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1.  Classification of Alzheimer Diagnosis from ADNI Plasma Biomarker Data 
Research into modeling the progression of Alzheimer’s disease (AD) has made recent progress in identifying plasma proteomic biomarkers to identify the disease at the pre-clinical stage. In contrast with cerebral spinal fluid (CSF) biomarkers and PET imaging, plasma biomarker diagnoses have the advantage of being cost-effective and minimally invasive, thereby improving our understanding of AD and hopefully leading to early interventions as research into this subject advances. The Alzheimer’s Disease Neuroimaging Initiative* (ADNI) has collected data on 190 plasma analytes from individuals diagnosed with AD as well subjects with mild cognitive impairment and cognitively normal (CN) controls. We propose an approach to classify subjects as AD or CN via an ensemble of classifiers trained and validated on ADNI data. Classifier performance is enhanced by an augmentation of a selective biomarker feature space with principal components obtained from the entire set of biomarkers. This procedure yields accuracy of 89% and area under the ROC curve of 94%.
PMCID: PMC4295502  PMID: 25599092
Alzheimer’s Disease Neuroimaging Initiative (ADNI); feature clustering; feature augmentation
2.  The effects of the ketogenic diet on behavior and cognition 
Epilepsy research  2011;100(3):304-309.
Multiple forms of the ketogenic diet (KD) have been successfully used to treat drug-resistant epilepsy, however its mainstream use as a first-line therapy is still limited. Further investigation into its clinical efficacy as well as the molecular basis of activity is likely to assist in the reversal of any resistance to its implementation. In this review we shall attempt to elucidate the current state of experimental and clinical data concerning the neuroprotective and cognitive effects of the KD in both humans and animals. Generally, it has been shown by many research groups that effective implementation of KD exerts strong neuroprotective effects with respect to social behavior and cognition. We will also elucidate the role of KD in the interesting relationship between sleep, epilepsy and memory. Currently available evidence also indicates that, under appropriate control, and with further studies investigating any potential long-term side effects, the KD is also a relatively safe intervention, especially when compared to traditional anti-epileptic pharmacotherapeutics. In addition, due to its neuroprotective capacity, the KD may also hold potential benefit for the treatment of other neurological or neurodegenerative disorders.
PMCID: PMC4112040  PMID: 21872440
Cognition; Memory; Learning; Sleep; Children; Dietary treatment
3.  Metabolic Dysfunction in Alzheimer’s Disease and Related Neurodegenerative Disorders 
Current Alzheimer research  2012;9(1):5-17.
Alzheimer’s disease and other related neurodegenerative diseases are highly debilitating disorders that affect millions of people worldwide. Efforts towards developing effective treatments for these disorders have shown limited efficacy at best, with no true cure to this day being present. Recent work, both clinical and experimental, indicates that many neurodegenerative disorders often display a coexisting metabolic dysfunction which may exacerbate neurological symptoms. It stands to reason therefore that metabolic pathways may themselves contain promising therapeutic targets for major neurodegenerative diseases. In this review, we provide an overview of some of the most recent evidence for metabolic dysregulation in Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease, and discuss several potential mechanisms that may underlie the potential relationships between metabolic dysfunction and etiology of nervous system degeneration. We also highlight some prominent signaling pathways involved in the link between peripheral metabolism and the central nervous system that are potential targets for future therapies, and we will review some of the clinical progress in this field. It is likely that in the near future, therapeutics with combinatorial neuroprotective and ‘eumetabolic’ activities may possess superior efficacies compared to less pluripotent remedies.
PMCID: PMC4097094  PMID: 22329649
Neurodegenerative diseases; metabolic dysfunction; bodyweight; diabetes; glucose homeostasis; insulin; leptin; ghrelin; adiponectin; glucagon-like peptide 1; Alzheimer’s disease; Huntington’s disease; Parkinson’s disease
4.  Metabolic and hormonal signatures in pre-manifest and manifest Huntington's disease patients 
Huntington's disease (HD) is an inherited neurodegenerative disorder typified by involuntary body movements, and psychiatric and cognitive abnormalities. Many HD patients also exhibit metabolic changes including progressive weight loss and appetite dysfunction. Here we have investigated metabolic function in pre-manifest and manifest HD subjects to establish an HD subject metabolic hormonal plasma signature. Individuals at risk for HD who have had predictive genetic testing showing the cytosine-adenine-guanine (CAG) expansion causative of HD, but who do not yet present signs and symptoms sufficient for the diagnosis of manifest HD are said to be “pre-manifest.” Pre-manifest and manifest HD patients, as well as both familial and non-familial controls, were evaluated for multiple peripheral metabolism signals including circulating levels of hormones, growth factors, lipids, and cytokines. Both pre-manifest and manifest HD subjects exhibited significantly reduced levels of circulating growth factors, including growth hormone and prolactin. HD-related changes in the levels of metabolic hormones such as ghrelin, glucagon, and amylin were also observed. Total cholesterol, HDL-C, and LDL-C were significantly decreased in HD subjects. C-reactive protein was significantly elevated in pre-manifest HD subjects. The observation of metabolic alterations, even in subjects considered to be in the pre-manifest stage of HD, suggests that in addition, and prior, to overt neuronal damage, HD affects metabolic hormone secretion and energy regulation, which may shed light on pathogenesis, and provide opportunities for biomarker development.
