bluuebunny/crossref_metadata_2025 huggingface dataset:
Elucidation of the Role of Clp Protease Components in Circadian Rhythm by Genetic Deletion and Overexpression in Cyanobacteria
Authors: Imai Keiko , Kitayama Yohko , Kondo Takao
Published: August 2013

In the cyanobacterium
Synechococcus elongatus
PCC7942, KaiA, KaiB, and KaiC are essential elements of the circadian clock, and Kai-based oscillation is thought to be the basic circadian timing mechanism. The Kai-based oscillator coupled with transcription/translation feedback and other intercellular factors maintains the stability of the 24-hour period
in vivo
. In this study, we showed that disruption of the Clp protease family genes
clpP1
,
clpP2
, and
clpX
and the overexpression of
clpP3
cause long-period phenotypes. There were no significant changes in the levels of the clock proteins in these mutants. The overexpression of
clpX
led to a decrease in
kaiBC
promoter activity, the disruption of the circadian rhythm, and eventually cell death. However, after the transient overexpression of
clpX
, the
kaiBC
gene expression rhythm recovered after a few days. The rhythm phase after recovery was almost the same as the phase before
clpX
overexpression. These results suggest that the core Kai-based oscillation was not affected by
clpX
overexpression. Moreover, we showed that the overexpression of
clpX
sequentially upregulated ribosomal protein subunit mRNA levels, followed by upregulation of other genes, including the clock genes. Additionally, we found that the disruption of
clpX
decreased the expression of the ribosomal protein subunits. Finally, we showed that the circadian period was prolonged following the addition of a translation inhibitor at a low concentration. These results suggest that translational efficiency affects the circadian period and that
clpX
participates in the control of translation efficiency by regulating the transcription of ribosomal protein genes.
ExbB Cytoplasmic Loop Deletions Cause Immediate, Proton Motive Force-Independent Growth Arrest
Authors: Bulathsinghala Charles M. , Jana Bimal , Baker Kristin R. , Postle Kathleen
Published: August 2013

The
Escherichia coli
TonB system consists of the cytoplasmic membrane proteins TonB, ExbB, and ExbD and multiple outer membrane active transporters for diverse iron siderophores and vitamin B
12
. The cytoplasmic membrane proteins harvest and transmit the proton motive force (PMF) to outer membrane transporters. This system, which spans the cell envelope, has only one component with a significant cytoplasmic presence, ExbB. Characterization of sequential 10-residue deletions in the ExbB cytoplasmic loop (residues 40 to 129; referred to as Δ10 proteins) revealed that it was required for all TonB-dependent activities, including interaction between the periplasmic domains of TonB and ExbD. Expression of eight out of nine of the Δ10 proteins at chromosomal levels led to immediate, but reversible, growth arrest. Arrest was not due to collapse of the PMF and did not require the presence of ExbD or TonB. All Δ10 proteins that caused growth arrest were dominant for that phenotype. However, several were not dominant for iron transport, indicating that growth arrest was an intrinsic property of the Δ10 variants, whether or not they could associate with wild-type ExbB proteins. The lack of dominance in iron transport also ruled out trivial explanations for growth arrest, such as high-level induction. Taken together, the data suggest that growth arrest reflected a changed interaction between the ExbB cytoplasmic loop and one or more unknown growth-regulatory proteins. Consistent with that, a large proportion of the ExbB cytoplasmic loop between transmembrane domain 1 (TMD1) and TMD2 is predicted to be disordered, suggesting the need for interaction with one or more cytoplasmic proteins to induce a final structure.
New Family of Tungstate-Responsive Transcriptional Regulators in Sulfate-Reducing Bacteria
Authors: Kazakov Alexey E. , Rajeev Lara , Luning Eric G. , Zane Grant M. , Siddartha Kavya , Rodionov Dmitry A. , Dubchak Inna , Arkin Adam P. , Wall Judy D. , Mukhopadhyay Aindrila , Novichkov Pavel S.
Published: August 2013

The trace elements molybdenum and tungsten are essential components of cofactors of many metalloenzymes. However, in sulfate-reducing bacteria, high concentrations of molybdate and tungstate oxyanions inhibit growth, thus requiring the tight regulation of their homeostasis. By a combination of bioinformatic and experimental techniques, we identified a novel regulator family, tungstate-responsive regulator (TunR), controlling the homeostasis of tungstate and molybdate in sulfate-reducing deltaproteobacteria. The effector-sensing domains of these regulators are similar to those of the known molybdate-responsive regulator ModE, while their DNA-binding domains are homologous to XerC/XerD site-specific recombinases. Using a comparative genomics approach, we identified DNA motifs and reconstructed regulons for 40 TunR family members. Positional analysis of TunR sites and putative promoters allowed us to classify most TunR proteins into two groups: (i) activators of
modABC
genes encoding a high-affinity molybdenum and tungsten transporting system and (ii) repressors of genes for toluene sulfonate uptake (TSUP) family transporters. The activation of
modA
and
modBC
genes by TunR in
Desulfovibrio vulgaris
Hildenborough was confirmed
in vivo
, and we discovered that the activation was diminished in the presence of tungstate. A predicted 30-bp TunR-binding motif was confirmed by
in vitro
binding assays. A novel TunR family of bacterial transcriptional factors controls tungstate and molybdate homeostasis in sulfate-reducing deltaproteobacteria. We proposed that TunR proteins participate in protection of the cells from the inhibition by these oxyanions. To our knowledge, this is a unique case of a family of bacterial transcriptional factors evolved from site-specific recombinases.
In Vitro
Evolution of an Archetypal Enteropathogenic Escherichia coli Strain
Authors: Nisa Shahista , Hazen Tracy H. , Assatourian Lillian , Nougayrède Jean-Philippe , Rasko David A. , Donnenberg Michael S.
Published: August 2013

