate for the first time that proteasomal inhibition restores the apoptosis sensitivity of CD28
null
T cells in ACS.

Conclusions—

We show that CD28
null
T cells in ACS harbor marked defects in molecules that regulate T-cell apoptosis, which tips the balance in favor of antiapoptotic signals and endows these cells with resistance to apoptosis. We demonstrate that the inhibition of proteasomal activity allows CD28
null
T cells to regain sensitivity to apoptosis. A better understanding of the molecular switches that control the apoptosis sensitivity of CD28
null
T cells may reveal novel strategies for targeted elimination of these T cells in ACS patients.
Demand management: an audit of chemical pathology test rejections by an electronic gate-keeping system at an academic hospital in Cape Town
Authors: Smit Ida , Zemlin Annalise E , Erasmus Rajiv T
Published: December 2014

Background

Demand management is an area of laboratory activity, which is becoming increasingly important. Within the health-care system, demand management can be defined as the use of health resources to maximise its utility. Tygerberg Hospital has introduced an electronic gate-keeping system. Chemistry tests which generate the highest cost are subjected to this system and may be automatically rejected according to a set of rules. This study aimed: (1) to identify the number of chemistry tests rejected by the eGK; (2) to identify which of these rejected tests were subsequently restored and (3) to assess the impact of rejections on clinical outcome and cost-saving.

Methods

A retrospective audit was conducted to determine the number of chemistry tests rejected and subsequently restored over a 6-month period. The case-notes of patients for whom requested tests previously rejected had been restored were randomly selected and investigated to assess clinical impact. Any cost-saving was calculated.

Results

A total of 68,480 tests were subjected to gate-keeping, and 4605 tests (6.7%) were rejected while 679 (14.7%) of these were restored by the requestor phoning the laboratory after obtaining authorisation. After examining a subset of clinical notes it was found that in most cases (80%), patient care was unaffected. The total cost saved was £25,387.

Conclusions

The majority of the rejected tests were unnecessary and following rejection, real savings were made. Electronic gate-keeping is a simple, effective and sustainable method of demand management.
Evaluating relational repair work with infants and mothers impacted by family violence
Authors: Bunston Wendy , Eyre Kathy , Carlsson Anthony , Pringle Kristen
Published: December 2014

This paper describes the delivery of a therapeutic infant/mother group work intervention program called The Peek-a-Boo ClubTM, which ran from mid-2005 until early 2012. It examines the importance of intervening early with infants and mothers impacted by family violence. The intervention used an ‘infant led’ approach to facilitate the repair of relational ruptures in the infant/mother attachment as a consequence of experiencing family violence. It provides an overview of the intervention and work undertaken to enhance the quality of the attachment between mothers and their infants. In particular, it presents the demographic data of 128 mothers and their infants who participated over a specific period (2007–2011) and then the results of a small quantitative pre- versus post-pilot evaluation of 30 groups over this same time period. Further qualitative data is also included. The challenges and complexities involved in collecting data from this ‘hard to reach’, ambivalent and vulnerable client group are discussed. The results of the evaluation indicate some limitations in the methodology, however, overall The Peek-a-Boo ClubTM program was associated with improved scores on outcome measures assessing infant, mother and infant–mother functioning. Though only a small study, it supports intervening early to assist mothers and infants impacted by family violence in order to repair relational disruption, and encourage mother’s availability to respond sensitively to their infant’s efforts in managing affect regulation. A more comprehensive, tailored and systematic evaluation of such interventions is recommended.
Identifying Adolescent Patients at Risk for Sexually Transmitted Infections
Authors: Victor Elizabeth C. , Chung Richard , Thompson Robert J.
Published: December 2014

This study examined the association between survey responses to health behaviors, personality/psychosocial factors, and self-reported sexually transmitted infections (STIs) to create a brief survey to identify youth at risk for contracting STIs. Participants included 200 racially diverse 14- to 18-year-old patients from a pediatric primary care clinic. Two sexual behavior variables and one peer norm variable were used to differentiate subgroups of individuals at risk of contracting a STI based on reported history of STIs using probability (decision tree) analyses. These items, as well as sexual orientation and having ever had oral sex, were used to create a brief sexual health screening (BSHS) survey. Each point increase in total BSHS score was associated with exponential growth in the percentage of sexually active adolescents reporting STIs. Findings suggest that the BSHS could serve as a useful tool for clinicians to quickly and accurately detect sexual risk among adolescent patients.
US transatlantic leadership after Iraq
Authors: Paquin Jonathan , Beauregard Philippe
Published: December 2014