PMCID: PMC4066441  PMID: 25002850
Huntington's disease; pre-manifest; metabolic hormones; lipids; peripheral energy metabolism
5.  Higher TNF-α, IGF-1, and Leptin Levels are Found in Tasters than Non-Tasters 
Taste perception is controlled by taste cells that are present in the tongue that produce and secrete various metabolic hormones. Recent studies have demonstrated that taste receptors in tongue, gut, and pancreas are associated with local hormone secretion. The aim of this study was to determine whether there is a link between taste sensitivity and levels of circulating metabolic hormones in humans and whether taste sensitivity is potentially related to peripheral metabolic regulation. Thirty-one subjects were recruited and separated into tasters and non-tasters based on their phenol thiocarbamide (PTC) bitter taste test results. Fasting plasma and saliva were collected and levels of hormones and cytokines were assayed. We observed significant differences in both hormone levels and hormone-body mass index (BMI) correlation between tasters and non-tasters. Tasters had higher plasma levels of leptin (p = 0.05), tumor necrosis factor-α (TNF-α) (p = 0.04), and insulin-like growth factor 1 (IGF-1) (p = 0.03). There was also a trend toward increased IGF-1 levels in the saliva of tasters (p = 0.06). We found a positive correlation between plasma levels of glucose and BMI (R = 0.4999, p = 0.04) exclusively in non-tasters. In contrast, plasma C-peptide levels were found to be positively correlated to BMI (R = 0.5563, p = 0.03) in tasters. Saliva TNF-α levels were negatively correlated with BMI in tasters (R = −0.5908, p = 0.03). Our findings demonstrate that there are differences in circulating levels of leptin, TNF-α, and IGF-1 between tasters and non-tasters. These findings indicate that in addition to the regulation of food consumption, taste perception also appears to be tightly linked to circulating metabolic hormone levels. People with different taste sensitivity may respond differently to the nutrient stimulation. Further work investigating the link between taste perception and peripheral metabolic control could potentially lead to the development of novel therapies for obesity or Type 2 diabetes.
PMCID: PMC4114300  PMID: 25120532
taste; TNF-α; leptin; IGF-1; BMI
6.  Systems-Level G Protein-Coupled Receptor Therapy Across a Neurodegenerative Continuum by the GLP-1 Receptor System 
With our increasing appreciation of the true complexity of diseases and pathophysiologies, it is clear that this knowledge needs to inform the future development of pharmacotherapeutics. For many disorders, the disease mechanism itself is a complex process spanning multiple signaling networks, tissues, and organ systems. Identifying the precise nature and locations of the pathophysiology is crucial for the creation of systemically effective drugs. Diseases once considered constrained to a limited range of organ systems, e.g., central neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’ disease (HD), the role of multiple central and peripheral organ systems in the etiology of such diseases is now widely accepted. With this knowledge, it is increasingly clear that these seemingly distinct neurodegenerative disorders (AD, PD, and HD) possess multiple pathophysiological similarities thereby demonstrating an inter-related continuum of disease-related molecular alterations. With this systems-level appreciation of neurodegenerative diseases, it is now imperative to consider that pharmacotherapeutics should be developed specifically to address the systemic imbalances that create the disorders. Identification of potential systems-level signaling axes may facilitate the generation of therapeutic agents with synergistic remedial activity across multiple tissues, organ systems, and even diseases. Here, we discuss the potentially therapeutic systems-level interaction of the glucagon-like peptide 1 (GLP-1) ligand–receptor axis with multiple aspects of the AD, PD, and HD neurodegenerative continuum.
