bluuebunny/crossref_metadata_2025 huggingface dataset:
Burkholderia xenovorans RcoM

Bx

-1, a Transcriptional Regulator System for Sensing Low and Persistent Levels of Carbon Monoxide
Authors: Kerby Robert L. , Roberts Gary P.
Published: August 2012

The single-component RcoM transcription factor couples an N-terminally bound heme cofactor with a C-terminal “LytTR” DNA-binding domain. Here the RcoM

Bx

-1 protein from
Burkholderia xenovorans
LB400 was heterologously expressed and then purified in a form with minimal bound CO (∼10%) and was found to stably bind this effector with a nanomolar affinity. DNase I protection assays demonstrated that the CO-associated form binds with a micromolar affinity to two ∼60-bp DNA regions, each comprised of a novel set of three direct-repeat binding sites spaced 21 bp apart on center. Binding to each region was independent, while binding to the triplet binding sites within a region was cooperative, depended upon spacing and sequence, and was marked by phased DNase I hyperactivity and protection patterns consistent with considerable changes in the DNA conformation of the nucleoprotein complex. Each protected binding site spanned a conserved motif (5′-TTnnnG-3′) that was present, in triplicate, in putative RcoM-binding regions of more than a dozen organisms.
In vivo
screens confirmed the functional importance of the conserved “TTnnnG” motif residues and their triplet arrangement and were also used to determine an improved binding motif [5′-CnnC(C/A)(G/A)TTCAnG-3′] that more closely corresponds to canonical LytTR domain/DNA-binding sites. A low-affinity but CO-dependent binding of RcoM

Bx

-1 to a variety of DNA probes was demonstrated
in vitro
. We posit that for the RcoM

Bx

-1 protein, the high CO affinity combined with multiple low-affinity DNA-binding events constitutes a transcriptional “accumulating switch” that senses low but persistent CO levels.
Dynamics of the SetCD-Regulated Integration and Excision of Genomic Islands Mobilized by Integrating Conjugative Elements of the SXT/R391 Family
Authors: Daccord Aurélie , Mursell Mathias , Poulin-Laprade Dominic , Burrus Vincent
Published: August 2012

Mobilizable genomic islands (MGIs) are small genomic islands that are mobilizable by SXT/R391 integrating conjugative elements (ICEs) due to similar origins of transfer. Their site-specific integration and excision are catalyzed by the integrase that they encode, but their conjugative transfer entirely depends upon the conjugative machinery of SXT/R391 ICEs. In this study, we report the mechanisms that control the excision and integration processes of MGIs. We found that while the MGI-encoded integrase Int
MGI
is sufficient to promote MGI integration, efficient excision from the host chromosome requires the combined action of Int
MGI
and of a novel recombination directionality factor, RdfM. We determined that the genes encoding these proteins are activated by SetCD, the main transcriptional activators of SXT/R391 ICEs. Although they share the same regulators, we found that unlike
rdfM
,
int
MGI
has a basal level of expression in the absence of SetCD. These findings explain how an MGI can integrate into the chromosome of a new host in the absence of a coresident ICE and shed new light on the cross talk that can occur between mobilizable and mobilizing elements that mobilize them, helping us to understand part of the rules that dictate horizontal transfer mechanisms.
Role of EscP (Orf16) in Injectisome Biogenesis and Regulation of Type III Protein Secretion in Enteropathogenic Escherichia coli
Authors: Monjarás Feria Julia , García-Gómez Elizabeth , Espinosa Norma , Minamino Tohru , Namba Keiichi , González-Pedrajo Bertha
Published: August 2012

Enteropathogenic
Escherichia coli
employs a type III secretion system (T3SS) to translocate virulence effector proteins directly into enterocyte host cells, leading to diarrheal disease. The T3SS is encoded within the chromosomal locus of enterocyte effacement (LEE). The function of some of the LEE-encoded proteins remains unknown. Here we investigated the role of the Orf16 protein in T3SS biogenesis and function. An
orf16
deletion mutant showed translocator and effector protein secretion profiles different from those of wild-type cells. The
orf16
null strain produced T3S structures with abnormally long needles and filaments that caused weak hemolysis of red blood cells. Furthermore, the number of fully assembled T3SSs was also reduced in the
orf16
mutant, indicating that Orf16, though not essential, is required for efficient T3SS assembly. Analysis of protein secretion revealed that Orf16 is a T3SS-secreted substrate and regulates the secretion of the inner rod component EscI. Both pulldown and yeast two-hybrid assays showed that Orf16 interacts with the C-terminal domain of an inner membrane component of the secretion apparatus, EscU; the inner rod protein EscI; the needle protein EscF; and the multieffector chaperone CesT. These results suggest that Orf16 regulates needle length and, along with EscU, participates in a substrate specificity switch from early substrates to translocators. Taken together, our results suggest that Orf16 acts as a molecular measuring device in a way similar to that of members of the
Yersinia
YscP and flagellar FliK protein family. Therefore, we propose that this protein be renamed EscP.
An Extracelluar Protease, SepM, Generates Functional Competence-Stimulating Peptide in Streptococcus mutans UA159
Authors: Hossain Mohammad Shahnoor , Biswas Indranil
Published: August 2012