This article explores whether the United States has been able to exert transatlantic leadership since its head-on diplomatic collision with several European capitals over the 2003 Iraq war. Considering that the decision to invade Iraq was made by the Bush administration, this article also explores whether there has been consistency between the Bush and Obama administrations over transatlantic leadership. To answer these questions, this article reports on a computer-assisted content analysis of the 415 official statements issued by the core transatlantic allies, namely the United States, France, and Britain, in response to four major crises that have occurred in the Middle East and North Africa in the post-Iraq era. This analysis provides qualitative and quantitative evidence leading to four main conclusions. Firstly, US leadership has endured in the post-Iraq era. Secondly, in most cases, France and Britain have aligned their diplomatic positions with those of the United States. Thirdly, the analysis confirms that there is a special Anglo-American relationship. Fourthly and lastly, there has been consistency between the Bush and Obama administrations, with the exception of the US response to the Libyan crisis, which suggests the emergence of a US ‘leading from behind’ transatlantic strategy.
Sociological quasi-labs: The case for deductive scenario development
Authors: Ruser Alexander
Published: December 2014

This article questions why sociology still lacks elaborate methods of scientific forecasting. Following a critical discussion of two main obstacles for sociological futures research – complexity and interaction – the deductive scenario development approach is introduced. Since scenario planning stresses plausibility not probability, it enables sociologists to create logical, not likely pictures of alternative futures. As a way of producing scientific results concerning future developments as well as testing scientific models, deductive scenario planning is labelled as sociological quasi-labs.
Projecting selves: From insecurity to reflection?
Authors: Graziosi Mariolina
Published: December 2014

This article addresses the question of the individual self under conditions of increased insecurity and risks. Considering the centrality of the relationship between ontological and social levels, it explores how the individual can develop ontological trust and the roles ego, self and subjectivity play in its formation. Surveying socio-historical data on the structure of personality and social change and examining the classical debates of Freud, Jung, Kierkegaard, Mead, Parsons and De Beauvoir, it finds an increasing prevalence of the regressive ego and fragmented personality. Searching for ways out of this predicament, the article argues that the development of a strong subjectivity can replace a regressive ego with a reflexive ego rooted in a social-existential project that would favour the formation of the ethical person able to face the social-existential contradictions of the present. The article proposes a new notion of the future as an individual project that encompasses both actions and values. It concludes by discussing personality development under the new social conditions of an absent moral centre.
Obsolescence, presentification, revolution: Sociotechnical discourse as site for in fieri futures
Authors: Pellegrino Giuseppina
Published: December 2014

Technological discourse constitutes a crucial resource in the process of future making and construction of new technologies. Narratives of such a discourse are characterized by projection of hopes and horrors through binary frameworks that enforce technological determinism. Sources and actors of technological discourse are multiple (the mass media, professional communities, institutions, enterprises). The article aims to identify different conceptions of technology as well as different durations of future traceable in technological discourse. On the one hand, technology appears as an autonomous, atemporal force; on the other hand, it appears split between a short and a long time frame. Whereas the short-term future is shaped by the culture of capitalism with its fast-paced consumption focused on obsolescence and presentification, the long-term future is more influenced by the binary frames recurrent in both popular media and science fiction. This long future horizon in technological discourse is concerned with the expectation of a radical revolution. It fosters scenarios where anger and desire, horror and hope coexist and compete with each other. The article proposes obsolescence, presentification and revolution as three concurrent modes of production of the sociotechnical discourse on the future.
Transculturality in the internet: Culture flows and virtual publics
Authors: Schachtner Christina
Published: December 2014

This article addresses the flows that criss-cross virtual space: flows of information, images and communication. These are cultural flows, or, to use the terminology of Schütz and Luckmann, intersubjective meaning-contexts consisting of values, social rules, world views and patterns of behaviour that are becoming increasingly dissociated from their national base thanks to digital media. They are articulated, passed on, qualified, rejected or transformed into something else in online discourse. The idea of culture as a ‘self-contained sphere’ has become obsolete in an age of digital media. Terms such as transculturality and transnationality reflect socio-cultural developments that are closely linked with the evolution of digital media. This article commences with a theoretical overview of how the social is formed under the influence of cultural flows, asking whether transcultural tendencies are emerging in new virtual publics and how such tendencies can be identified. Empirical findings drawn from a larger project on ‘Subject constructions and digital culture’ show how differences are dealt with according to the divergent tendencies of homogenization, particularization and hybridization.