PMCID: PMC4150252  PMID: 25225492
transcriptomics and proteomics; pharmacotherapeutics; pathophysiology; heptahelical G protein-coupled receptor
7.  The effects of aging on the BTBR mouse model of autism spectrum disorder 
Autism spectrum disorder (ASD) is a complex heterogeneous neurodevelopmental disorder characterized by alterations in social functioning, communicative abilities, and engagement in repetitive or restrictive behaviors. The process of aging in individuals with autism and related neurodevelopmental disorders is not well understood, despite the fact that the number of individuals with ASD aged 65 and older is projected to increase by over half a million individuals in the next 20 years. To elucidate the effects of aging in the context of a modified central nervous system, we investigated the effects of age on the BTBR T + tf/j mouse, a well characterized and widely used mouse model that displays an ASD-like phenotype. We found that a reduction in social behavior persists into old age in male BTBR T + tf/j mice. We employed quantitative proteomics to discover potential alterations in signaling systems that could regulate aging in the BTBR mice. Unbiased proteomic analysis of hippocampal and cortical tissue of BTBR mice compared to age-matched wild-type controls revealed a significant decrease in brain derived neurotrophic factor and significant increases in multiple synaptic markers (spinophilin, Synapsin I, PSD 95, NeuN), as well as distinct changes in functional pathways related to these proteins, including “Neural synaptic plasticity regulation” and “Neurotransmitter secretion regulation.” Taken together, these results contribute to our understanding of the effects of aging on an ASD-like mouse model in regards to both behavior and protein alterations, though additional studies are needed to fully understand the complex interplay underlying aging in mouse models displaying an ASD-like phenotype.
PMCID: PMC4150363  PMID: 25225482
ASD; autism; BDNF; aging; synaptic marker; neuroprotection; neurodevelopmental disorder; BTBR
8.  The role of Thyrotropin Releasing Hormone in aging and neurodegenerative diseases 
Thyrotropin releasing hormone (TRH) is primarily known as the central regulator of the hypothalamic-pituitary-thyroid (HPT) axis. However, TRH also exerts a variety of central nervous system effects independent from its activity in the HPT axis. With advancing age, decreases in TRH synthesis, expression, and activity have been demonstrated. Associated with this emerging evidence suggests that TRH is implicated in neurodegenerative diseases of aging, including Alzheimer’s disease and Parkinson’s disease. TRH and its synthetic analogs have been recognized as trophic factors in neurons of the diencephalon and spinal cord, and as neuroprotectants against oxidative stress, glutamate toxicity, caspase-induced cell death, DNA fragmentation, and inflammation. In this review, we will provide an overview of some of the roles of TRH, outside of the HPT axis, associated with pathological aging and neurodegeneration and we shall discuss the potential of TRH and TRH analogs for the treatment of neurodegenerative diseases.
PMCID: PMC3817016  PMID: 24199031
Thyrotropin Releasing Hormone; Alzheimer’s Disease; Parkinson’s Disease; Neuroprotection; Aging
9.  Altered Lipid and Salt Taste Responsivity in Ghrelin and GOAT Null Mice 
PLoS ONE  2013;8(10):e76553.
Taste perception plays an important role in regulating food preference, eating behavior and energy homeostasis. Taste perception is modulated by a variety of factors, including gastric hormones such as ghrelin. Ghrelin can regulate growth hormone release, food intake, adiposity, and energy metabolism. Octanoylation of ghrelin by ghrelin O-acyltransferase (GOAT) is a specific post-translational modification which is essential for many biological activities of ghrelin. Ghrelin and GOAT are both widely expressed in many organs including the gustatory system. In the current study, overall metabolic profiles were assessed in wild-type (WT), ghrelin knockout (ghrelin−/−), and GOAT knockout (GOAT−/−) mice. Ghrelin−/− mice exhibited decreased food intake, increased plasma triglycerides and increased ketone bodies compared to WT mice while demonstrating WT-like body weight, fat composition and glucose control. In contrast GOAT−/− mice exhibited reduced body weight, adiposity, resting glucose and insulin levels compared to WT mice. Brief access taste behavioral tests were performed to determine taste responsivity in WT, ghrelin−/− and GOAT−/− mice. Ghrelin and GOAT null mice possessed reduced lipid taste responsivity. Furthermore, we found that salty taste responsivity was attenuated in ghrelin−/− mice, yet potentiated in GOAT−/− mice compared to WT mice. Expression of the potential lipid taste regulators Cd36 and Gpr120 were reduced in the taste buds of ghrelin and GOAT null mice, while the salt-sensitive ENaC subunit was increased in GOAT−/− mice compared with WT mice. The altered expression of Cd36, Gpr120 and ENaC may be responsible for the altered lipid and salt taste perception in ghrelin−/− and GOAT−/− mice. The data presented in the current study potentially implicates ghrelin signaling activity in the modulation of both lipid and salt taste modalities.
