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1.  Targeting Extracellular DNA to Deliver IGF-1 to the Injured Heart 
Scientific Reports  2014;4:4257.
There is a great need for the development of therapeutic strategies that can target biomolecules to damaged myocardium. Necrosis of myocardium during a myocardial infarction (MI) is characterized by extracellular release of DNA, which can serve as a potential target for ischemic tissue. Hoechst, a histological stain that binds to double-stranded DNA can be conjugated to a variety of molecules. Insulin-like growth factor-1 (IGF-1), a small protein/polypeptide with a short circulating-half life is cardioprotective following MI but its clinical use is limited by poor delivery, as intra-myocardial injections have poor retention and chronic systemic presence has adverse side effects. Here, we present a novel delivery vehicle for IGF-1, via its conjugation to Hoechst for targeting infarcted tissue. Using a mouse model of ischemia-reperfusion, we demonstrate that intravenous delivery of Hoechst-IGF-1 results in activation of Akt, a downstream target of IGF-1 and protects from cardiac fibrosis and dysfunction following MI.
PMCID: PMC3945489  PMID: 24604065
2.  Mapping of the Mouse Olfactory System with Manganese-Enhanced Magnetic Resonance Imaging and Diffusion Tensor Imaging 
Brain structure & function  2012;218(2):527-537.
As the power of studying mouse genetics and behavior advances, research tools to examine systems level connectivity in the mouse are critically needed. In this study, we compared statistical mapping of the olfactory system in adult mice using manganese-enhanced MRI (MEMRI) and diffusion tensor imaging (DTI) with probabilistic tractography. The primary goal was to determine whether these complementary techniques can determine mouse olfactory bulb connectivity consistent with known anatomical connections. For MEMRI, 3D T1 weighted images were acquired before and after bilateral nasal administration of MnCl2 solution. Concomitantly, high resolution diffusion-tensor images were obtained ex vivo from a second group of mice and processed with a probabilistic tractography algorithm originating in the olfactory bulb. Incidence maps were created by co-registering and overlaying data from the two scan modalities. The resulting maps clearly show pathways between the olfactory bulb and amygdala, piriform cortex, caudate putamen, and olfactory cortex in both the DTI and MEMRI techniques that are consistent with the known anatomical connections. These data demonstrate that MEMRI and DTI are complementary, high-resolution neuroimaging tools that can be applied to mouse genetic models of olfactory and limbic system connectivity.
PMCID: PMC3425724  PMID: 22527121
Statistical Mapping; Diffusion Tensor Imaging (DTI); Manganese Enhanced MRI (MEMRI); Olfactory System; Mouse
3.  Organ-wide Telomeric Status in Diseased and Disease-free Prostatic Tissues 
The Prostate  2010;70(13):1471-1479.
Telomere attrition occurs early in the development of prostatic adenocarcinoma. However, little is known about either telomere status in benign prostatic hyperplasia (BPH), or the spatial and organ-wide distribution of potential telomere aberrations throughout all areas of prostatic glands affected by cancer or BPH.
Slot blot titration assay was used to determine telomere DNA content (TC), a proxy for telomere length, in macrodissected tissue consisting of 54 normal samples from 5 disease-free prostates, 128 BPH samples from 4 non-cancerous prostates, and 45 tumor, 73 BPH, and 4 prostatic intraepithelial neoplasia (PIN) samples from 5 cancerous prostates.
Compared to TC in normal prostate samples (n=54; TC mean=0.98), tumor samples displayed telomere attrition (n=45; TC mean=0.67). TC in PIN samples was similar to tumors. BPH samples from cancerous prostates were similar to TC in tumors and also displayed telomere shortening (n=73; TC mean=0.76), whereas BPH samples from non-cancerous prostates displayed longer telomeres (n=128; TC mean=1.06). In prostates affected by adenocarcinoma, areas of potential telomere attrition occurred in histologically normal tissues through the entire gland. However, three-dimensional zoning revealed a pattern of increasing TC as a function of distance from the primary (index) tumor.
Spatial distributions of TC in prostate specimens indicate a complex “field effect” with varying contributions from both cancer and BPH. The observation that telomere length variations occur in fields of histologically normal tissues surrounding the tumor is of clinical importance, as it may have implications for the diagnosis and focal therapy of prostate cancer.
PMCID: PMC3920907  PMID: 20687220
Telomere alterations; prostate cancer; benign prostatic hyperplasia; field cancerization
4.  Comparative Genomics of Cultured and Uncultured Strains Suggests Genes Essential for Free-Living Growth of Liberibacter 
PLoS ONE  2014;9(1):e84469.