Enteropathogenic
Escherichia coli
(EPEC) is a leading cause of infantile diarrhea in developing countries. EPEC strain E2348/69 is used worldwide as a prototype to study EPEC genetics and disease. However, isolates of E2348/69 differ phenotypically, reflecting a history of
in vitro
selection. To identify the genomic and phenotypic changes in the prototype strain, we sequenced the genome of the nalidixic acid-resistant (Nal
r
) E2348/69 clone. We also sequenced a recent
nleF
mutant derived by one-step PCR mutagenesis from the Nal
r
strain. The sequencing results revealed no unintended changes between the mutant and the parent strain. However, loss of the pE2348-2 plasmid and 3 nonsynonymous mutations were found in comparison to the published streptomycin-resistant (Str
r
) E2348/69 reference genome. One mutation is a conservative amino acid substitution in
ftsK
. Another, in
gyrA
, is a mutation known to result in resistance to nalidixic acid. The third mutation converts a stop codon to a tryptophan, predicted to result in the fusion of
hflD
, the lysogenization regulator, to
purB
. The
purB
gene encodes an adenylosuccinate lyase involved in purine biosynthesis. The Nal
r
clone has a lower growth rate than the Str
r
isolate when cultured in minimal media, a difference which is corrected upon addition of adenine or by genetic complementation with
purB
. Addition of adenine or genetic complementation also restored the invasion efficiency of the Nal
r
clone. This report reconciles longstanding inconsistencies in phenotypic properties of an archetypal strain and provides both reassurance and cautions regarding intentional and unintentional evolution
in vitro
.
Bacterial Floras and Biofilms of Malignant Wounds Associated with Breast Cancers
Authors: Fromantin Isabelle , Seyer Damien , Watson Sarah , Rollot Florence , Elard Jacqueline , Escande Marie Christine , De Rycke Yann , Kriegel Irène , Larreta Garde Véronique
Published: August 2013

The risk of infections and the appearance of symptoms (e.g., odors) represent the main troubles resulting from malignant wounds. The aim of this study was to characterize the balance of bacterial floras and the relationships between biofilms and bacteria and the emergence of symptoms. Experimental research was carried out for 42 days on malignant wounds associated with breast cancer. Investigations of bacterial floras (aerobes, aero-anaerobes, and anaerobes), detection of the presence of biofilms by microscopic epifluorescence, and clinical assessment were performed. We characterized biofilms in 32 malignant wounds associated with breast cancer and bacterial floras in 25 such wounds. A mixed group of floras, composed of 54 different bacterial types, was identified, with an average number per patient of 3.6 aerobic species and 1.7 anaerobic species; the presence of strict anaerobic bacterial strains was evidenced in 70% of the wounds; biofilm was observed in 35% of the cases. Odor was a reliable indicator of colonization by anaerobes, even when this symptom was not directly linked to any of the identified anaerobic bacteria. Bacteria are more likely to be present during myelosuppression and significantly increase the emergence of odors and pain when present at amounts of >10
5
· g
−1
. The presence of biofilms was not associated with clinical signs or with precise types of bacteria. No infections occurred during the 42-day evaluation period. This study provides a dynamic description of the bacterial floras of tumoral wounds. The study results highlight the absolute need for new therapeutic options that are effective for use on circulating bacteria as well as on bacteria organized in biofilm.
Directed Evolution of a Virus Exclusively Utilizing Human Epidermal Growth Factor Receptor as the Entry Receptor
Authors: Dai Hong-Sheng , Liu Zheng , Jiang Wen , Kuhn Richard J.
Published: August 2013

Rational design and directed evolution are powerful tools to generate and improve protein function; however, their uses are mostly limited to enzyme and antibody engineering. Here we describe a directed-evolution strategy, named the tandem selection and enrichment system (TSES), and its use in generating virus with exclusive specificity for a particular cellular receptor. In TSES, evolving viruses are sequentially and iteratively transferred between two different host cells, one for selection of receptor specificity and the other for enrichment of the fittest virus. By combining rational design and TSES, we generated human epidermal growth factor receptor (EGFR)-specific virus 1 (ESV1). ESV1 has the backbone of Sindbis virus (SINV) and displays an EGF domain engrafted onto structural protein E2 after residue Pro192, together with eight amino acid changes stabilizing the E2-EGF chimera. ESV1 uses EGFR to initiate infection and has lost the capacity to interact with all known SINV receptors. A 12.2-Å cryoelectron microscopic (cryoEM) reconstruction of ESV1 reveals that the E2-EGF fusion adopts a fixed conformation, with EGF sitting at the top of the E2 spike; The EGFR binding interface faces outward, and the EGF domain completely masks SINV receptor binding. The cryoEM structure of ESV1 explains the desirable properties of ESV1 and provides insights for its further modification. TSES expands the scope of directed evolution and can be easily extended to other targeting molecules and viral systems.
Transient Compartmentalization of Simian Immunodeficiency Virus Variants in the Breast Milk of African Green Monkeys
Authors: Ho Carrie , Wu Steven , Amos Joshua D. , Colvin Lisa , Smith Shannon D. , Wilks Andrew B. , DeMarco C. Todd , Brinkley Christie , Denny Thomas N. , Schmitz Joern E. , Rodrigo Allen G. , Permar Sallie R.
Published: August 2013