Cell-cell communication in Gram-positive bacteria often depends on the production of extracellular peptides. The cariogenic bacterium
Streptococcus mutans
employs so-called competence-stimulating peptide (CSP) to stimulate mutacin (bacteriocin) production and competence development through the activation of the ComDE two-component pathway. In
S. mutans
, CSP is secreted as a 21-residue peptide; however, mass spectrometric analysis of culture supernatant indicates the presence of an 18-residue proteolytically cleaved species. In this study, using a transposon mutagenesis screening, we identified a cell surface protease that is involved in the processing of 21-residue CSP to generate the 18-residue CSP. We named this protease SepM for
s
treptococcal
e
xtracellular
p
rotease required for
m
utacin production. We showed that the truncated 18-residue peptide is the biologically active form and that the specific postexport cleavage is a prerequisite to activate the ComDE two-component signal transduction pathway. We also showed that the CSP and the mutacins are exported outside the cell by the same ABC transporter, NlmTE. Our study further confirmed that the ComDE two-component system is absolutely necessary for mutacin production in
S. mutans
.
Analysing falls in coronary heart disease mortality in the West Bank between 1998 and 2009
Authors: Abu-Rmeileh Niveen M E , Shoaibi Azza , O'Flaherty Martin , Capewell Simon , Husseini Abdullatif
Published: August 2012

Objectives

To analyse coronary heart disease (CHD) mortality and risk factor trends in the West Bank, occupied Palestinian territory between 1998 and 2009.

Design

Modelling study using CHD IMPACT model.

Setting

The West Bank, occupied Palestinian territory.

Participants

Data on populations, mortality, patient groups and numbers, treatments and cardiovascular risk factor trends were obtained from national and local surveys, routine national and WHO statistics, and critically appraised. Data were then integrated and analysed using a previously validated CHD model.

Primary and secondary outcome measures

CHD deaths prevented or postponed are the main outcome.

Results

CHD death rates fell by 20% in the West Bank, between 1998 and 2009. Smoking prevalence was initially high in men, 51%, but decreased to 42%. Population blood pressure levels and total cholesterol levels also decreased. Conversely, body mass index rose by 1–2 kg/m2and diabetes increased by 2–8%. Population modelling suggested that more than two-thirds of the mortality fall was attributable to decreases in major risk factors, mainly total cholesterol, blood pressure and smoking. Approximately one-third of the CHD mortality decreases were attributable to treatments, particularly for secondary prevention and heart failure. However, the contributions from statins, surgery and angioplasty were consistently small.

Conclusions

CHD mortality fell by 20% between 1998 and 2009 in the West Bank. More than two-third of this fall was due to decreases in major risk factors, particularly total cholesterol and blood pressure. Our results clearly indicate that risk factor reductions in the general population compared save substantially more lives to specific treatments for individual patients. This emphasizes the importance of population-wide primary prevention strategies.
Seasonality, incidence and prognosis in atrial fibrillation and stroke in Denmark and New Zealand
Authors: Christensen Anette Luther , Rasmussen Lars Hvilsted , Baker Michael G , Lip Gregory Y H , Dethlefsen Claus , Larsen Torben Bjerregaard
Published: August 2012

Objectives

There are relatively few large studies of seasonal variation in the occurrence of stroke in patients with atrial fibrillation (AF). We investigated the seasonal variation in incidence rates of hospitalisation with stroke in patients from Denmark and New Zealand.

Design

Cohort study.

Setting

Nationwide hospital discharge data from Denmark and New Zealand.

Participants

243 381 (median age 75) subjects having a first-time hospitalisation with AF in Denmark and 51 480 (median age 76) subjects in New Zealand constituted the study population. Subjects with previous hospitalisation with stroke were excluded.

Primary and secondary effect measures

Peak-to-trough ratio of the seasonal variation in incidence rates of stroke in AF patients adjusted for an overall trend was primary effect measure and was assessed using a log-linear Poisson regression model. Secondary effect measures were incidence rate ratios of AF and 30-day case fatality for stroke patients.

Results

Incidence rates of AF per 1000 person-years in Denmark increased by 5.4% (95% CI 5.3% to 5.7%) for patients aged <65 and 5% (95% CI 4.9% to 5.1%) for patients aged ≥65, whereas the increase was 0.2% (95% CI −0.2% to 0.6%) for patients aged <65 and 2.6% (95% CI 2.4% to 2.8%) for patients aged ≥65 in New Zealand. In Denmark 36 088 subjects were hospitalised with stroke, and 7518 subjects in New Zealand, both showing peaks during winter with peak-to-trough ratios of 1.22 and 1.27, respectively and a decreasing trend. The 30-day case fatality risk for stroke patients having AF is now (2000–2008) about 20% in both countries.

Conclusions

Although incidence rates of hospitalisation with stroke in patients with AF have decreased in recent years, stroke remains a common AF complication with a high case fatality risk. The marked winter peak in incidence rates of hospitalisation with stroke in AF patients suggests that there are opportunities to reduce this complication. Further studies are necessary to identify how to optimise treatment of AF and prevention of stroke.
Prevalence, awareness, treatment and control of hypertension in a self-selected sub-Saharan African urban population: a cross-sectional study
Authors: Dzudie Anastase , Kengne André Pascal , Muna Walinjom F T , Ba Hamadou , Menanga Alain , Kouam Kouam Charles , Abah Joseph , Monkam Yves , Biholong Christian , Mintom Pierre , Kamdem Félicité , Djomou Armel , Ndjebet Jules , Wambo Cyrille , Luma Henry , Ngu Kathleen Blackett , Kingue Samuel
Published: August 2012

Objectives

Hypertension has been established as a major public health problem in Africa, but its specific contributions to disease burden are still incompletely understood. We report the prevalence and determinants of hypertension, detection, treatment and control rates among adults in major cities in Cameroon.

Design

Cross-sectional study.

Settings

Community-based multicentre study in major cities in Cameroon.

Participants

Participants were self-selected urban dwellers from the Center, Littoral, North-West and West Regions, who attended on 17 May 2011 a screening campaign advertised through mass media.

Primary and secondary outcomes measures

Hypertension defined as systolic (and/or diastolic) blood pressure (BP)≥ 140 (90) mm Hg, or ongoing BP-lowering medications.

