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1.  Impulsivity and Risk Taking in Bipolar Disorder and Schizophrenia 
Neuropsychopharmacology  2013;39(2):456-463.
Impulsive risk taking contributes to deleterious outcomes among clinical populations. Indeed, pathological impulsivity and risk taking are common in patients with serious mental illness, and have severe clinical repercussions including novelty seeking, response disinhibition, aggression, and substance abuse. Thus, the current study seeks to examine self-reported impulsivity (Barratt Impulsivity Scale) and performance-based behavioral risk taking (Balloon Analogue Risk Task) in bipolar disorder and schizophrenia. Participants included 68 individuals with bipolar disorder, 38 with schizophrenia, and 36 healthy controls. Self-reported impulsivity was elevated in the bipolar group compared with schizophrenia patients and healthy controls, who did not differ from each other. On the risk-taking task, schizophrenia patients were significantly more risk averse than the bipolar patients and controls. Aside from the diagnostic group differences, there was a significant effect of antipsychotic (AP) medication within the bipolar group: bipolar patients taking AP medications were more risk averse than those not taking AP medications. This difference in risk taking because of AP medications was not explained by history of psychosis. Similarly, the differences in risk taking between schizophrenia and bipolar disorder were not fully explained by AP effects. Implications for clinical practice and future research are discussed.
PMCID: PMC3870783  PMID: 23963117
antipsychotic medication; bipolar disorder; dopamine; impulsivity; psychiatry & behavioral sciences; psychopharmacology; risk-taking; schizophrenia; schizophrenia; antipsychotics; schizophrenia; psychopharmacology; behavioral sciences; dopamine
2.  Challenges in Developing a Validated Biomarker for Angiogenesis Inhibitors: The Motesanib Experience 
PLoS ONE  2014;9(10):e108048.
We sought to develop placental growth factor as a predictive pharmacodynamic biomarker for motesanib efficacy as first-line therapy in patients with advanced nonsquamous non–small-cell lung cancer.
Experimental Design
Placental growth factor was evaluated at baseline and study week 4 (after 3 weeks treatment) in an exploratory analysis of data from a randomized phase 2 study of motesanib 125 mg once daily plus carboplatin/paclitaxel and in a prespecified analysis of data from a randomized, double-blind phase 3 study of motesanib 125 mg once daily plus carboplatin/paclitaxel vs placebo plus carboplatin/paclitaxel (MONET1). Associations between fold-change from baseline in placental growth factor and overall survival were evaluated using Cox proportional hazards models.
In the phase 2 study, serum placental growth factor increased from baseline a mean 2.8-fold at study week 4. Patients with ≥2.2-fold change from baseline in placental growth factor (n = 18) had significantly longer overall survival than those with <2.2-fold change (n = 19; 22.9 vs 7.9 months; hazard ratio, 0.30; 95% CI, 0.12–0.74; P = 0.009). Consequently, placental growth factor was investigated as a pharmacodynamic biomarker in the phase 3 MONET1 study. There was no association between log-transformed placental growth factor fold-change from baseline to week 4 (continuous variable) and overall survival (hazard ratio, 0.98; 95% CI, 0.79–1.22; P = 0.868). MONET1 did not meet its primary endpoint of overall survival. Likewise, median overall survival was similar among patients with ≥2.0-fold change in placental growth factor (n = 229) compared with <2.0-fold change (n = 127; 14.8 vs 13.8 months; hazard ratio, 0.88; 95% CI, 0.67–1.15, P = 0.340).
Our results illustrate the challenges of successfully translating phase 2 biomarker results into phase 3 studies.
Trial Registration NCT00460317, NCT00369070
PMCID: PMC4196848  PMID: 25314641
3.  Delivery of Nox2-NADPH oxidase siRNA with polyketal nanoparticles for improving cardiac function following myocardial infarction 
Biomaterials  2013;34(31):7790-7798.
