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1.  Molecular bioluminescence imaging as a non-invasive tool for monitoring tumor growth and therapeutic response to MRI-guided laser ablation in a rat model of hepatocellular carcinoma 
Investigative radiology  2013;48(6):413-421.
To quantitatively compare tumor imaging by MRI and molecular bioluminescence imaging (BLI) and test the feasibility of monitoring the effect of MRI-guided laser ablation on tumor viability by 2D BLI and 3D DLIT in an orthotopic rat model of hepatocellular carcinoma (HCC).
Materials and Methods
This study was approved by the animal care committee. Rats underwent injection of N1S1 cells stably transfected with an empty vector (N=3) or a luciferase reporter (HSE-luc; N=4) into the liver. All rats underwent MR imaging to assess tumor establishment and volume and 2D BLI to assess tumor luminescence at day 7 with repeat MR imaging and 2D BLI and 3D diffuse luminescence tomography (3D DLIT) in select animals at day 14 and 21. MRI-guided laser ablation of the tumor was performed with pre and post-ablation 2D BLI and/or 3D DLIT (N=2). Tumors underwent histopathologic analysis to assess tumor viability.
MR imaging demonstrated hyperintense T2-weighted lesions at 3/3 and 4/4 sites in empty vector and HSE-luc rats, respectively. 2D BLI quantitation demonstrated 23.0 fold higher radiance in the HSE-luc group compared to the empty vector group at day 7 (p<0.01) and a significant correlation with tumor volume by MRI (r=0.86; p<0.03). Tumor dimensions by 3D DLIT and MRI demonstrated good agreement. 3D DLIT quantitation better agreed with the % of non-viable tumor by histopathology than 2D BLI quantitation following MRI-guided laser ablation.
Bioluminescence imaging is a feasible as non-invasive, quantitative tool for monitoring tumor growth and therapeutic response to thermal ablation in a rat model of HCC.
PMCID: PMC3904185  PMID: 23262791
Magnetic Resonance Imaging; Laser Ablation; Bioluminescence Imaging; Hepatocellular Carcinoma; Animal Model
2.  Chronic Stress Induces a Selective Decrease in AMPA Receptor-Mediated Synaptic Excitation at Hippocampal Temporoammonic-CA1 Synapses 
The Journal of Neuroscience  2013;33(40):15669-15674.
Chronic stress promotes depression, but how it disrupts cognition and mood remains unknown. Chronic stress causes atrophy of pyramidal cell dendrites in the hippocampus and cortex in human and animal models, and a depressive-like behavioral state. We now test the hypothesis that excitatory temporoammonic (TA) synapses in the distal dendrites of CA1 pyramidal cells in rats are altered by chronic unpredictable stress (CUS) and restored by chronic antidepressant treatment, in conjunction with the behavioral consequences of CUS. We observed a decrease in AMPAR-mediated excitation at TA-CA1 synapses, but not Schaffer collateral-CA1 synapses, after CUS, with a corresponding layer-specific decrease in GluA1 expression. Both changes were reversed by chronic fluoxetine. CUS also disrupted long-term memory consolidation in the Morris water maze, a function of TA-CA1 synapses. The decreases in TA-CA1 AMPAR-mediated excitation and performance in the consolidation test were correlated positively with decreases in sucrose preference, a measure of anhedonia. We conclude that chronic stress selectively decreases AMPAR number and function at specific synapses and suggest that this underlies various depressive endophenotypes. Our findings provide evidence that glutamatergic dysfunction is an underlying cause of depression and that current first-line antidepressant drugs act by restoring excitatory synaptic strength. Our findings suggest novel therapeutic targets for this debilitating disease.
PMCID: PMC3787493  PMID: 24089474
3.  AS30D Model of Hepatocellular Carcinoma: Tumorigenicity and Preliminary Characterization by Imaging, Histopathology, and Immunohistochemistry 
This study was designed to determine the tumorigenicity of the AS30D HCC cell line following orthotopic injection into rat liver and preliminarily characterize the tumor model by both magnetic resonance imaging (MRI) and ultrasound (US) as well as histopathology and immunohistochemistry.
AS30D cell line in vitro proliferation was assessed by using MTT assay. Female rats (N = 5) underwent injection of the AS30D cell line into one site in the liver. Rats subsequently underwent MR imaging at days 7 and 14 to assess tumor establishment and volume. One rat underwent US of the liver at day 7. Rats were euthanized at day 7 or 14 and livers were subjected to gross, histopathologic (H&E), and immunohistochemical (CD31) analysis to assess for tumor growth and neovascularization.