Results

In all, 2120 participants (1003 women) were included. Among them, 1007 (prevalence rate 47.5%) had hypertension, including 319 (awareness rate 31.7%) who were aware of their status. The prevalence of hypertension increased with age overall and by sex and region. Among aware hypertensive participants, 191 (treatment rate 59.9%) were on regular BP-lowering medication, and among those treated, 47 (controlled rate 24.6%) were at target BP levels (ie, systolic (and diastolic) BP<140 (90) mm Hg). In multivariable logistic regression analysis, male gender, advanced age, parental history of hypertension, diabetes mellitus, elevated waist and elevated body mass index (BMI) were the significant predictors of hypertension. Likewise, male gender, high BMI and physical inactivity were associated with poor control.

Conclusions

High prevalence of hypertension with low awareness, treatment and control were found in this urban population; these findings are significant and alarming with consideration to the various improvements in the access to healthcare and the continuing efforts to educate communities over the last few decades.
Do ω-3 or other fatty acids influence the development of ‘growing pains’? A prebirth cohort study
Authors: Golding Jean , Northstone Kate , Emmett Pauline , Steer Colin , Hibbeln Joseph R
Published: August 2012

Objectives

To assess whether the prevalence of growing pains varies with indicators of fatty acid exposure. Growing pains (limb pains of no obvious explanation) have been shown to be strongly linked to a family history of arthritis, and are thought to predict an increased risk of the development of arthritis in adulthood. Much has been made of the possibility of fatty acids, particularly the ω-3 fatty acids, playing a preventive role in the development of arthritis, but little research has been undertaken to determine whether such fatty acids might reduce the risk of growing pains. We aimed to assess whether the prevalence of growing pains varies with indicators of fatty acid exposures.

Design

Case–control study nested within a prospective longitudinal cohort comparing prenatal and postnatal diet, blood measures and variants in fatty acid desaturase (FADS) genes that influence the metabolism of fatty acids. Statistical analysis took account of factors such as gender, smoke exposure, maternal age and education, social class and parity.

Setting

Avon Longitudinal Study of Parents & Children.

Participants

All children born between 1 April 1991 and 31 December 1992 (approximately14 000) within the Avon area (only that part of Avon under the South-West Regional Health Authority). This project compared 1676 children who reported ‘growing pains’ at age 8 with 6155 with no such pain.

Primary outcome

Reported limb pains of no apparent origin.

Results

There was no indication that the affected children had diets that differed with regard to ω-3, plasma levels of fatty acids, or the FADS genetic variants. We also assessed fetal and infant exposure but neither maternal prenatal blood levels nor maternal dietary intake, or duration of breast feeding showed any significant relationships even after adjustment for confounders.

Conclusions

Thus, there is no evidence that ω-3 fatty acid status protects against the development of growing pains in childhood.
Retrospective study of the prevalence of postanaesthetic hypothermia in dogs
Authors: Redondo J. I. , Suesta P. , Serra I. , Soler C. , Soler G. , Gil L. , Gómez‐Villamandos R. J.
Published: August 2012

The anaesthetic records of 1525 dogs were examined to determine the prevalence of postanaesthetic hypothermia, its clinical predictors and consequences. Temperature was recorded throughout the anaesthesia. At the end of the procedure, details coded in were: hyperthermia (>39.50°C), normothermia (38.50°C–39.50°C), slight (38.49°C–36.50°C), moderate (36.49°C–34.00°C) and severe hypothermia (<34.00°C). Statistical analysis consisted of multiple regression to identify the factors that are associated with the temperature at the end of the procedure. Before premedication, the temperature was 38.7 ± 0.6°C (mean ± sd). At 60, 120 and 180 minutes from induction, the temperature was 36.7 ± 1.3°C, 36.1 ± 1.4°C and 35.8 ± 1.5°C, respectively. The prevalence of hypothermia was: slight, 51.5 per cent (95 per cent CI 49.0 to 54.0 per cent); moderate, 29.3 per cent (27.1–31.7 per cent) and severe: 2.8% (2.0–3.7%). The variables that associated with a decrease in the temperature recorded at the end of the anaesthesia were: duration of the preanesthetic time, duration of the anaesthesia, physical condition (ASA III and ASA IV dogs showed lower temperatures than ASA I dogs), the reason for anaesthesia (anaesthesia for diagnostic procedures or thoracic surgery reduce the temperature when compared with minor procedures), and the recumbency during the procedure (sternal and dorsal recumbencies showed lower temperatures than lateral recumbency). The temperature before premedication and the body surface (BS) were associated with a higher temperature at the end of the anaesthesia, and would be considered as protective factors.
Crystallisation pattern of vestibular mucus and its relation to vestibular electrical resistance in cycling sow
Authors: Luño V. , Gil L. , Jerez R. A. , Malo C. , Galé I. , de Blas I.
Published: August 2012

Changes in the genital mucus around the oestrus are used by different diagnostic methods to determine optimal fertilisation time. In the current study, the authors evaluated the different arborisation patterns found in vestibular mucus, and also established its relationship with vestibular resistance changes during oestrus. Thirty multiparous sows were checked by transrectal ultrasonography to determine ovulation time every 12 hours. Vestibular resistance was measured with a commercial resistance probe, and vestibular mucus ferning was also evaluated every 12 hours during the oestrus. Significant changes (P < 0.05) in vestibular resistance were detected, registering high variation among individuals. Maximum resistance data was reached between 12 and 24 hours after ovulation time in 83 per cent of the sows. Crystallisation samples were classified into three different patterns according to the fern‐like crystal degree. Arborisation peak occurred from 48 to 36 hours before the moment of ovulation, when vestibular resistance values increased gradually. In the optimal insemination moment, vestibular resistance increased significantly (P < 0.05) and vestibular mucus showed a low crystallisation pattern (P < 0.05). Combining several methods to measure genital mucus changes may predict the ovulation time and the best insemination moment.
Cyclin D2 rescues size and function of GATA4 haplo-insufficient hearts
Authors: Yamak Abir , Temsah Rana , Maharsy Wael , Caron Sophie , Paradis Pierre , Aries Anne , Nemer Mona
Published: August 2012