Myocardial infarction (MI) is the most common cause of heart failure (HF), the leading cause of death in the developed world. Oxidative stress due to excessive production of reactive oxygen species (ROS) plays a key role in the pathogenesis of cardiac remodeling leading to HF. NADPH oxidase with Nox2 as the catalytic subunit is a major source for cardiac ROS production. Nox2-NADPH expression is significantly increased in the infarcted myocardium, primarily in neutrophils, macrophages and myocytes. Moreover, mice lacking the Nox2 gene are protected from ischemic injury, implicating Nox2 as a potential therapeutic target. RNAi-mediated gene silencing holds great promise as a therapeutic owing to its high specificity and potency. However, in vivo delivery hurdles have limited its effective clinical use. Here, we demonstrate acid-degradable polyketal particles as delivery vehicles for Nox2-siRNA to the post-MI heart. In vitro, Nox2-siRNA particles are effectively taken up by macrophages and significantly knockdown Nox2 expression and activity. Following in vivo intramyocardial injection in experimental mice models of MI, Nox2-siRNA particles prevent upregulation of Nox2 and significantly recovered cardiac function. This study highlights the potential of polyketals as siRNA delivery vehicles to the MI heart and represents a viable therapeutic approach for targeting oxidative stress.
PMCID: PMC3766948  PMID: 23856052
nanoparticles; macrophages; myocardial infarction; gene expression; gene silencing
4.  Acute Preconditioning of Cardiac Progenitor Cells with Hydrogen Peroxide Enhances Angiogenic Pathways Following Ischemia-Reperfusion Injury 
Stem Cells and Development  2013;22(17):2414-2424.
There are a limited number of therapies available to prevent heart failure following myocardial infarction. One novel therapy that is currently being pursued is the implantation of cardiac progenitor cells (CPCs); however, their responses to oxidative stress during differentiation have yet to be elucidated. The objective of this study was to determine the effect of hydrogen peroxide (H2O2) treatment on CPC differentiation in vitro, as well as the effect of H2O2 preconditioning before implantation following ischemia-reperfusion (I/R) injury. CPCs were isolated and cloned from adult rat hearts, and then cultured in the absence or presence of H2O2 for 2 or 5 days. CPC survival was assessed with Annexin V, and cellular differentiation was evaluated through mRNA expression for cardiogenic genes. We found that 100 μM H2O2 decreased serum withdrawal-induced apoptosis by at least 45% following both 2 and 5 days of treatment. Moreover, 100 μM H2O2 treatment for 2 days significantly increased endothelial and smooth muscle markers compared to time-matched untreated CPCs. However, continued H2O2 treatment significantly decreased these markers. Left ventricular cardiac function was assessed 28 days after I/R and I/R with the implantation of Luciferase/GFP+ CPCs, which were preconditioned with 100 μM H2O2 for 2 days. Hearts implanted with Luciferase/GFP+ CPCs had significant improvement in both positive and negative dP/dT over I/R. Furthermore, cardiac fibrosis was significantly decreased in the preconditioned cells versus both I/R alone and I/R with control cells. We also observed a significant increase in endothelial cell density in the preconditioned CPC hearts compared to untreated CPC hearts, which also coincided with a higher density of Luciferase+ vessels. These findings suggest that preconditioning of CPCs with H2O2 for 2 days stimulates neoangiogenesis in the peri-infarct area following I/R injury and could be a viable therapeutic option to prevent heart failure.
PMCID: PMC3749704  PMID: 23544670
5.  Clinical and Metabolic Parameters in Non-Small Cell Lung Carcinoma and Colorectal Cancer Patients with and without KRAS Mutations 
Lung cancer (LC) and colorectal cancer (CRC) are the first and second deadliest types of cancer worldwide. EGFR-based therapy has been used in the treatment of these cancers with variable success. Presence of mutations in the KRAS driver oncogene, possibly induced by environmental factors such as carcinogens in diet and cigarette smoke, may confer worse prognosis and resistance to treatment for reasons not fully understood. Data on possible associations between KRAS mutational status and clinical and metabolic parameters, which may help in clinical management, as well as in identifying risk factors for developing these cancers, are limited in the current literature. We sequenced the KRAS gene and investigated the associations of variations in 108 patients with non-small cell lung carcinoma (NSCLC), the most common form of LC, and in 116 patients with CRC. All of the mutations originated from the guanosine nucleotide and over half of all transversions in NSCLC and CRC were c.34 G>T and c.35 G>T, respectively. c.35 G>A was the most frequent type of transition in both cancers. Excluding smoking, the clinical and metabolic parameters in patients carrying mutant and wild type KRAS were similar except that the CRC patients with transversion mutations were 8.6 years younger than those carrying the transitions (P < 0.01). Dyslipidemia, hypertension, family cancer history, and age of diagnosis older than 60 years were more frequent in NSCLC than CRC (P ≤ 0.04). These results suggest that most of the clinical and metabolic parameters investigated in this study are probably not associated with the more aggressive phenotype and differences in response to EGFR-based treatment previously reported in patients with KRAS mutations. However, the increased rates of abnormal metabolic parameters in patients with NSCLC in comparison to CRC indicate that these parameters may be more important in the management of NSCLC. CRC patients carrying transition mutations are older than those carrying transversions, suggesting that age may determine the type of KRAS mutation in CRC patients.