AS30D cell line demonstrated an in vitro doubling time of 33.2 ± 5.3 h. MR imaging demonstrated hyperintense T2-weighted and hypointense T1-weighted lesions with tumor induction in five of five and three of three sites at days 7 and 14, respectively. The mean (SD) tumor volume was 126.1 ± 36.2 mm3 at day 7 (N = 5). US of the liver demonstrated a well-circumscribed, hypoechoic mass and comparison of tumor dimensions agreed well with MRI. Analysis of H&E- and CD31-stained sections demonstrated moderate-high grade epithelial tumors with minimal tumor necrosis and evidence of diffuse intratumoral and peritumoral neovascularization by day 7.
AS30D HCC cell line is tumorigenic following orthotopic injection into rat liver and can be used to generate an early vascularizing, slower-growing rat HCC tumor model.
PMCID: PMC3904804  PMID: 22923329
Hepatocellular carcinoma; Rat model; Magnetic resonance imaging
4.  Development and Preliminary Testing of a Translational Model of Hepatocellular Carcinoma for Magnetic Resonance Imaging and Interventional Oncologic Investigations 
To develop a translational rat hepatocellular carcinoma (HCC) disease model for magnetic resonance imaging and image-guided interventional oncologic investigations.
Methods and Materials
Male rats underwent sham control surgery (N=6), selective bile duct ligation (SBDL; N=4) or common bile duct ligation (CBDL; N=6) with procedure optimization in 4 rats and N1S1 cell injection into 2–3 sites in the liver of 12 rats. All rats subsequently underwent MRI to assess tumor establishment and volume. Mesenteric angiography and percutaneous MR-guided laser ablation of the liver were performed in a subgroup of animals (N=4). Animal weight and liver tests were monitored. After harvesting, the livers were subjected to gross and microscopic analysis. Tumor volume and laboratory parameters were assessed between ligation groups.
MRI demonstrated hyperintense T2 and hypointense T1 lesions with tumor induction in 5/10 (50.0%), 7/8 (87.5%) and 12/12 (100%) sites in the control, SBDL and CBDL groups, respectively. Tumor volumes differed significantly by group (p<0.02). Mesenteric angiography demonstrated an enhancing tumor stain. Clinical and laboratory assessment revealed a significant decrease in weight (p = 0.01) and albumin (p<0.01) and increase in total bilirubin (p = 0.02) in CBDL rats but not SBDL rats (p=1.0). Histologic examination showed high-grade HCCs with local and vascular invasion within the context of early fibrosis in CBDL and SBDL rats. MR-guided laser ablation generated a 1–2 cm ablation zone with histology consistent with reversible and irreversible injury.
A biologically relevant rat hepatocellular carcinoma disease model was developed for MR imaging and preliminary interventional oncologic applications.
PMCID: PMC3904802  PMID: 22265247
5.  Development of Drugs for Epstein - Barr virus using High-Throughput in silico Virtual Screening 
Expert opinion on drug discovery  2010;5(12):10.1517/17460441.2010.524640.
Importance of the field
Epstein-Barr virus (EBV) is a ubiquitious human herpesvirus that is causally associated with endemic forms of Burkitt’s lymphoma (BL), nasopharyngeal carcinoma, and lymphoproliferative disease in immunosuppressed individuals. On a global scale, EBV infects over 90% of the adult population and is responsible for ~1% of all human cancers. To date, there is no efficacious drug or therapy for the treatment of EBV infection and EBV-related diseases.
Areas covered in this review
In this review, we discuss the existing anti-EBV inhibitors and those under development. We discuss the value of different molecular targets, including EBV lytic DNA replication enzymes, as well as proteins that are expressed exclusively during latent infection, like EBNA1 and LMP1. Since the atomic structure of the EBNA1 DNA binding domain has been described, it is an attractive target for in silico methods of drug design and small molecule screening. We discuss the use of computational methods that can greatly facilitate the development of novel inhibitors and how in silico screening methods can be applied to target proteins with known structures, like EBNA1, to treat EBV infection and disease.
What the reader will gain
The reader will be familiarized with the problems in targeting of EBV for inhibition by small molecules and how computational methods can greatly facilitate this process.
Take home message
Despite the impressive efficacy of nucleoside analogues for the treatment of herpesvirus lytic infection, there remain few effective treatments for latent infections. Since EBV-latent infection persists within and contributes to the formation of EBV-associated cancers, targeting EBV latent proteins is an unmet medical need. High throughput in silico screening can accelerate the process of drug discovery for novel and selective agents that inhibit EBV latent infection and associated disease.
PMCID: PMC3816986  PMID: 22822721
Epstein-Barr virus (EBV); DNA polymerase; LMP1; EBNA1; computational screening
6.  Local potentiation of excitatory synapses by serotonin and its alteration in rodent models of depression 
Nature neuroscience  2013;16(4):464-472.