Transcription factor GATA4 is a key regulator of cardiomyocyte growth, and differentiation and 50% reduction in GATA4 levels results in hypoplastic hearts. Search for GATA4 targets/effectors revealed cyclin D2 (CD2), a member of the D-type cyclins (D1, D2, and D3) that play a vital role in cell growth and differentiation as a direct transcriptional target and a mediator of GATA4 growth in postnatal cardiomyocytes. GATA4 associates with the CD2 promoter in cardiomyocytes and is sufficient to induce endogenous CD2 transcription and to dose-dependently activate the CD2 promoter in heterologous cells. Cardiomyocyte-specific overexpression of CD2 results in enhanced postnatal cardiac growth because of increased cardiomyocyte proliferation. When these transgenic mice are crossed with Gata4 heterozygote mice, they rescue the hypoplastic cardiac phenotype of Gata4+/− mice and enhance cardiomyocyte survival and heart function. The data uncover a role for CD2 in the postnatal heart as an effector of GATA4 in myocyte growth and survival. The finding that postnatal upregulation of a cell-cycle gene in GATA4 haplo-insufficient hearts may be protective opens new avenues for maintaining or restoring cardiac function in GATA4-dependent cardiac disease.
Treatment withN-acetyl-seryl-aspartyl-lysyl-proline prevents experimental autoimmune myocarditis in rats
Authors: Nakagawa Pablo , Liu Yunhe , Liao Tang-Dong , Chen Xiaojuan , González Germán E. , Bobbitt Kevin R. , Smolarek Derek , Peterson Ed L. , Kedl Ross , Yang Xiao-Ping , Rhaleb Nour-Eddine , Carretero Oscar A.
Published: August 2012

Myocarditis is commonly associated with cardiotropic infections and has been linked to development of autoimmunity. N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a naturally occurring tetrapeptide that prevents inflammation and fibrosis in hypertension and other cardiovascular diseases; however, its effect on autoimmune-mediated cardiac diseases remains unknown. We studied the effects of Ac-SDKP in experimental autoimmune myocarditis (EAM), a model of T cell-mediated autoimmune disease. This study was conducted to test the hypothesis that Ac-SDKP prevents autoimmune myocardial injury by modulating the immune responses. Lewis rats were immunized with porcine cardiac myosin and treated with Ac-SDKP or vehicle. In EAM, Ac-SDKP prevented both systolic and diastolic cardiac dysfunction, remodeling as shown by hypertrophy and fibrosis, and cell-mediated immune responses without affecting myosin-specific autoantibodies or antigen-specific T cell responses. In addition, Ac-SDKP reduced cardiac infiltration by macrophages, dendritic cells, and T cells, pro-inflammatory cytokines [interleukin (IL)-1α, tumor necrosis factor-α, IL-2, IL-17] and chemokines (cytokine-induced neutrophil chemoattractant-1, interferon-γ-induced protein 10), cell adhesion molecules (intercellular adhesion molecule-1, L-selectin), and matrix metalloproteinases (MMP). Ac-SDKP prevents autoimmune cardiac dysfunction and remodeling without reducing the production of autoantibodies or T cell responses to cardiac myosin. The protective effects of Ac-SDKP in autoimmune myocardial injury are most likely mediated by inhibition of 1) innate and adaptive immune cell infiltration and 2) expression of proinflammatory mediators such as cytokines, chemokines, adhesion molecules, and MMPs.
Oxygen dose responsiveness of human fetal airway smooth muscle cells
Authors: Hartman William R. , Smelter Dan F. , Sathish Venkatachalem , Karass Michael , Kim Sunchin , Aravamudan Bharathi , Thompson Michael A. , Amrani Yassine , Pandya Hitesh C. , Martin Richard J. , Prakash Y. S. , Pabelick Christina M.
Published: August 2012

Maintenance of blood oxygen saturation dictates supplemental oxygen administration to premature infants, but hyperoxia predisposes survivors to respiratory diseases such as asthma. Although much research has focused on oxygen effects on alveoli in the setting of bronchopulmonary dysplasia, the mechanisms by which oxygen affects airway structure or function relevant to asthma are still under investigation. We used isolated human fetal airway smooth muscle (fASM) cells from 18–20 postconceptual age lungs (canalicular stage) to examine oxygen effects on intracellular Ca2+ ([Ca2+]i) and cellular proliferation. fASM cells expressed substantial smooth muscle actin and myosin and several Ca2+ regulatory proteins but not fibroblast or epithelial markers, profiles qualitatively comparable to adult human ASM. Fluorescence Ca2+ imaging showed robust [Ca2+]i responses to 1 μM acetylcholine (ACh) and 10 μM histamine (albeit smaller and slower than adult ASM), partly sensitive to zero extracellular Ca2+. Compared with adult, fASM showed greater baseline proliferation. Based on this validation, we assessed fASM responses to 10% hypoxia through 90% hyperoxia and found enhanced proliferation at <60% oxygen but increased apoptosis at >60%, effects accompanied by appropriate changes in proliferative vs. apoptotic markers and enhanced mitochondrial fission at >60% oxygen. [Ca2+]i responses to ACh were enhanced for <60% but blunted at >60% oxygen. These results suggest that hyperoxia has dose-dependent effects on structure and function of developing ASM, which could have consequences for airway diseases of childhood. Thus detrimental effects on ASM should be an additional consideration in assessing risks of supplemental oxygen in prematurity.
TSC2 modulates cell adhesion and migration via integrin-α1β1
Authors: Moir Lyn M. , Black Judith L. , Krymskaya Vera P.
Published: August 2012