Natural hosts of simian immunodeficiency virus (SIV), African green monkeys (AGMs), rarely transmit SIV via breast-feeding. In order to examine the genetic diversity of breast milk SIV variants in this limited-transmission setting, we performed phylogenetic analysis on
envelope
sequences of milk and plasma SIV variants of AGMs. Low-diversity milk virus populations were compartmentalized from that in plasma. However, this compartmentalization was transient, as the milk virus lineages did not persist longitudinally.
Empty scrotum: undescended testis or ectopic?
Authors: Lopes Vieira Jose Filipe , Brahme Gayatri , Pandya Nikila , Desai Ashish
Published: August 2013

This case of ectopic testis highlights the importance of a thorough first newborn examination followed by senior review when appropriate, and the expertise required to interpret neonatal ultrasounds. Following a provisional diagnosis of unilateral undescended testicle in a newborn, an ultrasound scan was unable to correctly identify the condition, resulting in a delay in diagnosis. The condition was later recognised incidentally, which led to a successful surgical correction. The early clinical examination and recognition of ectopic testis allows a timely surgical intervention preventing potential complications.
Preoperative L1 and L2 paravertebral block is an effective postoperative analgesia for hip arthroscopy in a multimodal analgesic regimen
Authors: Ilkhchoui Yashar , Arndt Christopher D , Koshkin Eugene , Vagh Firoz
Published: August 2013

We describe the case of a 30-year-old woman with chronic hip pain secondary to avascular necrosis. She received preoperative L1–L2 paravertebral block for postoperative pain control after arthroscopic hip surgery. Preoperative paravertebral block at the level of L1–L2 provided an effective postoperative analgesia for hip arthroscopy when used in a multimodal approach.
The ‘gap’ in the ‘plasma osmolar gap’
Authors: Arora Alok
Published: August 2013

Ethylene glycol poisoning is a medical emergency that presents challenges for clinicians and clinical laboratories. If left untreated, it may cause morbidity and death, but effective therapy is available if diagnosed in time. The diagnosis of ethylene glycol poisoning is not always straightforward and the commonly quoted ‘plasma osmolar gap’ is not sufficiently sensitive to exclude a small ingestion and has been reported to be normal in a number of serious exposures. The ‘plasma osmolar gap’ cannot distinguish among ethanol, isopropyl alcohol, methanol or ethylene glycol. Thus, the measurement of serum ethylene glycol and, ideally, glycolic acid, its major toxic metabolite in serum, is definitive. This also holds true for methanol and its metabolite formic acid. Ethylene glycol metabolites target the kidney and lead to reversible oliguric or anuric injury, which in turn slows the elimination of ethylene glycol. The therapeutic options include reversal of metabolic acidosis, inhibition of alcohol dehydrogenase and early haemodialysis.
Lung sounds: how doctors draw crackles and wheeze
Authors: Scott Gregory , Presswood Edward J , Makubate Boikanyo , Cross Frank
Published: August 2013

Background

The content of medical records is a potential source of miscommunication between clinicians. Doctors' written entries have been criticised for their illegibility and ambiguity, but no studies have examined doctors' drawings that are commonly used for recording auscultation findings.

Objective

To compare doctors' drawings of auscultation findings, based on identical clinical information.

Methods

Doctors at the Royal London Hospital and a group of London based general practitioners (GPs) documented a respiratory examination with a drawing of the auscultation findings of bilateral mid and lower zone wheeze and right lower zone crackles. The graphical properties of each drawing were examined and the use of written captions and labels recorded. Drawings were classified into styles according to the use of symbols (defined as discrete characters or icons) and shading (cross-hatching, speckling or darkening) to depict the auscultation findings. The study was conducted between September and November 2011.

Results

Sixty-nine hospital doctors and 13 GPs participated. Ten drawing styles were identified from 78 completed drawings. Ten distinct symbols and a range of shading techniques were used. The most frequent style (21% of drawings) combined ‘X’ symbols representing crackles with musical notes for wheeze. There was inconsistency of representation across the drawings. Forty-seven (60%) drawings used an ‘X’ symbol exclusively to represent crackles, but six (8%) used ‘X’ only to represent wheeze, and 10 (13%) used ‘X’ to represent both findings. 91% of participants included captions or labels with their drawing.

Conclusions

There was wide variation in doctors' drawings of identical auscultation findings, and inconsistency in the meaning of symbols both between and within drawings. Doctors risk incorrectly interpreting each other's drawings when they are not effectively labelled. We recommend doctors consider using a written description instead, or draw different findings with distinct symbols or shading, labelling all findings clearly.
Where to deploy pre-exposure prophylaxis (PrEP) in sub-Saharan Africa?
Authors: Verguet Stéphane , Stalcup Meg , Walsh Julia A
Published: August 2013

Objective

Two randomised controlled trials showed that pre-exposure prophylaxis (PrEP) reduces HIV transmission between heterosexual men and women. We model the potential impact on transmission and cost-effectiveness of providing PrEP in sub-Saharan Africa.