The full genomes of two uncultured plant pathogenic Liberibacter, Ca. Liberibacter asiaticus and Ca. Liberibacter solanacearum, are publicly available. Recently, the larger genome of a closely related cultured strain, Liberibacter crescens BT-1, was described. To gain insights into our current inability to culture most Liberibacter, a comparative genomics analysis was done based on the RAST, KEGG, and manual annotations of these three organisms. In addition, pathogenicity genes were examined in all three bacteria. Key deficiencies were identified in Ca. L. asiaticus and Ca. L. solanacearum that might suggest why these organisms have not yet been cultured. Over 100 genes involved in amino acid and vitamin synthesis were annotated exclusively in L. crescens BT-1. However, none of these deficiencies are limiting in the rich media used to date. Other genes exclusive to L. crescens BT-1 include those involved in cell division, the stringent response regulatory pathway, and multiple two component regulatory systems. These results indicate that L. crescens is capable of growth under a much wider range of conditions than the uncultured Liberibacter strains. No outstanding differences were noted in pathogenicity-associated systems, suggesting that L. crescens BT-1 may be a plant pathogen on an as yet unidentified host.
PMCID: PMC3885570  PMID: 24416233
5.  Neonatal lesions of orbital frontal areas 11/13 in monkeys alter goal-directed behavior but spare fear conditioning and safety signal learning 
Recent studies in monkeys have demonstrated that damage to the lateral subfields of orbital frontal cortex (OFC areas 11/13) yields profound changes in flexible modulation of goal-directed behaviors and deficits in fear regulation. Yet, little consideration has been placed on its role in emotional and social development throughout life. The current study investigated the effects of neonatal lesions of the OFC on the flexible modulation of goal-directed behaviors and fear responses in monkeys. Infant monkeys received neonatal lesions of OFC areas 11/13 or sham-lesions during the first post-natal week. Modulation of goal-directed behaviors was measured with a devaluation task at 3–4 and 6–7 years. Modulation of fear reactivity by safety signals was assessed with the AX+/BX− fear-potentiated-startle paradigm at 6–7 years. Similar to adult-onset OFC lesions, selective neonatal lesions of OFC areas 11/13 yielded a failure to modulate behavioral responses guided by changes in reward value, but spared the ability to modulate fear responses in the presence of safety signals. These results suggest that these areas play a critical role in the development of behavioral adaptation during goal-directed behaviors, but not or less so, in the development of the ability to process emotionally salient stimuli and to modulate emotional reactivity using environmental contexts, which could be supported by other OFC subfields, such as the most ventromedial subfields (i.e., areas 14/25). Given similar impaired decision-making abilities and spared modulation of fear after both neonatal lesions of either OFC areas 11 and 13 or amygdala (Kazama et al., 2012; Kazama and Bachevalier, 2013), the present results suggest that interactions between these two neural structures play a critical role in the development of behavioral adaptation; an ability essential for the self-regulation of emotion and behavior that assures the maintenance of successful social relationships.
PMCID: PMC3940964  PMID: 24624054
orbitofrontal cortex (OFC); flexible decision-making; safety-signal processing; non-human primate development; areas 11 and 13
6.  Effect of bone marrow-derived extracellular matrix on cardiac function after ischemic injury 
Biomaterials  2012;33(31):10.1016/j.biomaterials.2012.07.010.
Ischemic heart disease is a leading cause of death, with few options to retain ventricular function following myocardial infarction. Hematopoietic-derived progenitor cells contribute to angiogenesis and tissue repair following ischemia reperfusion injury. Motivated by the role of bone marrow extracellular matrix (BM-ECM) in supporting the proliferation and regulation of these cell populations, we investigated BM-ECM injection in myocardial repair. In BM-ECM isolated from porcine sternum, we identified several factors important for myocardial healing, including vascular endothelial growth factor, basic fibroblast growth factor-2, and platelet-derived growth factor-BB. We further determined that BM-ECM serves as an adhesive substrate for endothelial cell proliferation. Bone marrow ECM was injected in a rat model of myocardial infarction, with and without a methylcellulose carrier gel. After one day, reduced infarct area was noted in rats receiving BM-ECM injection. After seven days we observed improved fractional shortening, decreased apoptosis, and significantly lower macrophage counts in the infarct border. Improvements in fractional shortening, sustained through 21 days, as well as decreased fibrotic area, enhanced angiogenesis, and greater c-kit-positive cell presence were associated with BM-ECM injection. Notably, the concentrations of BM-ECM growth factors were 103–108 fold lower than typically required to achieve a beneficial effect, as reported in pre-clinical studies that have administered single growth factors alone.
PMCID: PMC3849033  PMID: 22819498
Bone marrow; Growth factors; ECM (extracellular matrix); Heart; Thermally responsive material; Porcine tissue
7.  Early Intervention May Prevent the Development of PTSD: A Randomized Pilot Civilian Study with Modified Prolonged Exposure 
Biological psychiatry  2012;72(11):957-963.
Posttraumatic stress disorder is a major public health concern with long term sequelae. There are no accepted interventions delivered in the immediate aftermath of trauma. This study tested an early intervention aimed at modifying the memory to prevent the development of PTSD prior to memory consolidation.