Recent evidence suggests that the rare and progressive lung disease lymphangioleiomyomatosis (LAM) is metastatic in nature. Dysfunction of the tumor suppressor genes tuberous sclerosis complex (TSC), in particular mutational inactivation of TSC2, enhances both cell proliferation and migration. Although substantial progress has been made in understanding the role of TSC2 in abnormal LAM cell proliferation and its pharmacological targeting, the mechanisms underlying the enhanced migratory capacity in LAM are not well understood. In this study, we examined the role of TSC2 in cell attachment, spreading, and migration, processes that contribute to the metastatic phenotype. Here we show that loss of TSC2 increased both the attachment and spreading of mouse embryonic fibroblasts to the extracellular matrix proteins collagen type I and fibronectin and that reexpression of TSC2 reduced these effects. Integrin-α1β1 modulated cell migration with the β1-subunit involved in cell attachment and spreading as shown by using functional blocking antibodies. Loss of TSC2 increased integrin-α1 expression, and inhibition of this integrin subunit reduced cell migration. The enhanced attachment and spreading were independent of the intracellular signaling pathways mammalian target of rapamycin complex 1 and Rho-associated kinase, as pharmacological inhibition with rapamycin or Y27632, respectively, was without effect. Together, these data demonstrate that TSC2 controls cell migration, attachment, and spreading through the α1β1-integrin receptor and thus suggest a potential therapeutic target for the treatment of increased cell invasiveness in LAM.
Arylsulfonamide KCN1 InhibitsIn VivoGlioma Growth and Interferes with HIF Signaling by Disrupting HIF-1α Interaction with Cofactors p300/CBP
Authors: Yin Shaoman , Kaluz Stefan , Devi Narra S. , Jabbar Adnan A. , de Noronha Rita G. , Mun Jiyoung , Zhang Zhaobin , Boreddy Purushotham R. , Wang Wei , Wang Zhibo , Abbruscato Thomas , Chen Zhengjia , Olson Jeffrey J. , Zhang Ruiwen , Goodman Mark M. , Nicolaou K.C. , Van Meir Erwin G.
Published: August 2012

Purpose: The hypoxia-inducible factor-1 (HIF-1) plays a critical role in tumor adaptation to hypoxia, and its elevated expression correlates with poor prognosis and treatment failure in patients with cancer. In this study, we determined whether 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-phenylbenzenesulfonamide, KCN1, the lead inhibitor in a novel class of arylsulfonamide inhibitors of the HIF-1 pathway, had antitumorigenic properties in vivo and further defined its mechanism of action.

Experimental Design: We studied the inhibitory effect of systemic KCN1 delivery on the growth of human brain tumors in mice. To define mechanisms of KCN1 anti-HIF activities, we examined its influence on the assembly of a functional HIF-1α/HIF-1β/p300 transcription complex.

Results: KCN1 specifically inhibited HIF reporter gene activity in several glioma cell lines at the nanomolar level. KCN1 also downregulated transcription of endogenous HIF-1 target genes, such as VEGF, Glut-1, and carbonic anhydrase 9, in a hypoxia-responsive element (HRE)-dependent manner. KCN1 potently inhibited the growth of subcutaneous malignant glioma tumor xenografts with minimal adverse effects on the host. It also induced a temporary survival benefit in an intracranial model of glioma but had no effect in a model of melanoma metastasis to the brain. Mechanistically, KCN1 did not downregulate the levels of HIF-1α or other components of the HIF transcriptional complex; rather, it antagonized hypoxia-inducible transcription by disrupting the interaction of HIF-1α with transcriptional coactivators p300/CBP.

Conclusions: Our results suggest that the new HIF pathway inhibitor KCN1 has antitumor activity in mouse models, supporting its further translation for the treatment of human tumors displaying hypoxia or HIF overexpression. Clin Cancer Res; 18(24); 6623–33. ©2012 AACR.
Phase I Study of Vorinostat in Combination with Temozolomide in Patients with High-Grade Gliomas: North American Brain Tumor Consortium Study 04-03
Authors: Lee Eudocia Q. , Puduvalli Vinay K. , Reid Joel M. , Kuhn John G. , Lamborn Kathleen R. , Cloughesy Timothy F. , Chang Susan M. , Drappatz Jan , Yung W. K. Alfred , Gilbert Mark R. , Robins H. Ian , Lieberman Frank S. , Lassman Andrew B. , McGovern Renee M. , Xu Jihong , Desideri Serena , Ye Xiabu , Ames Matthew M. , Espinoza-Delgado Igor , Prados Michael D. , Wen Patrick Y.
Published: August 2012

Purpose: A phase I, dose-finding study of vorinostat in combination with temozolomide (TMZ) was conducted to determine the maximum tolerated dose (MTD), safety, and pharmacokinetics in patients with high-grade glioma (HGG).

Experimental Design: This phase I, dose-finding, investigational study was conducted in two parts. Part 1 was a dose-escalation study of vorinostat in combination with TMZ 150 mg/m2/day for 5 days every 28 days. Part 2 was a dose-escalation study of vorinostat in combination with TMZ 150 mg/m2/day for 5 days of the first cycle and 200 mg/m2/day for 5 days of the subsequent 28-day cycles.

Results: In part 1, the MTD of vorinostat administered on days 1 to 7 and 15 to 21 of every 28-day cycle, in combination with TMZ, was 500 mg daily. Dose-limiting toxicities (DLT) included grade 3 anorexia, grade 3 ALT, and grade 5 hemorrhage in the setting of grade 4 thrombocytopenia. In part 2, the MTD of vorinostat on days 1 to 7 and 15 to 21 of every 28-day cycle, combined with TMZ, was 400 mg daily. No DLTs were encountered, but vorinostat dosing could not be escalated further due to thrombocytopenia. The most common serious adverse events were fatigue, lymphopenia, thrombocytopenia, and thromboembolic events. There were no apparent pharmacokinetic interactions between vorinostat and TMZ. Vorinostat treatment resulted in hyperacetylation of histones H3 and H4 in peripheral mononuclear cells.