PMCID: PMC3790684  PMID: 24124572
10.  Long-Term Artificial Sweetener Acesulfame Potassium Treatment Alters Neurometabolic Functions in C57BL/6J Mice 
PLoS ONE  2013;8(8):e70257.
With the prevalence of obesity, artificial, non-nutritive sweeteners have been widely used as dietary supplements that provide sweet taste without excessive caloric load. In order to better understand the overall actions of artificial sweeteners, especially when they are chronically used, we investigated the peripheral and central nervous system effects of protracted exposure to a widely used artificial sweetener, acesulfame K (ACK). We found that extended ACK exposure (40 weeks) in normal C57BL/6J mice demonstrated a moderate and limited influence on metabolic homeostasis, including altering fasting insulin and leptin levels, pancreatic islet size and lipid levels, without affecting insulin sensitivity and bodyweight. Interestingly, impaired cognitive memory functions (evaluated by Morris Water Maze and Novel Objective Preference tests) were found in ACK-treated C57BL/6J mice, while no differences in motor function and anxiety levels were detected. The generation of an ACK-induced neurological phenotype was associated with metabolic dysregulation (glycolysis inhibition and functional ATP depletion) and neurosynaptic abnormalities (dysregulation of TrkB-mediated BDNF and Akt/Erk-mediated cell growth/survival pathway) in hippocampal neurons. Our data suggest that chronic use of ACK could affect cognitive functions, potentially via altering neuro-metabolic functions in male C57BL/6J mice.
PMCID: PMC3737213  PMID: 23950916
11.  Modulation of taste sensitivity by GLP-1 signaling in taste buds 
Modulation of sensory function can help animals adjust to a changing external and internal environment. Even so, mechanisms for modulating taste sensitivity are poorly understood. Using immunohistochemical, biochemical and behavioral approaches, we found that the peptide hormone glucagon-like peptide-1 (GLP-1) and its receptor (GLP-1R) are expressed in mammalian taste buds. Furthermore, we found that GLP-1 signaling plays an important role in the modulation of taste sensitivity: GLP-1R knockout mice exhibit a dramatic reduction in sweet taste sensitivity as well as an enhanced sensitivity to umami-tasting stimuli. Together, these findings suggest a novel paracrine mechanism for the hormonal modulation of taste function in mammals.
PMCID: PMC3731136  PMID: 19686117
glucagon-like peptide-1; hormone; sweet; umami; glutamate
12.  Plurigon: three dimensional visualization and classification of high-dimensionality data 
High-dimensionality data is rapidly becoming the norm for biomedical sciences and many other analytical disciplines. Not only is the collection and processing time for such data becoming problematic, but it has become increasingly difficult to form a comprehensive appreciation of high-dimensionality data. Though data analysis methods for coping with multivariate data are well-documented in technical fields such as computer science, little effort is currently being expended to condense data vectors that exist beyond the realm of physical space into an easily interpretable and aesthetic form. To address this important need, we have developed Plurigon, a data visualization and classification tool for the integration of high-dimensionality visualization algorithms with a user-friendly, interactive graphical interface. Unlike existing data visualization methods, which are focused on an ensemble of data points, Plurigon places a strong emphasis upon the visualization of a single data point and its determining characteristics. Multivariate data vectors are represented in the form of a deformed sphere with a distinct topology of hills, valleys, plateaus, peaks, and crevices. The gestalt structure of the resultant Plurigon object generates an easily-appreciable model. User interaction with the Plurigon is extensive; zoom, rotation, axial and vector display, feature extraction, and anaglyph stereoscopy are currently supported. With Plurigon and its ability to analyze high-complexity data, we hope to see a unification of biomedical and computational sciences as well as practical applications in a wide array of scientific disciplines. Increased accessibility to the analysis of high-dimensionality data may increase the number of new discoveries and breakthroughs, ranging from drug screening to disease diagnosis to medical literature mining.
PMCID: PMC3717481  PMID: 23885241
Plurigon; three dimensional; data visualization; data classification; multivariate data vectors; algorithms; systems biology; bioinformatics
13.  The stromal factors SDF1α, sFRP1 and VEGFD induce dopaminergic neuron differentiation of human pluripotent stem cells 
Journal of Neuroscience Research  2012;90(7):1367-1381.