Methods

We use a deterministic, compartmental model of HIV transmission to evaluate the potential of a 5-year PrEP intervention targeting the adult population of 42 sub-Saharan African countries. We examine the incremental impact of adding PrEP at pre-existing levels of male circumcision and antiretroviral therapy (ART). The base case assumes efficacy of 68%; adherence at 80%; country coverage at 10% of the HIV-uninfected adult population; and annual costs of PrEP and ART at US$200 and US$880 per person, respectively.

Results

After 5 years, 390 000 HIV infections (95% UR 190 000 to 630 000) would be prevented, 24% of these in South Africa. HIV infections averted per 100 000 people (adult) would range from 500 in Lesotho to 10 in Somalia. Incremental cost-effectiveness would be US$5800/disability-adjusted life year (DALY) (95% UR 3100 to 13500). Cost-effectiveness would range from US$500/DALY in Lesotho to US$44 600/DALY in Eritrea.

Conclusions

In a general adult population, PrEP is a high-cost intervention which will have maximum impact and be cost-effective only in countries that have high levels of HIV burden and low levels of male circumcision in the population. Hence, PrEP will likely be most effective in Southern Africa as a targeted intervention added to existing strategies to control the HIV pandemic.
Ecological correlates of variable organ sizes and fat loads in the most northerly wintering shorebirds
Authors: Ruthrauff D.R. , Dekinga A. , Gill R.E. , Summers R.W. , Piersma T.
Published: August 2013

Shorebirds at northern latitudes during the nonbreeding season typically carry relatively large lipid stores and exhibit an up-regulation of lean tissues associated with digestion and thermogenesis. Intraspecific variation in these tissues across sites primarily reflects differences in environmental conditions. Rock (Calidris ptilocnemis (Coues, 1873)) and Purple (Calidris maritima (Brünnich, 1764)) sandpipers are closely related species having the most northerly nonbreeding distributions among shorebirds, living at latitudes up to 61°N in Cook Inlet, Alaska, and up to ∼71°N in northern Norway, respectively. Cook Inlet is the coldest known site used by nonbreeding shorebirds, and the region’s mudflats annually experience extensive coverage of foraging sites by sea and shore-fast ice. Accordingly, Rock Sandpipers increase their fat stores to nearly 20% of body mass during winter. In contrast, Purple Sandpipers exploit predictably ice-free rocky intertidal foraging sites and maintain low (<6.5%) fat stores. Rock Sandpipers increase the mass of lean tissues from fall to winter, including contour feathers, stomach, and liver components. They also have greater lean pectoralis and supracoracoideus muscle and liver and kidney tissues compared with Purple Sandpipers in winter. This demonstrates a combined emphasis on digestive processes and thermogenesis, whereas Purple Sandpipers primarily augment organs associated with digestive processes. The high winter fat loads and increased lean tissues of Rock Sandpipers in Cook Inlet reflect the region’s persistent cold and abundant but sporadically unavailable food resources.
Damage to bridges due to the 27 February 2010 Chile earthquake
Authors: Mitchell Denis , Huffman Sharlie , Tremblay Robert , Saatcioglu Murat , Palermo Dan , Tinawi René , Lau David
Published: August 2013

This paper provides a summary of the damage to bridges in the Mw 8.8 Chile earthquake of 27 February 2010. Lessons from the different types of structural damage observed on concrete and steel bridges are discussed. The important roles played by soil liquefaction, settlement and embankment failures are highlighted. Aspects such as shear failure of steel piles, shear failure of concrete substructure elements, failures and severe buckling of steel braces, failures of shear keys and restrainers at supports, and damage to girders due to lack of diaphragms are described. Many examples of loss of superstructure support are presented. Skew supports and multi-span simply supported bridges were particularly susceptible to loss of support. Several aspects of the Chilean bridge design code are discussed and compared with North American codes (CSA S6 and AASHTO).
Solid and drift geology in forensic investigations
Authors: Ruffell Alastair
Published: August 2013

Forensic geology, forensic geoscience and geoforensics (see the text for definitions) are largely concerned with searches for buried objects or the use of sediment and soil analysis as evidence. Geophysics, remote sensing, geostatistics, geochemistry and geomorphology are considered part of these disciplines. Traditional outcrop geology is not often considered, with very few publications considering the role of mapped and logged geology. This work comprises a review of the few published works, followed by a range of case studies that demonstrate how traditional, outcrop-scale drift and solid geology can be used in forensic geology. These include: using geology in the search for buried explosives and human remains; the logging of solid and drift geology for analysis of disputed rock quality; and modelling groundwater flow from an illegal landfill site. Geological mapping may assist in limiting a search area or in understanding what failed in a search or forensic investigation. The scale of forensic geology discussed bridges the gap between the two traditions of forensic geology outlined above: that of searching large areas of ground and trace evidence analysis.
p90 ribosomal S6 kinase 3 contributes to cardiac insufficiency in α-tropomyosin Glu180Gly transgenic mice
Authors: Passariello Catherine L. , Gayanilo Marjorie , Kritzer Michael D. , Thakur Hrishikesh , Cozacov Zoharit , Rusconi Francesca , Wieczorek David , Sanders Michael , Li Jinliang , Kapiloff Michael S.
Published: August 2013