The causes of major depression remain unknown. Antidepressants elevate monoamine concentrations, particularly serotonin, but it remains uncertain which downstream events are critical to their therapeutic effects. We report that endogenous serotonin selectively potentiated excitatory synapses formed by the temporoammonic (TA) pathway with CA1 pyramidal cells via activation of 5-HT1BRs, without affecting nearby Schaffer collateral synapses. This potentiation was expressed postsynaptically by AMPA-type glutamate receptors and required calmodulin-dependent protein kinase-mediated phosphorylation of GluA1 subunits. Because they share common expression mechanisms, long-term potentiation and serotonin-induced potentiation occluded each other. Long-term consolidation of spatial learning, a function of TA-CA1 synapses, was enhanced by 5-HT1BR antagonists. Serotonin-induced potentiation was quantitatively and qualitatively altered in a rat model of depression, restored by chronic antidepressants, and required for the ability of chronic antidepressants to reverse stress-induced anhedonia. Changes in serotonin-mediated potentiation, and its recovery by antidepressants, implicate excitatory synapses as a locus of plasticity in depression.
PMCID: PMC3609911  PMID: 23502536
8.  Porcine Ex Vivo Liver Phantom for Dynamic Contrast-Enhanced Computed Tomography: Development and Initial Results 
Investigative radiology  2011;46(9):586-593.
To demonstrate the feasibility of developing a fixed, dual-input, biological liver phantom for dynamic contrast-enhanced computed tomography (CT) imaging and to report initial results of use of the phantom for quantitative CT perfusion imaging.
Materials and Methods
Porcine livers were obtained from completed surgical studies and perfused with saline and fixative. The phantom was placed in a body-shaped, CT-compatible acrylic container and connected to a perfusion circuit fitted with a contrast injection port. Flow-controlled contrast-enhanced imaging experiments were performed using a 128-slice and 64 slice, dual-source multidetector CT scanners. CT angiography protocols were employed to obtain portal venous and hepatic arterial vascular enhancement, reproduced over a period of four to six months. CT perfusion protocols were employed at different input flow rates to correlate input flow with calculated tissue perfusion, to test reproducibility and demonstrate the feasibility of simultaneous dual input liver perfusion. Histologic analysis of the liver phantom was also performed.
CT angiogram 3D reconstructions demonstrated homogenous tertiary and quaternary branching of the portal venous system out to the periphery of all lobes of the liver as well as enhancement of the hepatic arterial system to all lobes of the liver and gallbladder throughout the study period. For perfusion CT, the correlation between the calculated mean tissue perfusion in a volume of interest and input pump flow rate was excellent (R2 = 0.996) and color blood flow maps demonstrated variations in regional perfusion in a narrow range. Repeat perfusion CT experiments demonstrated reproducible time-attenuation curves and dual-input perfusion CT experiments demonstrated that simultaneous dual input liver perfusion is feasible. Histologic analysis demonstrated that the hepatic microvasculature and architecture appeared intact and well preserved at the completion of four to six months of laboratory experiments and contrast enhanced imaging.
We have demonstrated successful development of a porcine liver phantom using a flow-controlled extracorporeal perfusion circuit. This phantom exhibited reproducible dynamic contrast-enhanced CT of the hepatic arterial and portal venous system over a four to six month period.
PMCID: PMC3164269  PMID: 21610506
Perfusion Imaging; Computed Tomography; Biological Phantom; Porcine
9.  Homeostatic increase in excitability in area CA1 after Schaffer collateral transection in vivo 
Epilepsia  2011;52(9):1656-1665.
Epilepsy is a significant long-term consequence of traumatic brain injury (TBI) and is likely to result from multiple mechanisms. One feature that is common to many forms of TBI is denervation. We asked whether chronic partial denervation in vivo would lead to a homeostatic increase in the excitability of a denervated cell population.
To answer this question, we took advantage of the unique anatomy of the hippocampus where the input to the CA1 neurons, the Schaffer collaterals, could be transected in vivo with preservation of their outputs and only minor cell death.
Key findings
We observed a delayed increase in neuronal excitability, as apparent in extracellular recordings from hippocampal brain slices prepared 14 days (but not 3 days) postlesion. Although population spikes in slices from control and lesioned animals were comparable under resting conditions, application of solutions that were mildly proconvulsive (high K+, low Mg2+, low concentrations of bicuculline) produced increases in the number of population spikes in slices from lesioned rats, but not in slices from unlesioned sham controls. Denervation did not produce changes in several markers of GABAergic synaptic inhibition, including the number of GABAergic neurons, α1 GABAA receptor subunits, the vesicular GABA transporter, or miniature inhibitory postsynaptic currents.