Human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons hold potential for treating Parkinson’s disease (PD) through cell replacement therapy. Generation of DA neurons from hESCs has been achieved by co-culture with the stromal cell line PA6, a source of stromal cell-derived inducing activity (SDIA). However, the factor(s) produced by stromal cells that constitute SDIA are largely undefined. We previously reported that medium conditioned by PA6 cells can generate functional DA neurons from NTera2 human embryonal carcinoma stem cells. Here we show that PA6-conditioned medium can induce DA neuronal differentiation in both NTera2 cells and the hESC I6 cell line. To identify the factor(s) responsible for SDIA, we used large-scale microarray analysis of gene expression combined with mass spectrometric analysis of PA6-conditioned medium (CM). The candidate factors, hepatocyte growth factor (HGF), stromal cell-derived factor-1 α (SDF1α), secreted frizzled-related protein 1 (sFRP1), and vascular endothelial growth factor D (VEGFD) were identified and their concentrations in PA6 CM were established by immunoaffinity capillary electrophoresis. Upon addition of SDF1α, sFRP1 and VEGFD to the culture medium we observed an increase in the number of cells expressing tyrosine hydroxylase (a marker for DA neurons) and beta-III tubulin (a marker for immature neurons) in both the NTera2 and I6 cell lines. These results indicate that SDF1α, sFRP1 and VEGFD are major components of SDIA, and suggest the potential use of these defined factors to elicit DA differentiation of pluripotent human stem cells for therapeutic intervention in PD.
PMCID: PMC3350575  PMID: 22535492
dopaminergic neurons; neuronal differentiation; stromal cell derived inducing activity; embryonic stem cells
14.  Effective correction of experimental errors in quantitative proteomics using stable isotope labeling by amino acids in cell culture (SILAC) 
Journal of Proteomics  2012;75(12):3720-3732.
Accurate and reliable quantitative proteomics in cell culture has been considerably facilitated by the introduction of the stable isotope labeling by amino acids in cell culture (SILAC), combined with high resolution mass spectrometry. There are however several major sources of quantification errors that commonly occur with SILAC techniques, i.e. incomplete incorporation of isotopic amino acids, arginine-to-proline conversion, and experimental errors in final sample mixing. Dataset normalization is a widely adopted solution to such errors, however this may not completely prevent introducing incorrect expression ratios. Here we demonstrate that a label-swap replication of SILAC experiments was able to effectively correct experimental errors by averaging ratios measured in individual replicates using quantitative proteomics and phosphoproteomics of ligand treatment of neural cell cultures. Furthermore, this strategy was successfully applied to a SILAC triplet experiment, which presents a much more complicated experimental matrix, affected by both incomplete labeling and arginine-to-proline conversion. Based on our results, we suggest that SILAC experiments should be designed to incorporate label-swap replications for enhanced reliability in expression ratios.
PMCID: PMC3394155  PMID: 22575385
SILAC; incomplete isotope labeling; arginine-to-proline conversion; label-swap replication; receptor
15.  Discovery- and Target-Based Protein Quantification Using iTRAQ and Pulsed Q Collision Induced Dissociation (PQD) 
Journal of Proteomics  2012;75(8):2480-2487.
Pulsed Q collision-induced dissociation (PQD) was developed in part to facilitate detection of low-mass reporter ions using labeling reagents (e.g. iTRAQ) on LTQ platforms. It has generally been recognized that the scan speed and sensitivity of an LTQ are superior than those of an Orbitrap using the higher-energy collisional dissociation (HCD). However, the use of PQD in quantitative proteomics is limited, primarily due to the meager reproducibility of reporter ion ratios. Optimizations of PQD for iTRAQ quantification using LTQ have been reported, but a universally applicable strategy for quantifying the less abundant proteins has not been fully established. Adjustments of the AGC target, µscan, or scan speed offer only incremental improvements in reproducibility. From our experience, however, satisfactory coefficients of variation (CVs) of reporter ion ratios were difficult to achieve using the discovery-based approach. As an alternative, we implemented a target-based approach that obviates data dependency to allow repetitive data acquisitions across chromatographic peaks. Such a strategy generates enough data points for more reliable quantification. Using cAMP treatment in S49 cell lysates and this target-based approach, we were able to validate differentially expressed proteins, which were initially identified as potential candidates using the discovery-based PQD. The target-based strategy also yielded results comparable to those obtained from HCD in an Orbitrap. Our findings should aid LTQ users who desire to explore iTRAQ quantitative proteomics but have limited access to the more costly Orbitrap or other instruments.