Conclusion: Vorinostat in combination with temozolomide is well tolerated in patients with HGG. A phase I/II trial of vorinostat with radiotherapy and concomitant TMZ in newly diagnosed glioblastoma is underway. Clin Cancer Res; 18(21); 6032–9. ©2012 AACR.
Novel Role of Proline-Rich Nonreceptor Tyrosine Kinase 2 in Vascular Wall Remodeling After Balloon Injury
Authors: Gadepalli Ravisekhar , Singh Nikhlesh K. , Kundumani-Sridharan Venkatesh , Heckle Mark R. , Rao Gadiparthi N.
Published: August 2012

Objective—

To investigate the role of Pyk2, a proline-rich nonreceptor tyrosine kinase, in G protein−coupled receptor agonist, thrombin-induced human aortic smooth muscle cell growth and migration, and injury-induced vascular wall remodeling.

Methods and Results—

Thrombin, a G protein–coupled receptor agonist, activated Pyk2 in a time-dependent manner and inhibition of its stimulation attenuated thrombin-induced human aortic smooth muscle cell migration and proliferation. Thrombin also activated Grb2-associated binder protein 1, p115 Rho guanine nucleotide exchange factor, Rac1, RhoA, and p21-activated kinase 1 (Pak1) and interference with stimulation of these molecules attenuated thrombin-induced human aortic smooth muscle cell migration and proliferation. In addition, adenovirus-mediated expression of dominant negative Pyk2 inhibited thrombin-induced Grb2-associated binder protein 1, p115 rho guanine nucleotide exchange factor, Rac1, RhoA and Pak1 stimulation. Balloon injury also caused activation of Pyk2, Grb2-associated binder protein 1, p115 rho guanine nucleotide exchange factor, Rac1, RhoA, and Pak1 in the carotid artery of rat, and these responses were sensitive to inhibition by the dominant negative Pyk2. Furthermore, inhibition of Pyk2 activation resulted in reduced recruitment of smooth muscle cells onto the luminal surface and their proliferation in the intimal region leading to suppression of neointima formation.

Conclusion—

Together, these results demonstrate for the first time that Pyk2 plays a crucial role in G protein–coupled receptor agonist thrombin-induced human aortic smooth muscle cell growth and migration, as well as balloon injury–induced neointima formation.
Simultaneous Exposure of Sites in von Willebrand Factor for Glycoprotein Ib Binding and ADAMTS13 Cleavage
Authors: Chen Junmei , Ling Minhua , Fu Xiaoyun , López José A. , Chung Dominic W.
Published: August 2012

Objective—

Platelet-bound von Willebrand factor (VWF) was recently demonstrated to be a better substrate for ADAMTS13, suggesting that 1 conformational change exposes both the glycoprotein Ibα binding site in the A1 domain and the ADAMTS13 cleavage site in the A2 domain. Because ristocetin induces VWF to bind glycoprotein Ibα in the absence of shear stress, we evaluated whether it could also enhance ADAMTS13 proteolysis of VWF.

Methods and Results—

We used several VWF sources: plasma, purified plasma VWF, recombinant VWF fragments encompassing A1A2A3, A1A2, and 2 A2 domains, 1 containing a ristocetin-binding site (Asp1459–His1472) and the other lacking it. Ristocetin accelerated ADAMTS13 cleavage of multimeric VWF and of each of the recombinant VWF fragments except for the A2 domain lacking the ristocetin-binding site. We also examined the effect of ristocetin on the conformation of the A2 domain by assessing its effect on the susceptibility of Met1606 at the ADAMTS13 cleavage site to be oxidized by hypochlorous acid. Ristocetin markedly enhanced oxidation of Met1606 and Met1521 of the A2 domain.

Conclusion—

These data indicate that exposure of the sites for glycoprotein Ibα and ADAMTS13 are coupled, explaining why platelet-bound VWF is a better ADAMTS13 substrate and why enhanced proteolysis is often observed in type 2B von Willebrand disease.
The Induction of Yes-Associated Protein Expression After Arterial Injury Is Crucial for Smooth Muscle Phenotypic Modulation and Neointima Formation
Authors: Wang Xiaobo , Hu Guoqing , Gao Xiangwei , Wang Yong , Zhang Wei , Harmon Erin Yund , Zhi Xu , Xu Zhengping , Lennartz Michelle R. , Barroso Margarida , Trebak Mohamed , Chen Ceshi , Zhou Jiliang
Published: August 2012

Objective—

Abnormal proliferation and migration of vascular smooth muscle cells (SMCs) are the key events in the progression of neointima formation in response to vascular injury. The goal of this study is to investigate the functional role of a potent oncogene yes-associated protein (YAP) in SM phenotypic modulation in vitro and in vivo.

Methods and Results—

In vitro cell culture and in vivo in both mouse and rat arterial injury models YAP expression is significantly induced and correlated with the vascular SMC synthetic phenotype. Overexpression of YAP promotes SMC migration and proliferation while attenuating SM contractile gene expression. Conversely, knocking down endogenous YAP in SMCs upregulates SM gene expression but attenuates SMC proliferation and migration. Consistent with this, knocking down YAP expression in a rat carotid balloon injury model and genetic deletion of YAP, specifically, in vascular SMCs in mouse after carotid artery ligation injury attenuates injury-induced SM phenotypic switch and neointima formation.