We conclude that chronic partial denervation does lead to a delayed homeostatic increase in neuronal excitability, and may therefore contribute to the long-term neurological consequences of traumatic brain injury.
PMCID: PMC3169701  PMID: 21635239
traumatic brain injury; axonal injury; hippocampus; lesion; plasticity
10.  Dendritic Hold and Read: A Gated Mechanism for Short Term Information Storage and Retrieval 
PLoS ONE  2012;7(5):e37542.
Two contrasting theories have been proposed to explain the mechanistic basis of short term memory. One theory posits that short term memory is represented by persistent neural activity supported by reverberating feedback networks. An alternate, more recent theory posits that short term memory can be supported by feedforward networks. While feedback driven memory can be implemented by well described mechanisms of synaptic plasticity, little is known of possible molecular and cellular mechanisms that can implement feedforward driven memory. Here we report such a mechanism in which the memory trace exists in the form of glutamate-bound but Mg2+-blocked NMDA receptors on the thin terminal dendrites of CA1 pyramidal neurons. Because glutamate dissociates from subsets of NMDA receptors very slowly, excitatory synaptic transmission can leave a silent residual trace that outlasts the electrical activity by hundreds of milliseconds. Read-out of the memory trace is possible if a critical level of these bound-but-blocked receptors accumulates on a dendritic branch that will allow these quasi-stable receptors to sustain a regenerative depolarization when triggered by an independent gating signal. This process is referred to here as dendritic hold and read (DHR). Because the read-out of the input is not dependent on repetition of the input and information flows in a single-pass manner, DHR can potentially support a feedforward memory architecture.
PMCID: PMC3358290  PMID: 22629416
11.  Development of a High-Throughput Screen for Inhibitors of Epstein-Barr Virus EBNA1 
Journal of Biomolecular Screening  2010;15(9):1107-1115.
Latent infection with Epstein-Barr Virus (EBV) is a carcinogenic cofactor in several lymphoid and epithelial cell malignancies. At present, there are no small molecule inhibitors that specifically target EBV latent infection or latency-associated oncoproteins. EBNA1 is an EBV-encoded sequence-specific DNA-binding protein that is consistently expressed in EBV-associated tumors and required for stable maintenance of the viral genome in proliferating cells. EBNA1 is also thought to provide cell survival function in latently infected cells. In this work we describe the development of a biochemical high-throughput screening (HTS) method using a homogenous fluorescence polarization (FP) assay monitoring EBNA1 binding to its cognate DNA binding site. An FP-based counterscreen was developed using another EBV-encoded DNA binding protein, Zta, and its cognate DNA binding site. We demonstrate that EBNA1 binding to a fluorescent labeled DNA probe provides a robust assay with a Z-factor consistently greater than 0.6. A pilot screen of a small molecule library of ~14,000 compounds identified 3 structurally related molecules that selectively inhibit EBNA1, but not Zta. All three compounds had activity in a cell-based assay specific for the disruption of EBNA1 transcription repression function. One of the compounds was effective in reducing EBV genome copy number in Raji Burkitt lymphoma cells. These experiments provide a proof-of-concept that small molecule inhibitors of EBNA1 can be identified by biochemical high-throughput screening of compound libraries. Further screening in conjunction with medicinal chemistry optimization may provide a selective inhibitor of EBNA1 and EBV latent infection.
PMCID: PMC3310380  PMID: 20930215
13.  Mood disorder susceptibility gene CACNA1C modifies mood-related behaviors in mice and interacts with sex to influence behavior in mice and diagnosis in humans 
Biological psychiatry  2010;68(9):801-810.
Recent genome-wide association studies have associated polymorphisms in the gene CACNA1C, which codes for Cav1.2, with a bipolar disorder and depression diagnosis.
The behaviors of wild type and Cacna1c heterozygous mice of both sexes were evaluated in a number of tests. Based upon sex differences in our mouse data, we assessed a gene x sex interaction for diagnosis of mood disorders in human subjects. Data from the NIMH-BP Consortium and the GenRED Consortium were examined utilizing a combined dataset that included 2,021 mood disorder cases (1,223 females) and 1,840 controls (837 females).
In both male and female mice, Cacna1c haploinsufficiency is associated with lower exploratory behavior, decreased response to amphetamine, and antidepressant-like behavior in the forced swim and tail suspension tests. Female, but not male, heterozygous mice displayed decreased risk-taking behavior or increased anxiety in multiple tests, greater attenuation of amphetamine-induced hyperlocomotion, decreased development of learned helplessness, and a decreased acoustic startle response indicating a sex-specific role of Cacna1c. In humans, sex-specific genetic association was seen for two intronic single nucleotide polymorphisms (SNPs), rs2370419 and rs2470411, in CACNA1C, with effects in females (OR=1.64, 1.32), but not in males (OR=0.82, 0.86). The interactions by sex were significant after correction for testing 190 SNPs (P=1.4 x 10−4, 2.1 x 10−4; Pcorrected=0.03, 0.04), and were consistent across two large data sets.