PMCID: PMC3321122  PMID: 22397766
Pulsed Q collision-induced dissociation (PQD); linear ion trap; triple quadrupole (QqQ); higher energy collisional dissociation (HCD); iTRAQ (Isobaric Tag for Relative and Absolute Quantification)
16.  Age-Related Changes in Mouse Taste Bud Morphology, Hormone Expression, and Taste Responsivity 
Normal aging is a complex process that affects every organ system in the body, including the taste system. Thus, we investigated the effects of the normal aging process on taste bud morphology, function, and taste responsivity in male mice at 2, 10, and 18 months of age. The 18-month-old animals demonstrated a significant reduction in taste bud size and number of taste cells per bud compared with the 2- and 10-month-old animals. The 18-month-old animals exhibited a significant reduction of protein gene product 9.5 and sonic hedgehog immunoreactivity (taste cell markers). The number of taste cells expressing the sweet taste receptor subunit, T1R3, and the sweet taste modulating hormone, glucagon-like peptide-1, were reduced in the 18-month-old mice. Concordant with taste cell alterations, the 18-month-old animals demonstrated reduced sweet taste responsivity compared with the younger animals and the other major taste modalities (salty, sour, and bitter) remained intact.
PMCID: PMC3410661  PMID: 22056740
Taste buds; Aging; Sweet taste; Glucagon-like peptide-1; T1R3
17.  Effective use of latent semantic indexing and computational linguistics in biological and biomedical applications 
Text mining is rapidly becoming an essential technique for the annotation and analysis of large biological data sets. Biomedical literature currently increases at a rate of several thousand papers per week, making automated information retrieval methods the only feasible method of managing this expanding corpus. With the increasing prevalence of open-access journals and constant growth of publicly-available repositories of biomedical literature, literature mining has become much more effective with respect to the extraction of biomedically-relevant data. In recent years, text mining of popular databases such as MEDLINE has evolved from basic term-searches to more sophisticated natural language processing techniques, indexing and retrieval methods, structural analysis and integration of literature with associated metadata. In this review, we will focus on Latent Semantic Indexing (LSI), a computational linguistics technique increasingly used for a variety of biological purposes. It is noted for its ability to consistently outperform benchmark Boolean text searches and co-occurrence models at information retrieval and its power to extract indirect relationships within a data set. LSI has been used successfully to formulate new hypotheses, generate novel connections from existing data, and validate empirical data.
PMCID: PMC3558626  PMID: 23386833
latent semantic indexing; data mining; computational linguistics; molecular interactions; drug discovery
18.  VennPlex–A Novel Venn Diagram Program for Comparing and Visualizing Datasets with Differentially Regulated Datapoints 
PLoS ONE  2013;8(1):e53388.
With the development of increasingly large and complex genomic and proteomic data sets, an enhancement in the complexity of available Venn diagram analytical programs is becoming increasingly important. Current freely available Venn diagram programs often fail to represent extra complexity among datasets, such as regulation pattern differences between different groups. Here we describe the development of VennPlex, a program that illustrates the often diverse numerical interactions among multiple, high-complexity datasets, using up to four data sets. VennPlex includes versatile output features, where grouped data points in specific regions can be easily exported into a spreadsheet. This program is able to facilitate the analysis of two to four gene sets and their corresponding expression values in a user-friendly manner. To demonstrate its unique experimental utility we applied VennPlex to a complex paradigm, i.e. a comparison of the effect of multiple oxygen tension environments (1–20% ambient oxygen) upon gene transcription of primary rat astrocytes. VennPlex accurately dissects complex data sets reliably into easily identifiable groups for straightforward analysis and data output. This program, which is an improvement over currently available Venn diagram programs, is able to rapidly extract important datasets that represent the variety of expression patterns available within the data sets, showing potential applications in fields like genomics, proteomics, and bioinformatics.
PMCID: PMC3538763  PMID: 23308210
19.  Pancreas++: Automated Quantification of Pancreatic Islet Cells in Microscopy Images 
The microscopic image analysis of pancreatic Islet of Langerhans morphology is crucial for the investigation of diabetes and metabolic diseases. Besides the general size of the islet, the percentage and relative position of glucagon-containing alpha-, and insulin-containing beta-cells is also important for pathophysiological analyses, especially in rodents. Hence, the ability to identify, quantify and spatially locate peripheral, and “involuted” alpha-cells in the islet core is an important analytical goal. There is a dearth of software available for the automated and sophisticated positional quantification of multiple cell types in the islet core. Manual analytical methods for these analyses, while relatively accurate, can suffer from a slow throughput rate as well as user-based biases. Here we describe a newly developed pancreatic islet analytical software program, Pancreas++, which facilitates the fully automated, non-biased, and highly reproducible investigation of islet area and alpha- and beta-cell quantity as well as position within the islet for either single or large batches of fluorescent images. We demonstrate the utility and accuracy of Pancreas++ by comparing its performance to other pancreatic islet size and cell type (alpha, beta) quantification methods. Our Pancreas++ analysis was significantly faster than other methods, while still retaining low error rates and a high degree of result correlation with the manually generated reference standard.