Myocardial interstitial fibrosis is an important contributor to the development of heart failure. Type 3 p90 ribosomal S6 kinase (RSK3) was recently shown to be required for concentric myocyte hypertrophy under in vivo pathological conditions. However, the role of RSK family members in myocardial fibrosis remains uninvestigated. Transgenic expression of α-tropomyosin containing a Glu180Gly mutation (TM180) in mice of a mixed C57BL/6:FVB/N background induces a cardiomyopathy characterized by a small left ventricle, interstitial fibrosis, and diminished systolic and diastolic function. Using this mouse model, we now show that RSK3 is required for the induction of interstitial fibrosis in vivo. TM180 transgenic mice were crossed to RSK3 constitutive knockout ( RSK3−/−) mice. Although RSK3 knockout did not affect myocyte growth, the decreased cardiac function and mild pulmonary edema associated with the TM180 transgene were attenuated by RSK3 knockout. The improved cardiac function was consistent with reduced interstitial fibrosis in the TM180; RSK3−/− mice as shown by histology and gene expression analysis, including the decreased expression of collagens. The specific inhibition of RSK3 should be considered as a potential novel therapeutic strategy for improving cardiac function and the prevention of sudden cardiac death in diseases in which interstitial fibrosis contributes to the development of heart failure.
Chronic baroreflex activation restores spontaneous baroreflex control and variability of heart rate in obesity-induced hypertension
Authors: Iliescu Radu , Tudorancea Ionut , Irwin Eric D. , Lohmeier Thomas E.
Published: August 2013

The sensitivity of baroreflex control of heart rate is depressed in subjects with obesity hypertension, which increases the risk for cardiac arrhythmias. The mechanisms are not fully known, and there are no therapies to improve this dysfunction. To determine the cardiovascular dynamic effects of progressive increases in body weight leading to obesity and hypertension in dogs fed a high-fat diet, 24-h continuous recordings of spontaneous fluctuations in blood pressure and heart rate were analyzed in the time and frequency domains. Furthermore, we investigated whether autonomic mechanisms stimulated by chronic baroreflex activation and renal denervation—current therapies in patients with resistant hypertension, who are commonly obese—restore cardiovascular dynamic control. Increases in body weight to ∼150% of control led to a gradual increase in mean arterial pressure to 17 ± 3 mmHg above control (100 ± 2 mmHg) after 4 wk on the high-fat diet. In contrast to the gradual increase in arterial pressure, tachycardia, attenuated chronotropic baroreflex responses, and reduced heart rate variability were manifest within 1–4 days on high-fat intake, reaching 130 ± 4 beats per minute (bpm) (control = 86 ± 3 bpm) and ∼45% and <20%, respectively, of control levels. Subsequently, both baroreflex activation and renal denervation abolished the hypertension. However, only baroreflex activation effectively attenuated the tachycardia and restored cardiac baroreflex sensitivity and heart rate variability. These findings suggest that baroreflex activation therapy may reduce the risk factors for cardiac arrhythmias as well as lower arterial pressure.
Mitochondrial reactive oxygen species: which ROS signals cardioprotection?
Authors: Garlid Anders O. , Jaburek Martin , Jacobs Jeremy P. , Garlid Keith D.
Published: August 2013

Mitochondria are the major effectors of cardioprotection by procedures that open the mitochondrial ATP-sensitive potassium channel (mitoKATP), including ischemic and pharmacological preconditioning. MitoKATPopening leads to increased reactive oxygen species (ROS), which then activate a mitoKATP-associated PKCε, which phosphorylates mitoKATPand leaves it in a persistent open state (Costa AD, Garlid KD. Am J Physiol Heart Circ Physiol 295, H874–H882, 2008). The ROS responsible for this effect is not known. The present study focuses on superoxide (O2·−), hydrogen peroxide (H2O2), and hydroxyl radical (HO˙), each of which has been proposed as the signaling ROS. Feedback activation of mitoKATPprovides an ideal setting for studying endogenous ROS signaling. Respiring rat heart mitochondria were preincubated with ATP and diazoxide, together with an agent being tested for interference with this process, either by scavenging ROS or by blocking ROS transformations. The mitochondria were then assayed to determine whether or not the persistent phosphorylated open state was achieved. Dimethylsulfoxide (DMSO), dimethylformamide (DMF), deferoxamine, Trolox, and bromoenol lactone each interfered with formation of the ROS-dependent open state. Catalase did not interfere with this step. We also found that DMF blocked cardioprotection by both ischemic preconditioning and diazoxide. The lack of a catalase effect and the inhibitory effects of agents acting downstream of HO˙ excludes H2O2as the endogenous signaling ROS. Taken together, the results support the conclusion that the ROS message is carried by a downstream product of HO˙ and that it is probably a product of phospholipid oxidation.
Functional promoter variants in sphingosine 1-phosphate receptor 3 associate with susceptibility to sepsis-associated acute respiratory distress syndrome
Authors: Sun Xiaoguang , Ma Shwu-Fan , Wade Michael S. , Acosta-Herrera Marialbert , Villar Jesús , Pino-Yanes Maria , Zhou Tong , Liu Bin , Belvitch Patrick , Moitra Jaideep , Han Yoo-Jeong , Machado Roberto , Noth Imre , Natarajan Viswanathan , Dudek Steven M. , Jacobson Jeffrey R. , Flores Carlos , Garcia Joe G. N.
Published: August 2013