Our preclinical results support a role for CACNA1C in mood disorder pathophysiology, and the combination of human genetic and preclinical data support an interaction between sex and genotype.
PMCID: PMC2955812  PMID: 20723887
CACNA1C; bipolar disorder; major depression; Cav1.2; animal model; gender; sex differences
14.  Duty Hour Recommendations and Implications for Meeting the ACGME Core Competencies: Views of Residency Directors 
Mayo Clinic Proceedings  2011;86(3):185-191.
OBJECTIVE: To describe the views of residency program directors regarding the effect of the 2010 duty hour recommendations on the 6 core competencies of graduate medical education.
METHODS: US residency program directors in internal medicine, pediatrics, and general surgery were e-mailed a survey from July 8 through July 20, 2010, after the 2010 Accreditation Council for Graduate Medical Education (ACGME) duty hour recommendations were published. Directors were asked to rate the implications of the new recommendations for the 6 ACGME core competencies as well as for continuity of inpatient care and resident fatigue.
RESULTS: Of 719 eligible program directors, 464 (65%) responded. Most program directors believe that the new ACGME recommendations will decrease residents' continuity with hospitalized patients (404/464 [87%]) and will not change (303/464 [65%]) or will increase (26/464 [6%]) resident fatigue. Additionally, most program directors (249-363/464 [53%-78%]) believe that the new duty hour restrictions will decrease residents' ability to develop competency in 5 of the 6 core areas. Surgery directors were more likely than internal medicine directors to believe that the ACGME recommendations will decrease residents' competency in patient care (odds ratio [OR], 3.9; 95% confidence interval [CI], 2.5-6.3), medical knowledge (OR, 1.9; 95% CI, 1.2-3.2), practice-based learning and improvement (OR, 2.7; 95% CI, 1.7-4.4), interpersonal and communication skills (OR, 1.9; 95% CI, 1.2-3.0), and professionalism (OR, 2.5; 95% CI, 1.5-4.0).
CONCLUSION: Residency program directors' reactions to ACGME duty hour recommendations demonstrate a marked degree of concern about educating a competent generation of future physicians in the face of increasing duty hour standards and regulation.
The reactions of residency program directors to the ACGME duty hour recommendations demonstrate a marked degree of concern about educating a competent generation of future physicians in the face of increasing duty hour standards and regulation.
PMCID: PMC3046937  PMID: 21307391
15.  The Actin Binding Domain of βI-Spectrin Regulates the Morphological and Functional Dynamics of Dendritic Spines 
PLoS ONE  2011;6(1):e16197.
Actin microfilaments regulate the size, shape and mobility of dendritic spines and are in turn regulated by actin binding proteins and small GTPases. The βI isoform of spectrin, a protein that links the actin cytoskeleton to membrane proteins, is present in spines. To understand its function, we expressed its actin-binding domain (ABD) in CA1 pyramidal neurons in hippocampal slice cultures. The ABD of βI-spectrin bundled actin in principal dendrites and was concentrated in dendritic spines, where it significantly increased the size of the spine head. These effects were not observed after expression of homologous ABDs of utrophin, dystrophin, and α-actinin. Treatment of slice cultures with latrunculin-B significantly decreased spine head size and decreased actin-GFP fluorescence in cells expressing the ABD of α-actinin, but not the ABD of βI-spectrin, suggesting that its presence inhibits actin depolymerization. We also observed an increase in the area of GFP-tagged PSD-95 in the spine head and an increase in the amplitude of mEPSCs at spines expressing the ABD of βI-spectrin. The effects of the βI-spectrin ABD on spine size and mEPSC amplitude were mimicked by expressing wild-type Rac3, a small GTPase that co-immunoprecipitates specifically with βI-spectrin in extracts of cultured cortical neurons. Spine size was normal in cells co-expressing a dominant negative Rac3 construct with the βI-spectrin ABD. We suggest that βI-spectrin is a synaptic protein that can modulate both the morphological and functional dynamics of dendritic spines, perhaps via interaction with actin and Rac3.
