Validation of a Preclinical Drug Screening Platform for Pharmacoresistant Epilepsy

The successful identification of promising investigational therapies for the treatment of epilepsy can be credited to the use of numerous animal models of seizure and epilepsy for over 80 years. In this time, the maximal electroshock test in mice and rats, the subcutaneous pentylenetetrazol test in mice and rats, and more recently the 6 Hz assay in mice, have been utilized as primary models of electrically or chemically-evoked seizures in neurologically intact rodents. In addition, rodent kindling models, in which chronic network hyperexcitability has developed, have been used to identify new agents. It is clear that this traditional screening approach has greatly expanded the number of marketed drugs available to manage the symptomatic seizures associated with epilepsy. In spite of the numerous antiseizure drugs (ASDs) on the market today, the fact remains that nearly 30% of patients are resistant to these currently available medications. To address this unmet medical need, the National Institute of Neurological Disorders and Stroke (NINDS) Epilepsy Therapy Screening Program (ETSP) revised its approach to the early evaluation of investigational agents for the treatment of epilepsy in 2015 to include a focus on preclinical approaches to model pharmacoresistant seizures. This present report highlights the in vivo and in vitro findings associated with the initial pharmacological validation of this testing approach using a number of mechanistically diverse, commercially available antiseizure drugs, as well as several probe compounds that are of potential mechanistic interest to the clinical management of epilepsy.     

Hematological,biochemical, and morphological parameters as prognostic indicators for stranded common dolphins (Delphinus delphis) from Cape Cod,Massachusetts, U.S.A.

The current paucity of published blood values and other clinically relevant data for short‐beaked common dolphins, Delphinus delphis, hinders the ability of veterinarians and responders to make well‐informed diagnoses and disposition decisions regarding live strandings of this species. This study examined hematologic, clinical chemistry, and physical parameters from 26 stranded common dolphins on Cape Cod, Massachusetts, in light of their postrelease survival data to evaluate each parameter's efficacy as a prognostic indicator. Statistically and clinically significant differences were found between failed and survived dolphins, including lower hematocrit, hemoglobin, TCO₂, and bicarbonate and higher blood urea nitrogen, uric acid, and length‐to‐girth ratios in animals that failed. In general when compared to survivors, failed dolphins exhibited acidosis, dehydration, lower PCVs, and decreased body condition. Additionally, failed dolphins had the highest ALT, AST, CK, LDH, GGT, and lactate values. These blood values combined with necropsy findings indicate that there are likely a variety of factors affecting postrelease survival, including both preexisting illness and stranding‐induced conditions such as capture myopathy. Closer evaluation of these parameters for stranded common dolphins on point of care analyzers in the field may allow stranding personnel to make better disposition decisions in the future.     

Effects of gangliosides on the activity of the plasma membrane Ca-ATPase

Control of intracellular calcium concentrations ([Ca^2 +]_i) is essential for neuronal function, and the plasma membrane Ca^2 +-ATPase (PMCA) is crucial for the maintenance of low [Ca^2 +]_i. We previously reported on loss of PMCA activity in brain synaptic membranes during aging. Gangliosides are known to modulate Ca^2 + homeostasis and signal transduction in neurons. In the present study, we observed age-related changes in the ganglioside composition of synaptic plasma membranes. This led us to hypothesize that alterations in ganglioside species might contribute to the age-associated loss of PMCA activity. To probe the relationship between changes in endogenous ganglioside content or composition and PMCA activity in membranes of cortical neurons, we induced depletion of gangliosides by treating neurons with d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (d-PDMP). This caused a marked decrease in the activity of PMCA, which suggested a direct correlation between ganglioside content and PMCA activity. Neurons treated with neuraminidase exhibited an increase in GM1 content, a loss in poly-sialoganglioside content, and a decrease in PMCA activity that was greater than that produced by d-PDMP treatment. Thus, it appeared that poly-sialogangliosides had a stimulatory effect whereas mono-sialogangliosides had the opposite effect. Our observations add support to previous reports of PMCA regulation by gangliosides by demonstrating that manipulations of endogenous ganglioside content and species affect the activity of PMCA in neuronal membranes. Furthermore, our studies suggest that age-associated loss in PMCA activity may result in part from changes in the lipid environment of this Ca^2 + transporter.     