Patients (N=137) were randomly assigned to receive 3 sessions of an early intervention beginning in the emergency department (ED) compared to an assessment only control group. Posttraumatic stress reactions (PTSR) were assessed at 4 and 12 weeks post-injury and depression at baseline and week 4. The intervention consisted of modified prolonged exposure including imaginal exposure to the trauma memory, processing of traumatic material, and in vivo and imaginal exposure homework.
Patients were assessed an average of 11.79 hours post-trauma. Intervention participants reported significantly lower PTSR than the assessment group at 4 weeks post-injury, p < 0.01, and at 12 weeks post-injury, p < 0.05, and significantly lower depressive symptoms at Week 4 than the assessment group, p < 0.05. In a subgroup analysis the intervention was the most effective at reducing PTSD in rape victims at Week 4 (p=.004) and Week 12 (p=.05).
These findings suggest that the modified prolonged exposure intervention initiated within hours of the trauma in the ED is successful at reducing PTSR and depression symptoms one and three months after trauma exposure and is safe and feasible. This is the first behavioral intervention delivered immediately post-trauma that has been shown to be effective at reducing PTSR.
PMCID: PMC3467345  PMID: 22766415
early intervention; secondary prevention; PTSD; Acute Stress Disorder; prolonged exposure; memory consolidation
8.  A Naturally-Derived Cardiac Extracellular Matrix Enhances Cardiac Progenitor Cell Behavior In Vitro 
Acta biomaterialia  2012;8(12):4357-4364.
Myocardial infarction (MI) produces a collagen scar, altering the local microenvironment and impeding cardiac function. Cell therapy is a promising therapeutic option to replace the billions of myocytes lost following MI. Despite early successes, chronic function remains impaired and is likely a result of poor cellular retention, proliferation, and differentiation/maturation. While some efforts to deliver cells with scaffolds attempt to address these shortcomings, they lack the natural cues required for optimal cell function. The goal of this study was to determine whether a naturally-derived cardiac extracellular matrix (cECM) could enhance cardiac progenitor cell (CPC) function in vitro. CPCs were isolated via magnetic sorting of c-kit+ cells and were grown on plates coated with either cECM or collagen I (COL). Our results show an increase in early cardiomyocyte markers on cECM compared to COL, as well as corresponding protein expression later. CPCs show stronger serum-induced proliferation on cECM as compared to COL, as well as increased resistance to apoptosis following serum-starvation. Finally, a microfluidic adhesion assay demonstrated stronger adhesion of CPCs to cECM compared with COL. These data suggest that cECM may be optimal for CPC therapeutic delivery, as well as provide potential mechanisms for the shortcomings in naked cell therapy.
PMCID: PMC3488121  PMID: 22842035
ECM (extracellular matrix); Progenitor Cell; Cell adhesion; Cell proliferation; Gene expression
9.  Passive Pressure–Diameter Relationship and Structural Composition of Rat Mesenteric Lymphangions 
Lymphatic Research and Biology  2012;10(4):152-163.
Lymph flow depends on both the rate of lymph production by tissues and the extent of passive and active pumping. Here we aim to characterize the passive mechanical properties of a lymphangion in both mid-lymphangion and valve segments to assess regional differences along a lymphangion, as well as evaluating its structural composition.
Methods and Results
Mesenteric lymphatic vessels were isolated and cannulated in a microchamber for pressure–diameter (P-D) testing. Vessels were inflated from 0 to 20 cmH2O at a rate of 4 cmH2O/min, and vessel diameter was continuously tracked, using an inverted microscope, video camera, and custom LabVIEW program, at both mid-lymphangion and valve segments. Isolated lymphatic vessels were also pressure-fixed at 2 and 7 cmH2O and imaged using a nonlinear optical microscope (NLOM) to obtain collagen and elastin structural information. We observed a highly nonlinear P-D response at low pressures (3–5 cmH2O), which was modeled using a three-parameter constitutive equation. No significant difference in the passive P-D response was observed between mid-lymphangion and valve regions. NLOM imaging revealed an inner elastin layer and outer collagen layer at all locations. Lymphatic valve leaflets were predominantly elastin with thick axially oriented collagen bands at the insertion points.
We observed a highly nonlinear P-D response at low pressures (3–5 cmH2O) and developed the first constitutive equation to describe the passive P-D response for a lymphangion. The passive P-D response did not vary among regions, in agreement with the composition of elastin and collagen in the lymphatic wall.
PMCID: PMC3525898  PMID: 23145980
10.  Characterization of Superoxide Dismutases in Cardiac Progenitor Cells Demonstrates a Critical Role for Manganese Superoxide Dismutase 
Stem Cells and Development  2012;21(17):3136-3146.