The genetic mechanisms underlying the susceptibility to acute respiratory distress syndrome (ARDS) are poorly understood. We previously demonstrated that sphingosine 1-phosphate (S1P) and the S1P receptor S1PR3 are intimately involved in lung inflammatory responses and vascular barrier regulation. Furthermore, plasma S1PR3 protein levels were shown to serve as a biomarker of severity in critically ill ARDS patients. This study explores the contribution of single nucleotide polymorphisms (SNPs) of the S1PR3 gene to sepsis-associated ARDS. S1PR3 SNPs were identified by sequencing the entire gene and tagging SNPs selected for case-control association analysis in African- and ED samples from Chicago, with independent replication in a European case-control study of Spanish individuals. Electrophoretic mobility shift assays, luciferase activity assays, and protein immunoassays were utilized to assess the functionality of associated SNPs. A total of 80 variants, including 29 novel SNPs, were identified. Because of limited sample size, conclusive findings could not be drawn in African-descent ARDS subjects; however, significant associations were found for two promoter SNPs (rs7022797 −1899T/G; rs11137480 −1785G/C), across two ED samples supporting the association of alleles −1899G and −1785C with decreased risk for sepsis-associated ARDS. In addition, these alleles significantly reduced transcription factor binding to the S1PR3 promoter; reduced S1PR3 promoter activity, a response particularly striking after TNF-α challenge; and were associated with lower plasma S1PR3 protein levels in ARDS patients. These highly functional studies support S1PR3 as a novel ARDS candidate gene and a potential target for individualized therapy.
Perinatal nicotine-induced transgenerational asthma
Authors: Rehan Virender K. , Liu Jie , Sakurai Reiko , Torday John S.
Published: August 2013

Asthma is a major public health hazard worldwide. Its transgenerational inheritance has been inferred from epidemiological studies. More recently, using nicotine as a proxy for maternal smoking, we have demonstrated that an asthma-like phenotype can be inherited by rat offspring for up to two generations, i.e., multigenerationally, after the initial intrauterine exposure. We hypothesized that asthma transmission to offspring following perinatal nicotine exposure is not restricted up to F2 generation, but it also extends to subsequent generations. To test this hypothesis, using a well-established rat model of nicotine exposure-induced childhood asthma, we determined if perinatal nicotine exposure of F0 gestating dams would transmit asthma transgenerationally to F3 offspring. We now extend our findings to third-generation offspring, including abnormal pulmonary function, particularly as it relates to the occurrence in the upper airway exclusively in males, and to its effects on molecular functional markers (fibronectin and peroxisome proliferator-activated receptor γ), previously shown to be consistent with the asthma phenotype, herein expressed in fibroblasts isolated from the lung. These data, for the first time, demonstrate the transgenerational transmission of the asthma phenotype to F3 offspring following perinatal nicotine exposure of F0 dams.
Protein C and acute inflammation: a clinical and biological perspective
Authors: Christiaans Sarah C. , Wagener Brant M. , Esmon Charles T. , Pittet Jean Francois
Published: August 2013

The protein C system plays an active role in modulating severe systemic inflammatory processes such as sepsis, trauma, and acute respiratory distress syndrome (ARDS) via its anticoagulant and anti-inflammatory properties. Plasma levels of activated protein C (aPC) are lower than normal in acute inflammation in humans, except early after severe trauma when high plasma levels of aPC may play a mechanistic role in the development of posttraumatic coagulopathy. Thus, following positive results of preclinical studies, a clinical trial (PROWESS) with high continuous doses of recombinant human aPC given for 4 days demonstrated a survival benefit in patients with severe sepsis. This result was not confirmed by subsequent clinical trials, including the recently published PROWESS-SHOCK trial in patients with septic shock and a phase II trial with patients with nonseptic ARDS. A possible explanation for the major difference in outcome between PROWESS and PROWESS-SHOCK trials is that lung-protective ventilation was used for the patients included in the recent PROWESS-SHOCK, but not in the original PROWESS trial. Since up to 75% of sepsis originates from the lung, aPC treatment may not have added enough to the beneficial effect of lung-protective ventilation to show lower mortality. Thus whether aPC will continue to be used to modulate the acute inflammatory response in humans remains uncertain. Because recombinant human aPC has been withdrawn from the market, a better understanding of the complex interactions between coagulation and inflammation is needed before considering the development of new drugs that modulate both coagulation and acute inflammation in humans.
Vitamin D increases plasma renin activity independently of plasma Ca2+via hypovolemia and β-adrenergic activity
Authors: Atchison Douglas K. , Harding Pamela , Beierwaltes William H.
Published: August 2013

1, 25-Dihydroxycholechalciferol (calcitriol) and 19-nor-1, 25-dihydroxyvitamin D2(paricalcitol) are vitamin D receptor (VDR) agonists. Previous data suggest VDR agonists may actually increase renin-angiotensin activity, and this has always been assumed to be mediated by hypercalcemia. We hypothesized that calcitriol and paricalcitol would increase plasma renin activity (PRA) independently of plasma Ca2+via hypercalciuria-mediated polyuria, hypovolemia, and subsequent increased β-adrenergic sympathetic activity. We found that both calcitriol and paricalcitol increased PRA threefold ( P < 0.01). Calcitriol caused hypercalcemia, but paricalcitol did not. Both calcitriol and paricalcitol caused hypercalciuria (9- and 7-fold vs. control, P < 0.01) and polyuria (increasing 2.6- and 2.2-fold vs. control, P < 0.01). Paricalcitol increased renal calcium-sensing receptor (CaSR) expression, suggesting a potential cause of paricalcitol-mediated hypercalciuria and polyuria. Volume replacement completely normalized calcitriol-stimulated PRA and lowered plasma epinephrine by 43% ( P < 0.05). β-Adrenergic blockade also normalized calcitriol-stimulated PRA. Cyclooxygenase-2 inhibition had no effect on calcitriol-stimulated PRA. Our data demonstrate that vitamin D increases PRA independently of plasma Ca2+via hypercalciuria, polyuria, hypovolemia, and increased β-adrenergic activity.
Protective role of the endothelial isoform of nitric oxide synthase in ANG II-induced inflammatory responses in the kidney
Authors: Whiting Curtis , Castillo Alexander , Haque Mohammed Z. , Majid Dewan S. A.
Published: August 2013