PMCID: PMC3031527  PMID: 21297961
16.  Dynamic Regulation of the Kv2.1 Voltage-gated Potassium Channel during Brain Ischemia through Neuroglial Interaction 
The physiological significance of neuroglial interactions in the central nervous system has been emphasized in neurological conditions such as epilepsy and brain ischemia. The Kv2.1 voltage-gated potassium channel is unique in its ability to form large clusters in the plasma membrane of neuronal cell bodies. We have previously shown brain ischemia causes rapid dephosphorylation of Kv2.1 subunits and resultant activation of the ion channel function. However, the physiological significance of the channel clustering is unknown. Here we present evidence that clustered Kv2.1 channels in the neuronal plasma membrane are juxtaposed to axosomatic synapses and associated with astrocytic processes expressing high levels of glutamate transporters. In acute cortical slices, ischemic stress rapidly resulted in the dephosphorylation and dispersion of Kv2.1. Selective inhibition of metabolism in astrocytes was sufficient to induce Kv2.1 dephosphorylaion in neurons. Interestingly, these effects were blocked by the antagonists of ionotropic glutamate receptors, indicating the involvement of glutamate as the signal mediator between astrocytes and neurons. Furthermore, the pharmacological inhibition of glial glutamate transporter GLT-1 induced the similar Kv2.1 dephosphorylation, whereas exogeneous glutamate alone was not efficacious. These results suggest that ischemic stress rapidly causes the dysfunction of glutamate transporters in astrocytes and resultant accumulation of glutamate in the extracellular space. The elevated glutamate may subsequently activate ionotropic glutamate receptors and result in the dephosphorylation of Kv2.1 in neurons. These findings implicate that Kv2.1 clusters are strategically situated at neuroglial junctions to achieve the rapid modulation upon ischemic stress via glutamate signaling.
PMCID: PMC2951025  PMID: 18716211
Hypoxia; Astrocyte; Cerebral Cortex; Phosphorylation; Glutamate Transporter; Neuroprotection
17.  Discovery of Selective Inhibitors Against EBNA1 via High Throughput In Silico Virtual Screening 
PLoS ONE  2010;5(4):e10126.
Epstein-Barr Virus (EBV) latent infection is associated with several human malignancies and is a causal agent of lymphoproliferative diseases during immunosuppression. While inhibitors of herpesvirus DNA polymerases, like gancyclovir, reduce EBV lytic cycle infection, these treatments have limited efficacy for treating latent infection. EBNA1 is an EBV-encoded DNA-binding protein required for viral genome maintenance during latent infection.
Here, we report the identification of a new class of small molecules that inhibit EBNA1 DNA binding activity. These compounds were identified by virtual screening of 90,000 low molecular mass compounds using computational docking programs with the solved crystal structure of EBNA1. Four structurally related compounds were found to inhibit EBNA1-DNA binding in biochemical assays with purified EBNA1 protein. Compounds had a range of 20–100 µM inhibition of EBNA1 in fluorescence polarization assays and were further validated for inhibition using electrophoresis mobility shift assays. These compounds exhibited no significant inhibition of an unrelated DNA binding protein. Three of these compounds inhibited EBNA1 transcription activation function in cell-based assays and reduced EBV genome copy number when incubated with a Burkitt lymphoma cell line.
These experiments provide a proof-of-principle that virtual screening can be used to identify specific inhibitors of EBNA1 that may have potential for treatment of EBV latent infection.
PMCID: PMC2853575  PMID: 20405039
18.  Development of a small molecule serum and glucocorticoid-regulated kinase 1 antagonist and its evaluation as a prostate cancer therapeutic 
Cancer research  2008;68(18):7475-7483.
Androgens, through their actions on the androgen receptor (AR), are required for the development of the prostate and contribute to the pathological growth dysregulation observed in prostate cancers. Consequently, androgen ablation has become an essential component of the pharmacotherapy of prostate cancer. In this study, we explored the utility of targeting processes downstream of AR as an alternate approach for therapy. Specifically, we demonstrate that the serum and glucocorticoid-regulated kinase 1 (sgk1) gene is an androgen-regulated target gene in cellular models of prostate cancer. Furthermore, functional SGK1 protein, as determined by the phosphorylation of its target Nedd4-2, was also increased with androgen treatment. Importantly, we determined that RNAi-mediated knockdown of SGK1 expression attenuates androgen-mediated growth of the prostate cancer cell line, LNCaP. Given these findings, we explored the utility of SGK1 as a therapeutic target in prostate cancer by developing and evaluating a small molecule inhibitor of this enzyme. From these studies emerged GSK650394, a competitive inhibitor that quantitatively blocks the effect of androgens on LNCaP cell growth. Thus, in addition to androgen ablation, inhibition of pathways downstream of AR are likely to have therapeutic utility in prostate cancer.