Structural,Functional, and Inhibition Studies of a Gcn5-related N-Acetyltransferase (GNAT) Superfamily Protein PA4794: A NEW C-TERMINAL LYSINE PROTEIN ACETYLTRANSFERASE FROM PSEUDOMONAS AERUGINOSA*

The Gcn5-related N-acetyltransferase (GNAT) superfamily is a large group of evolutionarily related acetyltransferases, with multiple paralogs in organisms from all kingdoms of life. The functionally characterized GNATs have been shown to catalyze the transfer of an acetyl group from acetyl-coenzyme A (Ac-CoA) to the amine of a wide range of substrates, including small molecules and proteins. GNATs are prevalent and implicated in a myriad of aspects of eukaryotic and prokaryotic physiology, but functions of many GNATs remain unknown. In this work, we used a multi-pronged approach of x-ray crystallography and biochemical characterization to elucidate the sequence-structure-function relationship of the GNAT superfamily member PA4794 from Pseudomonas aeruginosa. We determined that PA4794 acetylates the Nϵ amine of a C-terminal lysine residue of a peptide, suggesting it is a protein acetyltransferase specific for a C-terminal lysine of a substrate protein or proteins. Furthermore, we identified a number of molecules, including cephalosporin antibiotics, which are inhibitors of PA4794 and bind in its substrate-binding site. Often, these molecules mimic the conformation of the acetylated peptide product. We have determined structures of PA4794 in the apo-form, in complexes with Ac-CoA, CoA, several antibiotics and other small molecules, and a ternary complex with the products of the reaction: CoA and acetylated peptide. Also, we analyzed PA4794 mutants to identify residues important for substrate binding and catalysis.     

ATP stimulates signal recognition particle (SRP)/FtsY-supported protein integration in chloroplasts

The signal recognition particle (SRP) and its receptor (FtsY in prokaryotes) are essential for cotranslational protein targeting to the endoplasmic reticulum in eukaryotes and the cytoplasmic membrane in prokaryotes. An SRP/FtsY-like protein targeting/integration pathway in chloroplasts mediates the posttranslational integration of the light-harvesting chlorophyll a/b-binding protein (LHCP) into thylakoid membranes. GTP, chloroplast SRP (cpSRP), and chloroplast FtsY (cpFtsY) are required for LHCP integration into thylakoid membranes. Here, we report the reconstitution of the LHCP integration reaction with purified recombinant proteins and salt-washed thylakoids. Our data demonstrate that cpSRP and cpFtsY are the only soluble protein components required for LHCP integration. In addition, our studies reveal that ATP, though not absolutely required, remarkably stimulates LHCP integration into salt-washed thylakoids. ATP stimulates LHCP integration by a mechanism independent of the thylakoidal pH gradient (DeltapH) and exerts no detectable effect on the formation of the soluble LHCP-cpSRP-targeting complex. Taken together, our results indicate the participation of a thylakoid ATP-binding protein in LHCP integration.     

Translocator Protein 18 kDa (TSPO) Is Regulated in White and Brown Adipose Tissue by Obesity

Translocator protein 18 kDa (TSPO) is an outer-mitochondrial membrane transporter which has many functions including participation in the mitochondrial permeability transition pore, regulation of reactive oxygen species (ROS), production of cellular energy, and is the rate-limiting step in the uptake of cholesterol. TSPO expression is dysregulated during disease pathologies involving changes in tissue energy demands such as cancer, and is up-regulated in activated macrophages during the inflammatory response. Obesity is associated with decreased energy expenditure, mitochondrial dysfunction, and chronic low-grade inflammation which collectively contribute to the development of the Metabolic Syndrome. Therefore, we hypothesized that dysregulation of TSPO in adipose tissue may be a feature of disease pathology in obesity. Radioligand binding studies revealed a significant reduction in TSPO ligand binding sites in mitochondrial extracts from both white (WAT) and brown adipose tissue (BAT) in mouse models of obesity (diet-induced and genetic) compared to control animals. We also confirmed a reduction in TSPO gene expression in whole tissue extracts from WAT and BAT. Immunohistochemistry in WAT confirmed TSPO expression in adipocytes but also revealed high-levels of TSPO expression in WAT macrophages in obese animals. No changes in TSPO expression were observed in WAT or BAT after a 17 hour fast or 4 hour cold exposure. Treatment of mice with the TSPO ligand PK11195 resulted in regulation of metabolic genes in WAT. Together, these results suggest a potential role for TSPO in mediating adipose tissue homeostasis.     