In the present study, we examine the hypothesis that the nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays a protective role in the development of ANG II-induced hypertension and renal injury by minimizing oxidative stress and the inflammation induced by TNF-α. Systolic blood pressure (SBP) and renal injury responses to chronic infusions of ANG II (via implanted minipumps) were evaluated for 2 wk in wild-type (WT) and in eNOS knockout mice (KO) cotreated with or without a superoxide (O2−) scavenger, tempol (400 mg/l in the drinking water), or a TNF-α receptor blocker, etanercept (5 mg/kg/day ip). In study 1, when ANG II was given at a dose of 25 ng/min, it increased mean SBP in WT mice (Δ36 ± 3 mmHg; n = 7), and this effect was attenuated in mice pretreated with tempol (Δ24 ± 3 mmHg; n = 6). In KO mice ( n = 9), this dose of ANG II resulted in severe renal injury associated with high mortality. To avoid this high mortality in KO, study 2 was conducted with a lower dose of ANG II (10 ng/min) that increased SBP slightly in WT (Δ17 ± 7 mmHg; n = 6) but exaggeratedly in KO (Δ48 ± 12 mmHg, n = 6) associated with severe renal injury. Cotreatment with either tempol ( n = 6) or etanercept ( n = 6) ameliorated the hypertensive, as well as the renal injury responses in KO compared with WT. These data demonstrate a protective role for eNOS activity in preventing renal inflammatory injury and hypertension induced by chronic increases in ANG II.
Sensitivity to theta-burst timing permits LTP in dorsal striatal adult brain slice
Authors: Hawes Sarah L. , Gillani Fawad , Evans Rebekah C. , Benkert Elizabeth A. , Blackwell Kim T.
Published: August 2013

Long-term potentiation (LTP) of excitatory afferents to the dorsal striatum likely occurs with learning to encode new skills and habits, yet corticostriatal LTP is challenging to evoke reliably in brain slice under physiological conditions. Here we test the hypothesis that stimulating striatal afferents with theta-burst timing, similar to recently reported in vivo temporal patterns corresponding to learning, evokes LTP. Recording from adult mouse brain slice extracellularly in 1 mM Mg2+, we find LTP in dorsomedial and dorsolateral striatum is preferentially evoked by certain theta-burst patterns. In particular, we demonstrate that greater LTP is produced using moderate intraburst and high theta-range frequencies, and that pauses separating bursts of stimuli are critical for LTP induction. By altering temporal pattern alone, we illustrate the importance of burst-patterning for LTP induction and demonstrate that corticostriatal long-term depression is evoked in the same preparation. In accord with prior studies, LTP is greatest in dorsomedial striatum and relies on N-methyl-d-aspartate receptors. We also demonstrate a requirement for both Gq- and Gs/olf-coupled pathways, as well as several kinases associated with memory storage: PKC, PKA, and ERK. Our data build on previous reports of activity-directed plasticity by identifying effective values for distinct temporal parameters in variants of theta-burst LTP induction paradigms. We conclude that those variants which best match reports of striatal activity during learning behavior are most successful in evoking dorsal striatal LTP in adult brain slice without altering artificial cerebrospinal fluid. Future application of this approach will enable diverse investigations of plasticity serving striatal-based learning.
Contrasting distribution of physiological cell types in different regions of the bed nucleus of the stria terminalis
Authors: Rodríguez-Sierra Olga E. , Turesson Hjalmar K. , Pare Denis
Published: August 2013

We characterized the electroresponsive and morphological properties of neurons in the bed nucleus of the stria terminalis (BNST). Previously, Rainnie and colleagues distinguished three cell types in the anterolateral region of BNST (BNST-AL): low-threshold bursting cells (LTB; type II) and regular spiking neurons that display time-dependent (RS; type I) or fast (fIR; type III) inward rectification in the hyperpolarizing direction (Hammack SE, Mania I, Rainnie DG. J Neurophysiol 98: 638–56, 2007). We report that the same neuronal types exist in the anteromedial (AM) and anteroventral (AV) regions of BNST. In addition, we observed two hitherto unreported cell types: late-firing (LF) cells, only seen in BNST-AL, that display a conspicuous delay to firing, and spontaneously active (SA) neurons, only present in BNST-AV, firing continuously at rest. However, the feature that most clearly distinguished the three BNST regions was the incidence of LTB cells (approximately 40–70%) and the strength of their bursting behavior (both higher in BNST-AM and AV relative to AL). The incidence of RS cells was similar in the three regions (∼25%), whereas that of fIR cells was higher in BNST-AL (∼25%) than AV or AM (≤8%). With the use of biocytin, two dominant morphological cell classes were identified but they were not consistently related to particular physiological phenotypes. One neuronal class had highly branched and spiny dendrites; the second had longer but poorly branched and sparsely spiny dendrites. Both often exhibited dendritic varicosities. Since LTB cells prevail in BNST, it will be important to determine what inputs set their firing mode (tonic vs. bursting) and in what behavioral states.
A Novel Algorithm for Simplification of Complex Gene Classifiers in Cancer
Authors: Wilson Raphael A. , Teng Ling , Bachmeyer Karen M. , Bissonnette Mei Lin Z. , Husain Aliya N. , Parham David M. , Triche Timothy J. , Wing Michele R. , Gastier-Foster Julie M. , Barr Frederic G. , Hawkins Douglas S. , Anderson James R. , Skapek Stephen X. , Volchenboum Samuel L.
Published: August 2013