PMCID: PMC4198984  PMID: 25158139
KRAS; non-small cell lung carcinoma; colorectal cancer; transition; transversion
6.  Increased NQO1 but Not c-MET and Survivin Expression in Non-Small Cell Lung Carcinoma with KRAS Mutations 
Cigarette smoking is one of the most significant public health issues and the most common environmental cause of preventable cancer deaths worldwide. EGFR (Epidermal Growth Factor Receptor)-targeted therapy has been used in the treatment of LC (lung cancer), mainly caused by the carcinogens in cigarette smoke, with variable success. Presence of mutations in the KRAS (Kirsten rat sarcoma viral oncogene homolog) driver oncogene may confer worse prognosis and resistance to treatment for reasons not fully understood. NQO1 (NAD(P)H:quinone oxidoreductase), also known as DT-diaphorase, is a major regulator of oxidative stress and activator of mitomycins, compounds that have been targeted in over 600 pre-clinical trials for treatment of LC. We sequenced KRAS and investigated expression of NQO1 and five clinically relevant proteins (DNMT1, DNMT3a, ERK1/2, c-MET, and survivin) in 108 patients with non-small cell lung carcinoma (NSCLC). NQO1, ERK1/2, DNMT1, and DNMT3a but not c-MET and survivin expression was significantly more frequent in patients with KRAS mutations than those without, suggesting the following: (1) oxidative stress may play an important role in the pathogenesis, worse prognosis, and resistance to treatment reported in NSCLC patients with KRAS mutations, (2) selecting patients based on their KRAS mutational status for future clinical trials may increase success rate, and (3) since oxidation of nucleotides also specifically induces transversion mutations, the high rate of KRAS transversions in lung cancer patients may partly be due to the increased oxidative stress in addition to the known carcinogens in cigarette smoke.
PMCID: PMC4199031  PMID: 25222473
lung cancer; non-small cell lung carcinoma; oxidative stress; KRAS; mutation; NQO1; DNA methyl transferase; ERK1/2; c-MET; survivin
7.  Human hair shaft proteomic profiling: individual differences, site specificity and cuticle analysis 
PeerJ  2014;2:e506.
Hair from different individuals can be distinguished by physical properties. Although some data exist on other species, examination of the individual molecular differences within the human hair shaft has not been thoroughly investigated. Shotgun proteomic analysis revealed considerable variation in profile among samples from Caucasian, African–American, Kenyan and Korean subjects. Within these ethnic groups, prominent keratin proteins served to distinguish individual profiles. Differences between ethnic groups, less marked, relied to a large extent on levels of keratin associated proteins. In samples from Caucasian subjects, hair shafts from axillary, beard, pubic and scalp regions exhibited distinguishable profiles, with the last being most different from the others. Finally, the profile of isolated hair cuticle cells was distinguished from that of total hair shaft by levels of more than 20 proteins, the majority of which were prominent keratins. The cuticle also exhibited relatively high levels of epidermal transglutaminase (TGM3), accounting for its observed low degree of protein extraction by denaturants. In addition to providing insight into hair structure, present findings may lead to improvements in differentiating hair from various ethnic origins and offer an approach to extending use of hair in crime scene evidence for distinguishing among individuals.
PMCID: PMC4137660  PMID: 25165623
Ancestry; Body site; Corneocytes; Differential expression; Forensic evidence; Keratin associated proteins; Keratins; Proteomics; Transglutaminase
8.  Conditioning Increases the Gain of Contraction-Induced Sarcolemmal Substrate Transport in Ultra-Endurance Racing Sled Dogs 
PLoS ONE  2014;9(7):e103087.