PMCID: PMC2562281  PMID: 18794135
serum and glucocorticoid-regulated kinase 1; androgen receptor; prostate cancer
19.  Maladaptive homeostatic plasticity in a rodent model of central pain syndrome: Thalamic hyperexcitability after spinothalamic tract lesions 
Central pain syndrome (CPS) is defined as pain associated with a lesion of the central nervous system and is a common consequence of spinal cord injuries. We generated a rodent model of CPS by making unilateral electrolytic or demyelinating lesions centered on the spinothalamic tract in rats. Thermal hyperalgesia and mechanical allodynia occurred in both hind paws and forepaws by seven days postlesion and were maintained >31 days. Field potentials in the ventral posterior lateral nucleus (VPL) in thalamic brain slices from lesioned animals displayed an increased probability of burst responses. Ethosuximide, a T-type calcium channel blocker, eliminated busting in lesioned thalamic slices and attenuated lesion-induced hyperalgesia and allodynia. We conclude that CPS results from an increase in the excitability of thalamic nuclei that have lost normal ascending inputs as the result of a spinal cord injury and suggest that ethosuximide will relieve CPS by restoring normal thalamic excitability.
PMCID: PMC2627563  PMID: 19005061
Thalamus; excitability; denervation; deafferentation; spinothalamic; calcium channels
Brain research  2006;1119(1):26-39.
We have previously shown that the HSV-2 anti-apoptotic protein ICP10PK is delivered by the replication incompetent virus mutant ΔRR and prevents kainic acid (KA)-induced epileptiform seizures and neuronal cell loss in the mouse and rat models of temporal lobe epilepsy. The present studies used ΔRR and the ICP10PK deleted virus mutant ΔPK, to examine the mechanism of neuroprotection. ΔRR-infected neuronal cells expressed a chimeric protein in which ICP10PK was fused in frame to LacZ (p175) while retaining ICP10PK kinase activity. ΔPK-infected neuronal cells expressed a mutant ICP10 protein that is deleted in PK domain and is kinase negative (p95). p175 and p95 were expressed in CA3 (86±3%) and CA1 (69±7%) cells from ΔRR or ΔPK-infected organotypic hippocampal cultures (OHC) and 80–85% of the ICP10 positive cells co-stained with antibody to βIII Tubulin (neuronal marker). ΔRR, but not ΔPK, inhibited KA-induced cell death and caspase-3 activation in CA3 neurons and inhibition was seen whether ΔRR was delivered 2 days before, or 2 days after KA administration (95 % neuroprotection). Neuroprotection was associated with ERK and Akt activation and was abrogated by simultaneous treatment with the MEK (U0126) and PI3-K (LY294002) inhibitors. Increased expression of the anti-apoptotic protein Bag-1 and the transcription factor CREB, and decreased expression of the pro-apoptotic protein Bad were associated with ΔRR-mediated neuroprotection and the surviving neurons retained normal synaptic function. The data indicate that ΔRR is a promising platform for neuroprotection from excitotoxic injury.
PMCID: PMC2648139  PMID: 17020750
ICP10PK; kainic acid; organotypic hippocampal cultures; apoptosis
21.  Estradiol induces dendritic spines by enhancing glutamate release independent of transcription: A mechanism for organizational sex differences 
Neuron  2008;58(4):584-598.
The naturally occurring sex difference in dendritic spine number on hypothalamic neurons offers a unique opportunity to investigate mechanisms establishing synaptic patterning during perinatal sensitive periods. A major advantage of the model is the ability to treat neonatal females with estradiol to permanently induce the male phenotype. During the development of other systems, exuberant innervation is followed by activity-dependent pruning necessary for elimination of spurious synapses. In contrast, we demonstrate that estradiol-induced organization in the hypothalamus involves the induction of new synapses on dendritic spines. Activation of estrogen receptors by estradiol triggers a non-genomic activation of PI3 kinase that results in enhanced glutamate release from presynaptic neurons. Subsequent activation of ionotropic glutamate receptors activates MAP kinases inducing dendritic spine formation. These results reveal a trans-neuronal mechanism by which estradiol acts during a sensitive period to establish a profound and lasting sex difference in hypothalamic synaptic patterning.
PMCID: PMC2570057  PMID: 18498739
The Journal of emergency medicine  2007;33(3):233-239.
Syncope is a common presentation to the Emergency Department (ED); however, appropriate management and indications for hospitalization remain an ongoing challenge. The objective of this study was to determine if a predefined decision rule could accurately identify patients with syncope likely to have an adverse outcome or critical intervention. A prospective, observational, cohort study was conducted of consecutive ED patients aged 18 years or older presenting with syncope. A clinical decision rule was developed a priori to identify patients at risk if they met any of the following 8 criteria: 1) Signs and symptoms of acute coronary syndrome; 2) Signs of conduction disease; 3) Worrisome cardiac history; 4) Valvular heart disease by history or physical examination; 5) Family history of sudden death; 6) Persistent abnormal vital signs in the ED; 7) Volume depletion; 8) Primary central nervous system event. The primary outcome was either a critical intervention or an adverse outcome within 30 days. Among 362 patients enrolled with syncope, 293 (81%) patients completed their 30-day follow-up. Of these, 201 (69%) were admitted. There were 68 patients (23%) who had either a critical intervention or adverse outcome. The rule identified 66/68 patients who met the outcome for a sensitivity of 97% (95% confidence interval 93–100%) and specificity of 62% (56 – 69%). This pathway may be useful in identifying patients with syncope who are likely to have adverse outcome or critical interventions. Implementation and multicenter validation is needed before widespread application.