Transplantation of cardiac progenitor cells (CPCs) is currently in early clinical testing as a potential therapeutic strategy. Superoxide is increased in the ischemic myocardium and poor survival of cells is one of the major limitations of cell transplantation therapy. Superoxide dismutase (SOD) levels were analyzed in c-kit-positive CPCs isolated from rat myocardium to identify their roles in protection against oxidative stress-induced apoptosis in vitro. CPCs were subjected to oxidative stress using xanthine/xanthine oxidase (XXO) and little apoptosis was detected. CPCs contained significantly higher levels of SOD1 and SOD2 as compared with adult cardiac cell types, both at the protein and activity levels. Both SOD1 and SOD2 were increased by XXO at the mRNA and protein level, suggesting compensatory adaptation. Only knockdown of SOD2 and not SOD1 with siRNA sensitized the cells to XXO-apoptosis, despite only accounting for 10% of total SOD levels. Finally, we found XXO activated Akt within 10 min, and this regulated both SOD2 gene expression and protection against apoptosis. Rat CPCs are resistant to superoxide-induced cell death, primarily through higher levels of SOD2 compared to adult cardiac-derived cells. Exposure to superoxide increases expression of SOD2 in an Akt-dependent manner and regulates CPC survival during oxidative stress.
PMCID: PMC3495122  PMID: 22758933
11.  Inducible Renitence limits Listeria monocytogenes Escape from Vacuoles in Macrophages 
Membranes of endolysosomal compartments in macrophages are often damaged by physical or chemical effects of particles ingested through phagocytosis or by toxins secreted by intracellular pathogens. This study identified a novel inducible activity in macrophages which increases resistance of phagosomes, late endosomes and lysosomes to membrane damage. Pretreatment of murine macrophages with lipopolysaccharide, peptidoglycan, tumor necrosis factor-α or interferon-γ (IFN-γ) conferred protection against subsequent damage to intracellular membranes caused by photooxidative chemistries or by phagocytosis of ground silica or silica microspheres. Phagolysosome damage was partially dependent on reactive oxygen species, but was independent of the phagocyte oxidase. IFN-γ-stimulated macrophages from mice lacking the phagocyte oxidase inhibited escape from vacuoles by the intracellular pathogen Listeria monocytogenes (L.m.), which suggested a role for this inducible renitence (resistance to pressure) in macrophage resistance to infection by pathogens that damage intracellular membranes. Renitence and inhibition of L.m. escape were partially attributable to heat shock protein-70 (HSP70). Thus, renitence is a novel, inducible activity of macrophages which maintains or restores the integrity of endolysosomal membranes.
PMCID: PMC3478491  PMID: 23002437
12.  High Titers of IgE Antibody to Dust Mite Allergen and the Risk for Wheezing Among Asthmatic Children Infected with Rhinovirus 
The relevance of allergic sensitization, judged by titers of serum IgE antibodies, to the risk of an asthma exacerbation caused by rhinovirus is unclear.
To examine the prevalence of rhinovirus infections in relation to the atopic status of children treated for wheezing in Costa Rica, a country with an increased asthma burden.
The children enrolled (n=287) were 7 through 12 years old. They included 96 with acute wheezing, 65 with stable asthma, and 126 non-asthmatic controls. PCR methods, including gene sequencing to identify rhinovirus strains, were used to identify viral pathogens in nasal washes. Results were examined in relation to wheezing, total IgE, allergen-specific IgE antibody, and levels of expired nitric oxide (FENO).
Sixty-four percent of wheezing children compared to 13% of children with stable asthma and 17% of the non-asthmatic controls tested positive for rhinovirus (p<0.001 for both comparisons). Among wheezing subjects, 75% of the rhinoviruses detected were Group C strains. High titers of IgE antibodies to dust mite allergen (especially Dermatophagoides sp) were common and correlated significantly with levels of total IgE and FENO. The greatest risk for wheezing was observed among children with titers of IgE antibodies to dust mite ≥17.5 IU/ml who tested positive for rhinovirus (odds ratio for wheezing: 31.5; 95% CI 8.3–108, p<0.001).
High titers of IgE antibody to dust mite allergen were common and significantly increased the risk for acute wheezing provoked by rhinovirus among asthmatic children.