Conclusion—

YAP plays a novel integrative role in SM phenotypic modulation by inhibiting SM-specific gene expression while promoting SM proliferation and migration in vitro and in vivo. Blocking the induction of YAP would be a potential therapeutic approach for ameliorating vascular occlusive diseases.
Facio-Genital Dysplasia-5 Regulates Matrix Adhesion and Survival of Human Endothelial Cells
Authors: Nakhaei-Nejad Maryam , Haddad George , Zhang Qiu-Xia , Murray Allan G.
Published: August 2012

Objective—

The function of the endothelial cell (EC)-enriched Rho family guanine nucleotide exchange factor, facio-genital dysplasia-5 (FGD5), is poorly understood. We sought to determine whether FGD5 regulates endothelial cytoskeletal reorganization and angiogenesis.

Methods and Results—

We observed that FGD5 is expressed in primary human EC isolated from sites across the vasculature. Inhibition of FGD5 expression using RNA interference decreased the protein by ≈70%. In 3-dimensional vascular endothelial growth factor–stimulated angiogenesis in vitro, FGD5-deficient endothelial sprout protrusion was markedly blunted versus nonsilenced controls. FGD5 knockdown impaired adhesion to fibronectin and collagen IV and remodeling of matrix adhesion complexes. Similarly, monolayer electric impedance was decreased, and impedance increased at a slower rate after seeding FGD5-deficient cells versus controls, reflecting decreased EC spreading. Further, FGD5 plays a role in cell survival, because expression of cleaved caspase-3 was increased in FGD5-deficient EC after loss of cell−matrix contacts, and proapoptotic tumor necrosis factor-α stimulation elicited EC with subdiploid DNA content among FGD5-deficient EC. Mechanistically, the phosphatidylinositol 3-kinase/Akt pathway that regulates both adhesive and survival signal transduction pathways requires FGD5. Vascular endothelial growth factor–stimulated Akt phosphorylation and downstream forkhead box protein-O1 inactivation is inhibited by FGD5 loss.

Conclusion—

FGD5 regulates endothelial adhesion, survival, and angiogenesis by modulating phosphatidylinositol 3-kinase signaling.
Decreased Krev Interaction–Trapped 1 Expression Leads to Increased Vascular Permeability and Modifies Inflammatory Responses In Vivo
Authors: Corr Maripat , Lerman Irina , Keubel Julia M. , Ronacher Lisa , Misra Ravi , Lund Frances , Sarelius Ingrid H. , Glading Angela J.
Published: August 2012

Objective—

The regulation of vascular permeability, leukocyte trafficking, and the integrity of endothelial cell–cell contacts are closely linked by a complex mechanism of interregulation. Here, we investigate the role of Krev interaction–trapped 1 (KRIT1), an adherens junction accessory protein required for cell–cell junction stability, in regulating these vascular functions.

Methods and Results—

Krit1

+/−

mice exhibited an enhanced edematous response to the complex inflammatory stimuli found in the passive K/BxN model of inflammatory arthritis and the murine air pouch model, yet leukocyte infiltration was unchanged. Correspondingly, reduced KRIT1 expression increased baseline arteriole and venule permeability 2-fold over that of wild-type littermates, as measured by intravital microscopy of the intact cremaster muscle vascular network, but this increase was not accompanied by increased leukocyte extravasation or activation. Direct stimulation with tumor necrosis factor-α induced increased permeability in wild-type mice, but surprisingly no increase over baseline levels was observed in
Krit1

+/−

mice, despite extensive leukocyte activation. Finally, adoptive transfer of
Krit1

+/−

bone marrow failed to increase permeability in wild-type mice. However, reduced expression of KRIT1 in the hematopoietic lineage dampened the differences observed in baseline permeability.

Conclusion—

Taken together, our data indicate an integral role for KRIT1 in microvessel homeostasis and the vascular response to inflammation.
Inverse Solution Mapping of Epicardial Potentials
Authors: Sapp John L. , Dawoud Fady , Clements John C. , Horáček B. Milan
Published: August 2012

Background—

Catheter ablation of ventricular tachycardia (VT) is still one of the most challenging procedures in cardiac electrophysiology, limited, in part, by unmappable arrhythmias that are nonsustained or poorly tolerated. Calculation of the inverse solution from body surface potential mapping (sometimes known as ECG imaging) has shown tremendous promise and can rapidly map these arrhythmias, but we lack quantitative assessment of its accuracy in humans. We compared inverse solution mapping with computed tomography–registered electroanatomic epicardial contact catheter mapping to study the resolution of this technique, the influence of myocardial scar, and the ability to map VT.

Methods and Results—

For 4 patients undergoing epicardial catheter mapping and ablation of VT, 120-lead body surface potential mappings were obtained during implantable defibrillator pacing, catheter pacing from 79 epicardial sites, and induced VT. Inverse epicardial electrograms computed using individualized torso/epicardial surface geometries extracted from computed tomography images were compared with registered electroanatomic contact maps. The distance between estimated and actual epicardial pacing sites was 13±9 mm over normal myocardium with no stimulus-QRS delay but increased significantly over scar (
P
=0.013) or was close to scar (
P
=0.014). Contact maps during right ventricular pacing correlated closely to inverse solution isochrones. Maps of inverse epicardial potentials during 6 different induced VTs indicated areas of earliest activation, which correlated closely with clinically identified VT exit sites for 2 epicardial VTs.

Conclusions—

Inverse solution maps can identify sites of epicardial pacing with good accuracy, which diminishes over myocardial scar or over slowly conducting tissue. This approach can also identify epicardial VT exit sites and ventricular activation sequences.
Docosahexaenoic Acid, but Not Eicosapentaenoic Acid, Supplementation Reduces Vulnerability to Atrial Fibrillation
Authors: Ramadeen Andrew , Connelly Kim A. , Leong-Poi Howard , Hu Xudong , Fujii Hiroko , Laurent Gabriel , Domenichiello Anthony F. , Bazinet Richard P. , Dorian Paul
Published: August 2012

Background—

The potential health benefits of ω-3 polyunsaturated fatty acids (PUFAs) usually are studied using a combination of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). This combination reduces vulnerability to experimentally induced atrial fibrillation (AF). It is unknown whether EPA and DHA have differential effects when taken alone. Using a model of pacing-induced atrial hemodynamic overload, we investigated the individual effects of EPA and DHA on vulnerability to AF and atrial remodeling.