The clinical application of complex molecular classifiers as diagnostic or prognostic tools has been limited by the time and cost needed to apply them to patients. Using an existing 50-gene expression signature known to separate two molecular subtypes of the pediatric cancer rhabdomyosarcoma, we show that an exhaustive iterative search algorithm can distill this complex classifier down to two or three features with equal discrimination. We validated the two-gene signatures using three separate and distinct datasets, including one that uses degraded RNA extracted from formalin-fixed, paraffin-embedded material. Finally, to show the generalizability of our algorithm, we applied it to a lung cancer dataset to find minimal gene signatures that can distinguish survival. Our approach can easily be generalized and coupled to existing technical platforms to facilitate the discovery of simplified signatures that are ready for routine clinical use. Cancer Res; 73(18); 5625–32. ©2013 AACR.
Targeting the Tumor Microenvironment: From Understanding Pathways to Effective Clinical Trials
Authors: Fang Hua , DeClerck Yves A.
Published: August 2013

It is clear that tumor cells do not act alone but in close interaction with the extracellular matrix and with stromal cells in the tumor microenvironment (TME). As our understanding of tumor cell–stroma interactions increased over the last two decades, significant efforts have been made to develop agents that interfere with these interactions. Here, we discuss four different therapeutic strategies that target the TME, focusing on agents that are at the most advanced stage of preclinical or clinical development. We end this review by outlining some of the lessons we have learned so far from the development of TME-targeting agents. Cancer Res; 73(16); 4965–77. ©2013 AACR.
p63, Sharp1, and HIFs: Master Regulators of Metastasis in Triple-Negative Breast Cancer
Authors: Piccolo Stefano , Enzo Elena , Montagner Marco
Published: August 2013

Metastasis is the most significant cause of cancer-associated morbidity and mortality but remains poorly understood. Recent work revealed that metastasis of aggressive triple-negative breast cancers is suppressed by Sharp1, a factor that promotes degradation of hypoxia-inducible factors (HIF) and blunts HIF-induced malignant cell behavior. Cancer Res; 73(16); 4978–81. ©2013 AACR.
What's in a Label? Radioimmunotherapy for Metastatic Prostate Cancer
Authors: Simone Charles B. , Hahn Stephen M.
Published: August 2013

Prostate cancer has a highly tumor-restricted prostate-specific membrane antigen (PSMA) and may be the ideal solid-organ malignancy for treatment with radioimmunotherapy. Encouraging results using lutetium-177–labeled anti-PSMA monoclonal antibody J591 from a phase II study by Tawaga and colleagues support the continued clinical and preclinical development of radioimmunotherapy for solid tumors. Clin Cancer Res; 19(18); 4908–10. ©2013 AACR.
Inhibition of Cell Adhesion by a Cadherin-11 Antibody Thwarts Bone Metastasis
Authors: Lee Yu-Chen , Bilen Mehmet Asim , Yu Guoyu , Lin Song-Chang , Huang Chih-Fen , Ortiz Angelica , Cho Hyojin , Song Jian H. , Satcher Robert L. , Kuang Jian , Gallick Gary E. , Yu-Lee Li-Yuan , Huang Wilber , Lin Sue-Hwa
Published: August 2013

Cadherin-11 (CDH11) is a member of the cadherin superfamily mainly expressed in osteoblasts but not in epithelial cells. However, prostate cancer cells with a propensity for bone metastasis express high levels of cadherin-11 and reduced levels of E-cadherin. Downregulation of cadherin-11 inhibits interaction of prostate cancer cells with osteoblasts in vitro and homing of prostate cancer cells to bone in an animal model of metastasis. These findings indicate that targeting cadherin-11 may prevent prostate cancer bone metastasis. To explore this possibility, a panel of 21 monoclonal antibodies (mAb) was generated against the extracellular (EC) domain of cadherin-11. Two antibodies, mAbs 2C7 and 1A5, inhibited cadherin-11–mediated cell–cell aggregation in vitro using L-cells transfected with cadherin-11. Both antibodies demonstrated specificity to cadherin-11, and neither antibody recognized E-cadherin or N-cadherin on C4-2B or PC3 cells, respectively. Furthermore, mAb 2C7 inhibited cadherin-11–mediated aggregation between the highly metastatic PC3-mm2 cells and MC3T3-E1 osteoblasts. Mechanistically, a series of deletion mutants revealed a unique motif, aa 343-348, in the cadherin-11 EC3 domain that is recognized by mAb 2C7 and that this motif coordinated cell–cell adhesion. Importantly, administration of mAb 2C7 in a prophylactic setting effectively prevented metastasis of PC3-mm2 cells to bone in an in vivo mouse model. These results show that targeting the extracellular domain of cadherin-11 can limit cellular adhesion and metastatic dissemination of prostate cancer cells.

Implications: Monotherapy using a cadherin-11 antibody is a suitable option for the prevention of bone metastases. Mol Cancer Res; 11(11); 1401–11. ©2013 AACR.