PMCID: PMC3792652  PMID: 22560151
acute asthma; dust mite specific IgE; emergency room visits; viral respiratory tract infections; rhinovirus strain C; total serum IgE; inhaled allergens; exhaled nitric oxide (FENO)
13.  Effects of Substance P in the Amygdala, Ventromedial Hypothalamus and Periaqueductal Gray on Fear Potentiated Startle 
The neural pathways through which substance P (SP) influences fear and anxiety are poorly understood. However, the amygdala, a brain area repeatedly implicated in fear and anxiety processes, is known to contain large numbers of SP containing neurons and SP receptors. Several studies have implicated SP neurotransmission within the amygdala in anxiety processes. In the present study, we evaluated the effects of site-specific infusions of a SP receptor antagonist, GR 82334, on conditioned fear responses using the fear-potentiated startle paradigm. GR 82334 infusion into the basolateral (BLA) or the medial (MeA) nuclei of the amygdala, but not into the central nucleus (CeA) of the amygdala, dose-dependently reduced fear-potentiated startle. Similar effects were obtained with GR 82334 infusion into the ventromedial nucleus of the hypothalamus (VMH), to which the MeA projects, and into the rostral dorsolateral periaqueductal gray (PAG), to which the VMH projects, but not into the deep layers of the superior colliculus/deep mesencephalic nucleus (dSC/DpMe), an output of the CeA previously shown to be important for fear-potentiated startle. Consistent with previous findings, infusion of the AMPA receptor antagonist, NBQX, into the dSC/DpMe, but not into the PAG, did disrupt fear-potentiated startle. These findings suggest that multiple outputs from the amygdala play a critical role in fear-potentiated startle and that SP plays a critical, probably modulatory role, in the MeA to VMH to PAG to the startle pathway based on these and data from others.
PMCID: PMC3792658  PMID: 18418359
Amygdala; Hypothalamus; Periaqueductal Gray; Superior Colliculus; Midbrain; GR 82334; Morphine; Anxiety; CRH; Tachykinin
14.  Identification of the Mutation Responsible for the Temperature-Sensitive Lipopolysaccharide O-Antigen Defect in the Pseudomonas aeruginosa Cystic Fibrosis Isolate 2192 
Journal of Bacteriology  2013;195(7):1504-1514.
Pseudomonas aeruginosa in the lungs of cystic fibrosis (CF) patients is characterized by a series of genotypic and phenotypic changes that reflect the transition from acute to chronic infection. These include the overproduction of the exopolysaccharide alginate and the loss of complete lipopolysaccharide (LPS). LPS is a major component of the Gram-negative outer membrane and is composed of lipid A, core oligosaccharide, and O antigen. In this report, we show that the LPS defect of the P. aeruginosa chronic infection isolate 2192 is temperature sensitive. When grown at 25°C, 2192 expresses serotype O1 LPS with a moderate chain length and in reduced amounts relative to those of a wild-type serotype O1 laboratory strain (stO1). In contrast, 2192 expresses no LPS O antigen when grown at 37°C. This is the first time that a temperature-sensitive defect in O-antigen production has been reported. Using complementation analyses with a constructed wbpM deletion mutant of stO1, we demonstrate that the temperature-sensitive O-antigen production defect in 2192 is due to a mutation in wbpM, which encodes a UDP-4,6-GlcNAc dehydratase involved in O-antigen synthesis. The mutation, a deletion of a single amino acid (V636) from the extreme C terminus of WbpM, renders the protein less stable than its wild-type counterpart. This residue of WbpM, which is critical for stability and function, is located outside of the recognized domains of the protein and may provide insight into the structure-function relationship of this enzyme, which is found in all 20 serotypes of P. aeruginosa. We also identify a promoter of wbpM, map a transcriptional start site of wbpM, and show that mucoidy plays a role in the loss of expression of high-molecular-weight LPS in this CF isolate.
PMCID: PMC3624535  PMID: 23354750
15.  Cellular Encapsulation Enhances Cardiac Repair 
Stem cells for cardiac repair have shown promise in preclinical trials, but lower than expected retention, viability, and efficacy. Encapsulation is one potential strategy to increase viable cell retention while facilitating paracrine effects.
Methods and Results
Human mesenchymal stem cells (hMSC) were encapsulated in alginate and attached to the heart with a hydrogel patch in a rat myocardial infarction (MI) model. Cells were tracked using bioluminescence (BLI) and cardiac function measured by transthoracic echocardiography (TTE) and cardiac magnetic resonance imaging (CMR). Microvasculature was quantified using von Willebrand factor staining and scar measured by Masson's Trichrome. Post‐MI ejection fraction by CMR was greatly improved in encapsulated hMSC‐treated animals (MI: 34±3%, MI+Gel: 35±3%, MI+Gel+hMSC: 39±2%, MI+Gel+encapsulated hMSC: 56±1%; n=4 per group; P<0.01). Data represent mean±SEM. By TTE, encapsulated hMSC‐treated animals had improved fractional shortening. Longitudinal BLI showed greatest hMSC retention when the cells were encapsulated (P<0.05). Scar size at 28 days was significantly reduced in encapsulated hMSC‐treated animals (MI: 12±1%, n=8; MI+Gel: 14±2%, n=7; MI+Gel+hMSC: 14±1%, n=7; MI+Gel+encapsulated hMSC: 7±1%, n=6; P<0.05). There was a large increase in microvascular density in the peri‐infarct area (MI: 121±10, n=7; MI+Gel: 153±26, n=5; MI+Gel+hMSC: 198±18, n=7; MI+Gel+encapsulated hMSC: 828±56 vessels/mm2, n=6; P<0.01).