Methods and Results—

Thirty-four dogs were randomized into 3 groups, all of which underwent simultaneous atrial and ventricular pacing at 220 beats per minute for 14 days. One group received purified DHA (≈1 g/d) orally for 21 days beginning 7 days before pacing began. Similarly, 1 group received ≈1 g/d purified EPA. In a third (control) group (No-PUFAs), 8 dogs received ≈1 g/d olive oil, and 12 were unsupplemented. Electrophysiological and echocardiographic measurements were taken at baseline and 21 days. Atrial tissue samples were collected at 21 days for histological and molecular analyses. Persistent AF inducibility was significantly reduced by DHA compared with No-PUFAs median [25–75 percentiles], 0% [0%–3%] for DHA versus 3.1% [2.2%–11%] for No-PUFAs;
P
=0.007) but not by EPA (3.4% [1.9%–8.9%]). DHA also reduced atrial fibrosis compared with No-PUFAs (11±6% versus 20±4%, respectively;
P
<0.05), whereas EPA did not (15±5%;
P
>0.05).

Conclusions—

DHA is more effective than EPA in attenuating AF vulnerability and atrial remodeling in structural remodeling–induced AF.
Assessing the Risk of Bleeding in Patients With Atrial Fibrillation
Authors: Lip Gregory Y. H. , Banerjee Amitava , Lagrenade Isabelle , Lane Deirdre A. , Taillandier Sophie , Fauchier Laurent
Published: August 2012

Background—

Management decisions for thromboprophylaxis in atrial fibrillation need to balance the risk of stroke against serious hemorrhage. The objective of the present analysis is to compare the Hypertension, Abnormal renal/liver function, Stroke, Bleeding history or predisposition, Labile international normalized ratio, Elderly (>65 years), Drugs/alcohol concomitantly (HAS-BLED) score against other older bleeding risk scores and the new Anticoagulation and Risk Factors in Atrial Fibrillation score in an atrial fibrillation cohort.

Methods and Results—

Patients diagnosed with nonvalvular atrial fibrillation in a 4-hospital institution between 2000 and 2010 were identified. Independent risk factors of bleeding were investigated using Cox regression. The predictive value of several bleeding risk schema was assessed using the c-statistic and net reclassification improvement. Oral anticoagulation use was highest in moderate-risk patients (59.8%) but only slightly more than high-risk (50.1%) and low-risk (46.4%) patients. Those at higher bleeding risk (HAS-BLED ≥3) were also at highest risk of stroke/thromboembolism or stroke/thromboembolism/death, as well as bleeding and all-cause mortality. On multivariable analysis, independent predictors of bleeding were age ≥75 years and age ≥65 years, alcohol excess, anemia, and heart failure. All risk scores had only modest predictive ability for bleeding, whether on vitamin K antagonist or not (c-statistic ≈0.6). When the HAS-BLED score was compared with other bleeding risk scores, the net reclassification improvement was significantly improved against all other scores tested.

Conclusions—

Current oral anticoagulation prescribing patterns would suggest that bleeding risk estimation by clinicians is poor and that oral anticoagulation prescribing does not reflect bleeding risk per se. The HAS-BLED score performs well in relation to predicting bleeding events compared with older bleeding scores and the Anticoagulation and Risk Factors in Atrial Fibrillation score, with significantly improved reclassification using HAS-BLED compared with all other bleeding risk scores tested.
Clinical and Histological Significance of Gadolinium Enhancement in Carotid Atherosclerotic Plaque
Authors: Millon Antoine , Boussel Loic , Brevet Marie , Mathevet Jean-Louis , Canet-Soulas Emmanuelle , Mory Cyril , Scoazec Jean-Yves , Douek Philippe
Published: August 2012

Background and Purpose—

Although the ability of MRI to investigate carotid plaque composition is well established, the mechanism and the significance of plaque gadolinium (Gd) enhancement remain unknown. We evaluated clinical and histological significance of Gd enhancement of carotid plaque in patients undergoing endarterectomy for carotid stenosis.

Methods—

Sixty-nine patients scheduled for a carotid endarterectomy prospectively underwent a 3-T MRI. Carotid plaque enhancement was assessed on T1-weighted images performed before and 5 minutes after Gd injection. Enhancement was recorded according to its localization. Histological analysis was performed of the entire plaque and of the area with matched contrast enhancement on MR images.

Results—

Gd enhancement was observed in 59% patients. Three types of carotid plaques were identified depending on enhancement location (shoulder region, shoulder and fibrous cap, and central in the plaque). Fibrous cap rupture, intraplaque hemorrhage, and plaque Gd enhancement was significantly more frequent in symptomatic than in asymptomatic patients (
P
=0.043,
P
<0.0001, and
P
=0.034, respectively). After histological analysis, Gd enhancement was significantly associated with vulnerable plaque (American Heart Association VI,
P
=0.006), neovascularization (
P
<0.0001), macrophages (
P
=0.030), and loose fibrosis (
P
<0.0001). Prevalence of neovessels, macrophages, and loose fibrosis in the area of Gd enhancement was 97%, 87%, and 80%, respectively, and was different depending on the enhancement location in the plaque. Fibrous cap status and composition were different depending on the type of plaque.

Conclusions—

Gd enhancement of carotid plaque is associated with vulnerable plaque phenotypes and related to an inflammatory process.