Polyamine permeation and rectification of Kir4.1 channels

Inward rectifier K(+) (Kir) channels are expressed in multiple neuronal and glial cells. Recent studies have equated certain properties of exogenously expressed Kir4.1 channels with those of native K(+) currents in brain cells, as well as demonstrating the expression of Kir4.1 subunits in these tissues. There are nagging problems however with assigning native currents to Kir4.1 channels. One major concern is that in many native tissues, the putatively correlated currents show much weaker rectification than typically reported for cloned Kir4.1 channels. We have now examined the polyamine-dependence of Kir4.1 channels expressed at high density in Cosm6 cells, using inside-out membrane patches. The experiments reveal a complex and variable rectification that can help explain the variability reported for candidate Kir4.1 currents in native cells. Most importantly, rectification seems to be incomplete, even at high polyamine concentrations. In excised membrane patches, with high levels of expression, and high concentrations of spermine, there is approximately 15% residual conductance that is insensitive to spermine. From a biophysical perspective, this is a striking finding, and indicates either that a bound spermine fails to completely block permeation or that significant spermine permeation (i.e. 'punchthrough') is occurring. To examine this further, we have examined block by philanthotoxin (PhTx, essentially spermine with a bulky tail). PhTx block, while less potent, is more complete than spermine block. This leads us to propose that spermine 'punchthrough' may be significant in Kir4 channels, and that this may be a major contributor to the weak rectification observed under physiological conditions.     

Gender differences in clinical presentation and outcomes of epidemic Kaposi sarcoma in Uganda

Introduction

The incidence of Kaposi sarcoma (KS) has increased dramatically among women in sub-Saharan Africa since the onset of the HIV pandemic, but data on KS disease in women are limited. To identify gender-related differences in KS presentation and outcomes, we evaluated the clinical manifestations and response in men and women with AIDS-associated KS in Uganda.

Methods and Findings

HIV-infected adults with KS attending the Infectious Diseases Institute (IDI) and Uganda Cancer Institute (UCI) in Kampala, Uganda between 2004 and 2006 were included in a retrospective cohort. Evaluation of KS presentation was based on the clinical features described at the initial KS visit. Response was evaluated as the time to “improvement”, as defined by any decrease in lesion size, lesion number, or edema. The cohort consisted of 197 adults with HIV and KS: 55% (108/197) were women. At presentation, the median CD4 T-cell count was significantly lower in women (58 cells/mm³; IQR 11–156 cells/mm³) than men (124 cells/mm³; IQR 22–254 cells/mm³) (p = 0.02). Women were more likely than men to present with lesions of the face (OR 2.8, 95% CI, 1.4, 5.7; p = 0.005) and hard palate (OR 2.0, 95% CI, 1.1, 3.7; p = 0.02), and were less likely than men to have lower extremity lesions (OR 0.54, 95% CI, 0.3, 0.99; p = 0.05). Women were less likely than men to demonstrate clinical improvement (HR = 0.52, CI 0.31, 0.88; p = 0.01) in multivariate analysis.

Conclusions

The clinical presentation and response of KS differs between men and women in Uganda. These data suggest that gender affects the pathophysiology of KS, which may have implications for the prevention, diagnosis, and treatment of KS in both men and women. Prospective studies are needed to identify predictors of response and evaluate efficacy of treatment in women with KS, particularly in Africa where the disease burden is greatest.     