Alginate encapsulation improved retention of hMSCs and facilitated paracrine effects such as increased peri‐infarct microvasculature and decreased scar. Encapsulation of MSCs improved cardiac function post‐MI and represents a new, translatable strategy for optimization of regenerative therapies for cardiovascular diseases.
PMCID: PMC3835246  PMID: 24113327
angiogenesis; cardiovascular diseases; heart failure; ischemia; myocardial infarction
16.  Poststroke Hip Fracture: Prevalence, Clinical Characteristics, Mineral-Bone Metabolism, Outcomes, and Gaps in Prevention 
Stroke Research and Treatment  2013;2013:641943.
Objective. To assess the prevalence, clinical and laboratory characteristics, and short-term outcomes of poststroke hip fracture (HF). Methods. A cross-sectional study of 761 consecutive patients aged ≥60 years (82.3 ± 8.8 years; 75% females) with osteoporotic HF. Results. The prevalence of poststroke HF was 13.1% occurring on average 2.4 years after the stroke. The poststroke group compared to the rest of the cohort had a higher proportion of women, subjects with dementia, history of TIA, hypertension, coronary artery disease, secondary hyperparathyroidism, higher serum vitamin B12 levels (>350 pmol/L), walking aid users, and living in residential care facilities. The majority of poststroke HF patients had vitamin D insufficiency (68%) and excess bone resorption (90%). This group had a 3-fold higher incidence of postoperative myocardial injury and need for institutionalisation. In multivariate analysis, independent indicators of poststroke HF were female sex (OR 3.6), history of TIA (OR 5.2), dementia (OR 4.1), hypertension (OR 3.2), use of walking aid (OR 2.5), and higher vitamin B12 level (OR 2.3). Only 15% of poststroke patients received antiosteoporotic therapy prior to HF. Conclusions. Approximately one in seven HFs occurs in older stroke survivors and are associated with poorer outcomes. Early implementation of fracture prevention strategies is needed.
PMCID: PMC3800649  PMID: 24187647
17.  Detection Of Ras GTPase Protein Radicals Through Immuno-Spin Trapping* 
Free radical biology & medicine  2012;53(6):1339-1345.
Over the past decade immuno-spin trapping (IST) has been used to detect and identify protein radical sites in numerous heme and metalloproteins. To date, however, the technique has had little application toward non-metalloproteins. In this study, we demonstrate the successful application of IST in a system free of transition metals and present the first conclusive evidence of ·NO-mediated protein radical formation in the HRas GTPase. HRas is a non-metalloprotein that plays a critical role in regulating cell growth control. Protein radical formation in Ras GTPases has long been suspected of initiating premature release of bound guanine nucleotide. This action results in altered Ras activity both in vitro and in vivo. As described herein, successful application of IST may provide a means for detecting and identifying radical-mediated Ras activation in many different cancers and disease states where Ras GTPases play an important role.
PMCID: PMC3549333  PMID: 22819983
Ras GTPase; radical-mediated activation; protein radical; immuno-spin trapping
18.  Circulating folate, vitamin B12, homocysteine, vitamin B12 transport proteins and risk of prostate cancer: a case-control study, systematic review and meta-analysis 
Disturbed folate metabolism is associated with an increased risk of some cancers. Our objective was to determine whether blood levels of folate, vitamin B12 and related metabolites were associated with prostate cancer risk.
Matched case-control study nested within the UK population-based ProtecT study of PSA-detected prostate cancer in men aged 50–69 years. Plasma concentrations of folate, B12 (cobalamin), holo-haptocorrin, holo- and total-transcobalamin, and total homocysteine (tHcy) were measured in 1,461 cases and 1,507 controls. ProtecT study estimates for associations of folate, B12, and tHcy with prostate cancer risk were included in a meta-analysis, based on a systematic review.
In the ProtecT study, increased B12 and holo-haptocorrin concentrations showed positive associations with prostate cancer risk (highest vs lowest quartile of B12 odds ratio (OR)=1.17 (95% CI 0.95–1.43), P-for-trend=0.06; highest vs lowest quartile of holo-haptocorrin OR=1.27 (1.04–1.56), P-for-trend=0.01); folate, holo-transcobalamin and tHcy were not associated with prostate cancer risk. In the meta-analysis, circulating B12 levels were associated with an increased prostate cancer risk (pooled OR=1.10 (1.01–1.19) per 100 pmol/L increase in B12, P=0.002); the pooled OR for the association of folate with prostate cancer was positive (OR=1.11 (0.96–1.28) per 10 nmol/L, P=0.2) and conventionally statistically significant if ProtecT (the only case-control study) was excluded (OR=1.18 (1.00–1.40) per 10 nmol/L, P=0.02).
Vitamin B12 and (in cohort studies) folate were associated with increased prostate cancer risk.
Given current controversies over mandatory fortification, further research is needed to determine whether these are causal associations.