Endurance exercise relies on transsarcolemmal flux of substrates in order to avoid depletion of intramuscular reserves. Previous studies of endurance trained sled dogs have shown a remarkable capacity of these dogs to adapt rapidly to endurance exercise by decreasing the utilization of intramuscular reserves. The current study tested the hypothesis that the dogs' glycogen-sparing phenotype is due to increased sarcolemmal transport of glucose and fatty acids. Basal and exercise-induced transport of glucose and fatty acids into sarcolemmal vesicles was evaluated in racing sled dogs prior to and after 7 months of exercise conditioning. Sarcolemmal substrate transport capacity was measured using sarcolemmal vesicles and radiolabelled substrates, and transporter abundance was measured using Western blot quantification in whole muscle homogenates and the sarcolemmal vesicle preparations. Conditioning resulted in increased basal and exercise-induced transport of both glucose and palmitate. Neither acute exercise nor conditioning resulted in changes in muscle content of GLUT4 or FAT/CD36, but conditioning did result in decreased abundance of both transporters in the sarcolemmal vesicles used for the basal transport assays, and this decrease was further amplified in the vesicles used for the exercise-induced transport assays. These results demonstrate conditioning-induced increases in sarcolemmal transport of oxidizable substrates, as well as increased gain of exercise-induced sarcolemmal transport of these substrates. These results further indicate that increased sarcolemmal transport of oxidizable substrates may be due to either an increased intrinsic capacity of the existing transporters or to a different population of transporters from those investigated.
PMCID: PMC4116179  PMID: 25075856
9.  Induction of pluripotency in bone marrow mononuclear cells via polyketal nanoparticle-mediated delivery of mature microRNAs 
Biomaterials  2013;34(17):4235-4241.
Since the successful generation of induced pluripotent stem cells (iPSC) from adult somatic cells using integrating-viral methods, various methods have been tried for iPSC generation using non-viral and non-integrating technique for clinical applications. Recently, various non-viral approaches such as protein, mRNA, microRNA, and small molecule transduction were developed to avoid genomic integration and generate stem cell-like cells from mouse and human fibroblasts. Despite these successes, there has been no successful generation of iPSC from bone marrow (BM)-derived hematopoietic cells derived using non-viral methods to date. Previous reports demonstrate the ability of polymeric micro and nanoparticles made from polyketals to deliver various molecules to macrophages. MicroRNA-loaded nanoparticles were created using the olyketal polymer PK3 (PK3-miR) and delivered to somatic cells for 6 days, resulting in the formation of colonies. Isolated cells from these colonies were assayed and substantial induction of the pluripotency markers Oct4, Sox2, and Nanog were detected. Moreover, colonies transferred to feeder layers also stained positive for pluripotency markers including SSEA-1. Here, we demonstrate successful activation of pluripotency-associated genes in mouse BM-mononuclear cells using embryonic stem cell (ESC)-specific microRNAs encapsulated in the acid sensitive polyketal PK3. These reprogramming results demonstrate that a polyketal-microRNA delivery vehicle can be used to generate various reprogrammed cells without permanent genetic manipulation in an efficient manner.
PMCID: PMC3740735  PMID: 23489923
MicroRNAs; Stem cell; Pluripotency; Nanoparticles; Polyketal
10.  Scavenger Receptor A Modulates the Immune Response to Pulmonary Cryptococcus neoformans Infection 
Scavenger receptors represent an important class of pattern recognition receptors shown to mediate both beneficial and detrimental roles in host defense against microbial pathogens. The role of the major macrophage scavenger receptor, scavenger receptor A (SRA), in the immune response against the pathogenic fungus, Cryptococcus neoformans, is unknown. To evaluate the role of SRA in anticryptococcal host defenses, SRA+/+ mice and SRA−/− mice were infected intratracheally with C. neoformans. Results show that infection of SRA−/− mice resulted in a reduction in the pulmonary fungal burden at the efferent phase (3 wk) compared with SRA+/+ mice. Improved fungal clearance in SRA−/− mice was associated with decreased accumulation of eosinophils and greater accumulation of CD4+ T cells and CD11b+ dendritic cells. Additional parameters were consistent with enhanced anti-cryptococcal immunity in the infected SRA−/− mice: 1) increased expression of the costimulatory molecules CD80 and CD86 by lung APCs, 2) decreased expression of Th2 cytokines (IL-4 and IL-13) and IL-10 in lung leukocytes and in cryptococcal Ag-pulsed splenocytes, 3) diminished IgE production in sera, and 4) increased hallmarks of classical pulmonary macrophage activation. These effects were preceded by increased expression of early pro-Th1 genes in pulmonary lymph nodes at the afferent phase (1 wk). Collectively, our data show that SRA can be exploited by C. neoformans to interfere with the early events of the afferent responses that support Th1 immune polarization. This results in amplification of Th2 arm of the immune response and subsequently impaired adaptive control of C. neoformans in the infected lungs.
PMCID: PMC4007509  PMID: 23733871
11.  Functionalized Dendrimer-Based Delivery of Angiotensin Type 1 Receptor siRNA for Preserving Cardiac Function Following Infarction 
Biomaterials  2013;34(14):3729-3736.