PMCID: PMC3535421  PMID: 23293605
pancreas; islets of Langerhans; alpha-cells; beta-cells; quantification; software; algorithm
20.  Growth Inhibition by miR-519 via Multiple p21-Inducing Pathways 
Molecular and Cellular Biology  2012;32(13):2530-2548.
The microRNA miR-519 robustly inhibits cell proliferation, in turn triggering senescence and decreasing tumor growth. However, the molecular mediators of miR-519-elicited growth inhibition are unknown. Here, we systematically investigated the influence of miR-519 on gene expression profiles leading to growth cessation in HeLa human cervical carcinoma cells. By analyzing miR-519-triggered changes in protein and mRNA expression patterns and by identifying mRNAs associated with biotinylated miR-519, we uncovered two prominent subsets of miR-519-regulated mRNAs. One subset of miR-519 target mRNAs encoded DNA maintenance proteins (including DUT1, EXO1, RPA2, and POLE4); miR-519 repressed their expression and increased DNA damage, in turn raising the levels of the cyclin-dependent kinase (cdk) inhibitor p21. The other subset of miR-519 target mRNAs encoded proteins that control intracellular calcium levels (notably, ATP2C1 and ORAI1); their downregulation by miR-519 aberrantly elevated levels of cytosolic [Ca2+] storage in HeLa cells, similarly increasing p21 levels in a manner dependent on the Ca2+-activated kinases CaMKII and GSK3β. The rises in levels of DNA damage, the Ca2+ concentration, and p21 levels stimulated an autophagic phenotype in HeLa and other human carcinoma cell lines. As a consequence, ATP levels increased, and the level of activity of the AMP-activated protein kinase (AMPK) declined, further contributing to the elevation in the abundance of p21. Our results indicate that miR-519 promotes DNA damage, alters Ca2+ homeostasis, and enhances energy production; together, these processes elevate the expression level of p21, promoting growth inhibition and cell survival.
PMCID: PMC3434494  PMID: 22547681
21.  BRET Biosensor Analysis of Receptor Tyrosine Kinase Functionality 
Bioluminescence resonance energy transfer (BRET) is an improved version of earlier resonance energy transfer technologies used for the analysis of biomolecular protein interaction. BRET analysis can be applied to many transmembrane receptor classes, however the majority of the early published literature on BRET has focused on G protein-coupled receptor (GPCR) research. In contrast, there is limited scientific literature using BRET to investigate receptor tyrosine kinase (RTK) activity. This limited investigation is surprising as RTKs often employ dimerization as a key factor in their activation, as well as being important therapeutic targets in medicine, especially in the cases of cancer, diabetes, neurodegenerative, and respiratory conditions. In this review, we consider an array of studies pertinent to RTKs and other non-GPCR receptor protein–protein signaling interactions; more specifically we discuss receptor-protein interactions involved in the transmission of signaling communication. We have provided an overview of functional BRET studies associated with the RTK superfamily involving: neurotrophic receptors [e.g., tropomyosin-related kinase (Trk) and p75 neurotrophin receptor (p75NTR)]; insulinotropic receptors [e.g., insulin receptor (IR) and insulin-like growth factor receptor (IGFR)] and growth factor receptors [e.g., ErbB receptors including the EGFR, the fibroblast growth factor receptor (FGFR), the vascular endothelial growth factor receptor (VEGFR) and the c-kit and platelet-derived growth factor receptor (PDGFR)]. In addition, we review BRET-mediated studies of other tyrosine kinase-associated receptors including cytokine receptors, i.e., leptin receptor (OB-R) and the growth hormone receptor (GHR). It is clear even from the relatively sparse experimental RTK BRET evidence that there is tremendous potential for this technological application for the functional investigation of RTK biology.
PMCID: PMC3620488  PMID: 23577003
receptor tyrosine kinase; RTK; protein–protein interaction; neurotrophic; insulin receptor; insulin-like growth factor receptor; epidermal growth factor receptor; cytokine receptors
22.  Correction: Altered Hypothalamic Protein Expression in a Rat Model of Huntington's Disease 
PLoS ONE  2012;7(11):10.1371/annotation/677c26e3-ce52-4837-853a-63c4ed7d72c0.
PMCID: PMC3552740
23.  Altered Hypothalamic Protein Expression in a Rat Model of Huntington's Disease 
PLoS ONE  2012;7(10):e47240.