PMCID: PMC3759018  PMID: 20501771
folate; vitamin B12; cobalamin; transcobalamin; haptocorrin; homocysteine; folate-mediated one-carbon metabolism; prostate cancer
Molecular psychiatry  2012;18(3):308-319.
The lateral division of the bed nucleus of the stria terminalis (BNST), which forms part of the circuitry regulating fear and anxiety, contains a large number of neurons expressing corticotropin releasing factor (CRF), a neuropeptide that plays a prominent role in the etiology of fear- and anxiety-related psychopathologies. Stress increases CRF expression within BNST neurons, implicating these cells in stress- and anxiety-related behaviors. These experiments examined the effect of chronically enhanced CRF expression within BNST neurons on conditioned and unconditioned anxiety-related behavior by using a lentiviral vector containing a promoter that targets CRF gene over-expression (OE) to CRFergic cells. We found that BNST CRF over-expression did not affect unconditioned anxiety-like responses in the elevated plus maze or basal acoustic startle amplitude. CRF OE induced prior to training weakened sustained fear (conditioned anxiety); when induced after conditioning, CRF OE increased expression of the conditioned emotional memory. Increased BNST CRF expression did not affect plasma corticosterone concentration but did decrease CRFR1 receptor density within the BNST and CRFR2 receptor density within the dorsal portion of the caudal dorsal raphe nucleus. These data raise the possibility that the observed behavioral effects may be mediated by enhanced CRF receptor signaling or compensatory changes in CRF receptor density within these structures. Together, these studies demonstrate that CRF neurons within the lateral BNST modulate conditioned anxiety-like behaviors and also suggest that enhanced CRF expression within these neurons may contribute to inappropriate regulation of emotional memories.
PMCID: PMC3578178  PMID: 22290119
bed nucleus of the stria terminalis; corticotropin releasing factor; fear; anxiety; startle; HPA axis
20.  Inhibition of fear is differentially associated with cycling estrogen levels in women 
Although the prevalence of posttraumatic stress disorder (PTSD) is twice as high in women as it is in men, the role of estrogen in the risk for PTSD is not well understood. Deficits in fear inhibition and impaired safety signal learning may be biomarkers for PTSD. We examined menstrual cycle phase and serum estradiol levels in naturally cycling women while they were undergoing a novel conditioned inhibition procedure that measured their ability to discriminate between cues representing danger versus safety and to inhibit fear in the presence of safety cues.
Sample 1 included healthy participants in whom we compared inhibition of fear-potentiated startle during the follicular (lower estrogen) and luteal (higher estrogen) phases of the menstrual cycle. We used the same paradigm in a traumatized clinical population (sample 2) in whom we compared low versus high estradiol levels.
In both samples, we found that lower estrogen in cycling women was associated with impaired fear inhibition.
In the clinical sample, the low estradiol group was on average older than the high estradiol group owing to the random recruitment approach; we did not exclude participants based on hormonal status or menopause.
Our results suggest that the lower estrogen state during normal menstrual cycling may contribute to risk for anxiety disorders through dysregulated fear responses.
PMCID: PMC3756118  PMID: 23611176
Depression and anxiety  2012;30(3):217-224.
Previous work has shown that inhibition of fear is impaired in posttraumatic stress disorder (PTSD) resulting from both civilian and combat trauma. The purpose of the present study was to investigate the inhibition of learned fear in traumatized individuals diagnosed with either acute stress disorder (ASD) or PTSD. This is the first study to use a conditioned inhibition paradigm with traumatized individuals within a month of trauma exposure. We hypothesized that impaired fear inhibition would be evident in PTSD, but not ASD.
Using established translational, psychophysiological methods including fear-potentiated startle, and skin conductance, we examined fear acquisition, stimulus discrimination, and the transfer of learned safety in a Croatian population with ASD or PTSD. This cross-sectional study included three age-matched groups: healthy nontrauma controls (n = 27), a group with chronic PTSD (10 or more years since trauma exposure, n = 24), and a group with ASD (30 days or less since trauma exposure, n = 27).
The presence of trauma-related psychopathology, whether acute or chronic, was associated with an impaired ability to transfer learned safety based on fear-potentiated startle measures, while healthy control subjects showed significant fear inhibition in the presence of the safety cue compared to the danger cue, F(1,26) = 12.64, P = .001.
These data expand our previously observed findings of PTSD-associated fear inhibition deficits by demonstrating that trauma-related impairments in safety learning are evident within 30 days of trauma exposure.
PMCID: PMC3752410  PMID: 22907890
anxiety disorders; biological markers; PTSD; startle; trauma
22.  Conditioned fear extinction and reinstatement in a human fear-potentiated startle paradigm 
The purpose of this study was to analyze fear extinction and reinstatement in humans using fear-potentiated startle. Participants were fear conditioned using a simple discrimination procedure with colored lights as the conditioned stimuli (CSs) and an airblast to the throat as the unconditioned stimulus (US). Participants were extinguished 24 h after fear conditioning. Upon presentation of unsignaled USs after extinction, participants displayed significant fear reinstatement. In summary, these procedures produced robust fear-potentiated startle, significant CS+/CS− discrimination, within-session extinction, and significant reinstatement. This is the first demonstration of fear extinction and reinstatement in humans using startle measures.