Cardiovascular disease (CVD) is the leading cause of death throughout the world and much pathology is associated with upregulation of inflammatory genes. Gene silencing using RNA interference is a powerful tool in regulating gene expression, but its application in CVDs has been prevented by the lack of efficient delivery systems. We report here the development of tadpole dendrimeric materials for siRNA delivery in a rat ischemia-reperfusion (IR) model. Angiotensin II (Ang II) type 1 receptor (AT1R), the major receptor that mediates most adverse effects of Ang II, was chosen to be the silencing targeting. Among the three tadpole dendrimers synthesized, the oligo-arginine conjugated dendrimer loaded with siRNA demonstrated effective down-regulation in AT1R expression in cardiomyocytes in vitro. When the dendrimeric material was applied in vivo, the siRNA delivery prevented the increase in AT1R levels and significantly improved cardiac function recovery compared to saline injection or empty dendrimer treated groups after IR injury. These experiments demonstrate a potential treatment for dysfunction caused by IR injury and may represent an alternative to AT1R blockade.
PMCID: PMC3594544  PMID: 23433774
Dendrimer; Cardiomyocyte; Gene Expression; Ischemia-Reperfusion
12.  Over-Expression of Catalase in Myeloid Cells Confers Acute Protection Following Myocardial Infarction 
Cardiovascular disease is the leading cause of death in the United States and new treatment options are greatly needed. Oxidative stress is increased following myocardial infarction and levels of antioxidants decrease, causing imbalance that leads to dysfunction. Therapy involving catalase, the endogenous scavenger of hydrogen peroxide (H2O2), has been met with mixed results. When over-expressed in cardiomyocytes from birth, catalase improves function following injury. When expressed in the same cells in an inducible manner, catalase showed a time-dependent response with no acute benefit, but a chronic benefit due to altered remodeling. In myeloid cells, catalase over-expression reduced angiogenesis during hindlimb ischemia and prevented monocyte migration. In the present study, due to the large inflammatory response following infarction, we examined myeloid-specific catalase over-expression on post-infarct healing. We found a significant increase in catalase levels following infarction that led to a decrease in H2O2 levels, leading to improved acute function. This increase in function could be attributed to reduced infarct size and improved angiogenesis. Despite these initial improvements, there was no improvement in chronic function, likely due to increased fibrosis. These data combined with what has been previously shown underscore the need for temporal, cell-specific catalase delivery as a potential therapeutic option.
PMCID: PMC4057773  PMID: 24853285
oxidative stress; myocardial infarction; antioxidant therapy
13.  A Novel AX+/BX− Paradigm to Assess Fear Learning and Safety-Signal Processing with Repeated-Measure Designs 
Journal of neuroscience methods  2013;214(2):177-183.
One of the core symptoms of anxiety disorders, such as Post-Traumatic Stress Disorder is the failure to overcome feelings of danger despite being in a safe environment. This inability likely stems from an inability to fully process safety signals, which are cues in the environment, that enable healthy individuals to over-ride fear in aversive situations. Studies examining safety signal learning in rodents, humans, and non-human primates currently rely on between-groups designs. Because repeated-measures designs reduce the number of subjects required, and facilitate a broader range of safety signal studies, the current project sought to develop a repeated-measures safety-signal learning paradigm in non-human primates. Twelve healthy rhesus macaques of both sexes received three rounds of auditory fear-potentiated startle training and testing using an AX+/BX− design with all visual cues. Cue AX was paired with an aversive blast of air, whereas the same X cue in compound with another B cue (BX) signaled the absence of an air blast. Hence, cue B served as a safety signal. Once animals consistently discriminated between the aversive (AX+) and safe (BX−) cues, measured by greater startle amplitude in the presence of AX vs. BX, they were tested for conditioned inhibition by eliciting startle in the presence of a novel ambiguous combined cue (AB). Similar to previous AX+/BX− studies, healthy animals rapidly learned to discriminate between the AX+ and BX− cues as well as demonstrate conditioned inhibition in the presence of the combined AB cue (i.e. lower startle amplitude in the presence of AB vs AX). Additionally, animals performed consistently across three rounds of testing using three new cues each time. The results validate this novel method that will serve as a useful tool for better understanding the mechanisms for the regulation of fear and anxiety.
PMCID: PMC3644366  PMID: 23376500
14.  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
15.  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
16.  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
17.  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
18.  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
19.  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
20.  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
21.  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
22.  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
24.  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
25.  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

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