Huntington's disease (HD) is a neurodegenerative disorder, which is characterized by progressive motor impairment and cognitive alterations. Changes in energy metabolism, neuroendocrine function, body weight, euglycemia, appetite function, and circadian rhythm can also occur. It is likely that the locus of these alterations is the hypothalamus. We used the HD transgenic (tg) rat model bearing 51 CAG repeats, which exhibits similar HD symptomology as HD patients to investigate hypothalamic function. We conducted detailed hypothalamic proteome analyses and also measured circulating levels of various metabolic hormones and lipids in pre-symptomatic and symptomatic animals. Our results demonstrate that there are significant alterations in HD rat hypothalamic protein expression such as glial fibrillary acidic protein (GFAP), heat shock protein-70, the oxidative damage protein glutathione peroxidase (Gpx4), glycogen synthase1 (Gys1) and the lipid synthesis enzyme acylglycerol-3-phosphate O-acyltransferase 1 (Agpat1). In addition, there are significant alterations in various circulating metabolic hormones and lipids in pre-symptomatic animals including, insulin, leptin, triglycerides and HDL, before any motor or cognitive alterations are apparent. These early metabolic and lipid alterations are likely prodromal signs of hypothalamic dysfunction. Gaining a greater understanding of the hypothalamic and metabolic alterations that occur in HD, could lead to the development of novel therapeutics for early interventional treatment of HD.
PMCID: PMC3475691  PMID: 23094041
24.  Identification of Proteins and Phosphoproteins Using Pulsed Q Collision Induced Dissociation (PQD) 
Pulsed Q collision induced dissociation (PQD) was developed to facilitate detection of low-mass reporter ions from labeling reagents (e.g. iTRAQ) in peptide quantification using an LTQ mass spectrometer (MS). Despite the large number of linear ion traps worldwide, the use and optimization of PQD for protein identification have been limited, in part due to less effective ion fragmentation relative to the collision induced dissociation (CID). PQD expands the m/z coverage of fragment ions to the lower m/z range by circumventing the typical low mass cut-off of an ion trap MS. Since database searching relies on the matching between theoretical and observed spectra, it is not clear how ion intensity and peak number might affect the outcomes of a database search. In this report, we systematically evaluated the attributes of PQD mass spectra, performed intensity optimization, and assessed the benefits of using PQD on the identification of peptides and phosphopeptides from an LTQ. Based on head-to-head comparisons between CID (higher intensity) and PQD (better m/z coverage), peptides identified using PQD generally have Xcorr scores lower than those using CID. Such score differences were considerably diminished by the use of 0.1% m-nitrobenzyl alcohol (m-NBA) in mobile phases. The ion intensities of both CID and PQD were adversely affected by increasing m/z of the precursor, with PQD more sensitive than CID. In addition to negating the 1/3 rule, PQD enhances direct bond cleavage and generates patterns of fragment ions different from those of CID, particularly for peptides with a labile functional group (e.g. phosphopeptides). The higher energy fragmentation pathway of PQD on peptide fragmentation was further compared to those of CID and the quadrupole-type activation in parallel experiments.
PMCID: PMC3183836  PMID: 21952889
Pulsed Q collision induced dissociation (PQD); linear ion trap; triple quadrupole (QqQ); protein identification
25.  Metabolic Context Regulates Distinct Hypothalamic Transcriptional Responses to Antiaging Interventions 
The hypothalamus is an essential relay in the neural circuitry underlying energy metabolism that needs to continually adapt to changes in the energetic environment. The neuroendocrine control of food intake and energy expenditure is associated with, and likely dependent upon, hypothalamic plasticity. Severe disturbances in energy metabolism, such as those that occur in obesity, are therefore likely to be associated with disruption of hypothalamic transcriptomic plasticity. In this paper, we investigated the effects of two well-characterized antiaging interventions, caloric restriction and voluntary wheel running, in two distinct physiological paradigms, that is, diabetic (db/db) and nondiabetic wild-type (C57/Bl/6) animals to investigate the contextual sensitivity of hypothalamic transcriptomic responses. We found that, both quantitatively and qualitatively, caloric restriction and physical exercise were associated with distinct transcriptional signatures that differed significantly between diabetic and non-diabetic mice. This suggests that challenges to metabolic homeostasis regulate distinct hypothalamic gene sets in diabetic and non-diabetic animals. A greater understanding of how genetic background contributes to hypothalamic response mechanisms could pave the way for the development of more nuanced therapeutics for the treatment of metabolic disorders that occur in diverse physiological backgrounds.
PMCID: PMC3427989  PMID: 22934110

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