PMCID: PMC3746591  PMID: 17142300
23.  Effects of Neonatal Amygdala Lesions on Fear Learning, Conditioned Inhibition, and Extinction in Adult Macaques 
Behavioral neuroscience  2012;126(3):392-403.
Fear conditioning studies have demonstrated the critical role played by the amygdala in emotion processing. Although all lesion studies until now investigated the effect of adult-onset damage on fear conditioning, the current study assessed fear-learning abilities, as measured by fear-potentiated startle, in adult monkeys that had received neonatal neurotoxic amygdala damage or sham-operations. After fear acquisition, their abilities to learn and use a safety cue to modulate their fear to the conditioned cue, and, finally, to extinguish their response to the fear conditioned cue were measured with the AX+/BX− Paradigm. Neonatal amygdala damage retarded, but did not completely abolish, the acquisition of a learned fear. After acquisition of the fear signal, four of the six animals with neonatal amygdala lesions discriminated between the fear and safety cues and were also able to use the safety signal to reduce the potentiated-startle response and to extinguish the fear response when the air-blast was absent. In conclusion, the present results support the critical contribution of the amygdala during the early phases of fear conditioning that leads to quick, robust responses to potentially threatening stimuli, a highly adaptive process across all species and likely to be present in early infancy. The neonatal amygdala lesions also indicated the presence of amygdala-independent alternate pathways that are capable to support fear learning in the absence of a functional amygdala. This parallel processing of fear responses within these alternate pathways was also sufficient to support the ability to flexibly modulate the magnitude of the fear responses.
PMCID: PMC3740331  PMID: 22642884
safety-signal learning; post-traumatic stress disorder; emotion regulation; rhesus monkey
24.  Contingency Awareness and Fear Inhibition in a Human Fear-Potentiated Startle Paradigm 
Behavioral neuroscience  2006;120(5):995-1004.
Fear-potentiated startle is defined as an increase in the magnitude of the startle reflex in the presence of a stimulus that was previously paired with an aversive event. It has been proposed that a subject’s awareness of the contingencies in the experiment may affect fear-potentiated startle. The authors adapted a conditional discrimination procedure (AX+/BX−), previously validated in animals, to a human fear-potentiated startle paradigm in 50 healthy volunteers. This paradigm allows for an assessment of fear-potentiated startle during threat conditions as well as inhibition of fear-potentiated startle during safety conditions. A response keypad was used to assess contingency awareness on a trial-by-trial basis. Both aware and unaware subjects showed fear-potentiated startle. However, awareness was related to stimulus discrimination and fear inhibition.
PMCID: PMC3740393  PMID: 17014251
human startle response; contingency awareness; discrimination learning; fear inhibition
25.  Induction of colonic aberrant crypts in mice by feeding apparent N-nitroso compounds derived from hot dogs 
Nutrition and Cancer  2012;64(2):342-349.
Nitrite-preserved meats (e.g., hot dogs) may help cause colon cancer because they contain N-nitroso compounds. We tested whether purified hot-dog-derived total apparent N-nitroso compounds (ANC) could induce colonic aberrant crypts, which are putative precursors of colon cancer. We purified ANC precursors in hot dogs and nitrosated them to produce ANC. In preliminary tests, CF1 mice received 1 or 3 i.p. injections of 5mg azoxymethane (AOM)/kg. In Experiments 1 and 2, female A/J mice received ANC in diet. In Experiment 1, ANC dose initially dropped sharply because the ANC precursors had mostly decomposed but, later in Experiment 1 and throughout Experiment 2, ANC remained at 85 nmol/g diet. Mice were killed after 8 (AOM tests) or 17–34 (ANC tests) wk. Median numbers of aberrant crypts in the distal 2 cm of the colon for 1 and 3 AOM injections, CF1 controls, ANC (Experiment 1), ANC (Experiment 2),and untreated A/J mice were 31, 74, 12, 20, 12, and 5–6, with P < 0.01 for both ANC tests. Experiment 2 showed somewhat increased numbers of colonic mucin-depleted foci in the ANC-treated group. We conclude that hot-dog-derived ANC induced significant numbers of aberrant crypts in the mouse colon.
PMCID: PMC3733137  PMID: 22293095
Aberrant Crypt Foci; chemically induced; Animals; Azoxymethane; administration & dosage; toxicity; Carcinogens; toxicity; Colonic Neoplasms; chemically induced; Feces; chemistry; Female; Food Handling; Meat Products; analysis; toxicity; Mice; Nitrosation; Nitroso Compounds; analysis; toxicity; Sodium Nitrite; administration & dosage; metabolism

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