Structural determinants of the substrate and stereochemical specificity of phosphotriesterase

Bacterial phosphotriesterase (PTE) catalyzes the hydrolysis of a wide variety of organophosphate nerve agents and insecticides. Previous kinetic studies with a series of enantiomeric organophosphate triesters have shown that the wild type PTE generally prefers the S(P)-enantiomer over the corresponding R(P)-enantiomers by factors ranging from 1 to 90. The three-dimensional crystal structure of PTE with a bound substrate analogue has led to the identification of three hydrophobic binding pockets. To delineate the factors that govern the reactivity and stereoselectivity of PTE, the dimensions of these three subsites have been systematically altered by site-directed mutagenesis of Cys-59, Gly-60, Ser-61, Ile-106, Trp-131, Phe-132, His-254, His-257, Leu-271, Leu-303, Phe-306, Ser-308, Tyr-309, and Met-317. These studies have shown that substitution of Gly-60 with an alanine within the small subsite dramatically decreased k(cat) and k(cat)/K(a) for the R(P)-enantiomers, but had little influence on the kinetic constants for the S(P)-enantiomers of the chiral substrates. As a result, the chiral preference for the S(P)-enantiomers was greatly enhanced. For example, the value of k(cat)/K(a) with the mutant G60A for the S(P)-enantiomer of methyl phenyl p-nitrophenyl phosphate was 13000-fold greater than that for the corresponding R(P)-enantiomer. The mutation of I106, F132, or S308 to an alanine residue, which enlarges the small or leaving group subsites, caused a significant reduction in the enantiomeric preference for the S(P)-enantiomers, due to selective increases in the reaction rates for the R(P)-enantiomers. Enlargement of the large subsite by the construction of an H254A, H257A, L271A, or M317A mutant had a relatively small effect on k(cat)/K(a) for either the R(P)- or S(P)-enantiomers and thus had little effect on the overall stereoselectivity. These studies demonstrate that by modifying specific residues located within the active site of PTE, it is possible to dramatically alter the stereoselectivity and overall reactivity of the native enzyme toward chiral substrates.     

Antidiabetic Drug Metformin Prevents Progression of Pancreatic Cancer by Targeting in Part Cancer Stem Cells and mTOR Signaling

Epidemiologic studies have shown that diabetes mellitus is associated positively with increased risk of pancreatic ductal adenocarcinoma (PDAC), and recent meta-analysis studies showed that metformin, reduces the risk of pancreatic cancer (PC). We tested the effects of metformin on pancreatic intraepithelial neoplasia (PanIN) and their progression to PDAC in p48Cre/+.LSL-KrasG12D/+ transgenic mice. Mice fed control diet showed 80% and 62% incidence of PDAC in males and females, respectively. Male mice showed 20% and 26%, and female mice showed 7% and 0% PDAC incidence with 1000- and 2000-ppm metformin treatments, respectively. Both doses of metformin decreased pancreatic tumor weights by 34% to 49% (P < 0.03–0.001). The drug treatment caused suppression of PanIN 3 (carcinoma in situ) lesions by 28% to 39% (P < .002) and significant inhibition of carcinoma spread in the pancreas. The pancreatic tissue and/or serum of mice fed metformin showed a significant inhibition of mammalian target of rapamycin (mTOR), extracellular signal-regulated kinases (ERK), phosphorylated extracellular signal-regulated kinases (pErk), and insulin-like growth factor 1 (IGF-1) with an increase in phosphorylated 5′ adenosine monophosphate kinase (pAMPK), tuberous sclerosis complex 1 (TSC1, TSC2), C-protein and an autophagy related protein 2 (ATG2). The cancer stem cell (CSC) markers were significantly decreased (P < 0.04–0.0002) in the pancreatic tissue. These results suggest that biologic effects of metformin are mediated through decreased CSC markers cluster of differentiation 44 (CD44 and CD133), aldehyde dehydrogenase isoform 1 (ALDH1), and epithelial cell adhesion molecule (EPCAM) and modulation of the mTOR signaling pathway. Our preclinical data indicate that metformin has significant potential for use in clinical trials for PC chemoprevention.