PMCID: PMC2276584  PMID: 17976548
syncope; outcomes; decision; rule
23.  Glutamate-mediated excitotoxicity in neonatal hippocampal neurons is mediated by mGluR-induced release of Ca++ from intracellular stores and is prevented by estradiol 
The European journal of neuroscience  2006;24(11):3008-3016.
Hypoxic/ischemic (HI) brain injury in newborn full-term and premature infants is a common and pervasive source of life time disabilities in cognitive and locomotor function. In the adult, HI induces glutamate release and excitotoxic cell death dependent on NMDA receptor activation. In animal models of the premature human infant, glutamate is also released following HI, but neurons are largely insensitive to NMDA or AMPA/kainic acid (KA) receptor-mediated damage. Using primary cultured hippocampal neurons we have determined that glutamate increases intracellular calcium much more than kainic acid. Moreover, glutamate induces cell death by activating Type I metabotropic glutamate receptors (mGluRs). Pretreatment of neurons with the gonadal steroid estradiol reduces the level of the Type I metabotropic glutamate receptors and completely prevents cell death, suggesting a novel therapeutic approach to excitotoxic brain damage in the neonate.
PMCID: PMC2362502  PMID: 17156362
excitatory amino acids; imaging; metabotropic receptors; neuroprotection; perinatal brain injury; steroids
24.  Morphologic Blooming in Breast MRI as a Characterization of Margin for Discriminating Benign from Malignant Lesions 
Academic radiology  2006;13(11):1344-1354.
Develop a fully automated, objective method for evaluating morphology on breast MR and evaluate effectiveness of the new morphological method for detecting breast cancers.
We present a new automated method (Morphological Blooming) for identifying and classifying breast lesions on MR which measures margin sharpness, a characteristic related to blooming, defined as rapid enhancement, with a border that is initially sharp but becomes unsharp after seven minutes. Independent training sets (98 biopsy-proven lesions) and testing sets (179 breasts, 127 patients, acquired at 5 institutions) were used. Morphological Blooming was evaluated as a stand-alone feature and as an adjunct to kinetics using FROC (free-response ROC) and sensitivity analysis. Dependence of false positive (FP) rates on acquisition times and pathologies of contralateral breasts were evaluated.
Sensitivity of Morphological Blooming was 80% with 2.46 false positives (FP) per non-cancerous breast: FPs did not vary significantly by acquisition times. FPs varied significantly by pathologies of contralateral breasts (cancerous contralateral: 4.29 FP/breast; non-cancerous contralateral: 0.48 FP/breast; p<.0001). Evaluation of 45 cancers showed suspicious morphologies on 10/15 (67%) cancers with benign-like kinetics and suspicious kinetics on 5/10 (50%) cancers with benign-like morphologies.
We present a new, fully automated method of identifying and classifying margin sharpness of breast lesions on MR that can be used to direct radiologists’ attention to lesions with suspicious morphologies. Morphological Blooming may have important utility for assisting radiologists in identifying cancers with benign-like kinetics and discriminating normal tissues that exhibit cancer-like enhancement curves and for improving the performance of CAD systems.
PMCID: PMC1899409  PMID: 17070452
25.  Intranasal administration of the growth compromised HSV-2 vector ΔRR prevents kainate induced seizures and neuronal loss in rats and mice 
Identification of targets and delivery platforms for gene therapy of neurodegenerative disorders is a clinical challenge. We describe a novel paradigm in which the neuroprotective gene is the herpes simplex virus type 2 (HSV-2) anti-apoptotic gene ICP10PK and the vector is the growth compromised HSV-2 mutant ΔRR. ΔRR is delivered intranasally. It is not toxic in rats and mice. ICP10PK is expressed in the hippocampus of the ΔRR treated animals for at least 42 days in the absence of virus replication and late virus gene expression. Its expression is regulated by an AP-1 amplification loop. Intranasally delivered ΔRR prevents kainic acid (KA) induced seizures, neuronal loss and inflammation, in both rats and mice. The data suggest that ΔRR is a promising therapeutic platform for neurodegenerative diseases.
PMCID: PMC1513123  PMID: 16500153
HSV; ICP10PK; neuroprotection; gene therapy; kainic acid; seizures; glia

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