Archive for April, 2010

Roskamp Institute Poster Abstract from VA Reasearch Week

As I mentioned before, most of the PhD students and research assistants from our Institute participated in poster session  that was organized as a part of VA Research Week event series. Here you will find the abstract of our posters, enjoy 🙂

Development of the Barnes Maze as an Alternative to the Morris Water Maze Following TBI in Mice.

Scott Ferguson1,2, Benoit Mouzon1,2, John Phillips1, Vani Ganapapthi1, Alex Bishop1, Gogce Kayihan1,2, Venkatarajan Mathura1,2, Michael Mullan1,2 , Fiona Crawford1,2

1The Roskamp Institute, Sarasota, FL, USA; 2 James A Haley Veterans Administration, Tampa, FL, USA

Traumatic brain injury (TBI) is suffered by approximately 1.4 million people in the United States each year. TBI is the leading cause of death and disability in the most active population (under 45 years of age) in industrialized countries. Within the military, TBI is associated with 59% of blast-associated injuries seen at Walter Reed Army Medical Center, and between January 2003 and May 2005, 31% of all admissions to WRAMC had a brain injury. Apolipoprotein E (APOE) polymorphisms are known to impact the outcome after TBI, with the APOE4 allele (and concomitant ApoE4 expressed protein) associated with worse outcome than ApoE3 following TBI.   As part of our TBI research program we are exploring the molecular, neurobehavioral and neuropathological outcome after TBI in mouse models of injury, including APOE transgenic mice.

In order to evaluate differential outcomes of injury and its effects on motor skills and memory, we have optimized a series of neurobehavioral tests in mice.  The Rotarod test measures motor skill and learning via a programmable rotating bar.  Rotarod has shown the ability to distinguish between injured and uninjured mice, and has shown appropriate trends between differing levels of injury.  Morris water maze is a test of spatial memory and learning originally designed for rats but later adapted for mice.  APOE3 mice performed better than APOE4 mice in our Rotarod results, demonstrating an APOE genotype-dependent effect on motor function following TBI.  However, in the cognitive paradigm not only did we fail to detect any APOE genotype-dependent effects, we observed no significant differences in performance between injured and uninjured mice.  We therefore explored other cognitive paradigms for their ability to discriminate between injured and uninjured mice.

The Barnes maze is analogous to the Morris water maze in that it is also a test of spatial memory and learning, but because swimming is not involved it is associated with less stress than MWM typically induces in mice.  Others have shown that there is a strain-dependent effect on the ability of mice to learn the water maze task as well as the Barnes maze task.  Given that C57BL/6J mice are reported to perform better on the Barnes maze task, and this is the background strain utilized in our research, we optimized a paradigm of the Barnes maze for use in our TBI studies.  Our results show a statistically significant effect of injury on the spatial memory and learning of C57BL/6J wild type mice.  Future studies will re-examine the effect of APOE genotype on spatial memory following TBI using this test.

This research was funded by a Department of Defense award (W81XWH-07-1-0700) to Dr. Fiona Crawford and by the Roskamp Foundation

APP is Internalized After CD40 Ligation Which Increases Aβ Production

Ghania Ait-Ghezala, Jeremy Frieling, Myles Mullan, Claude-Henry Volmar, and Michael J. Mullan.

CD40, a member of the tumor necrosis factor receptor superfamily, and its cognate ligand CD40L are both elevated in the brains of Alzheimer’s disease (AD) patients compared to controls. We have shown that pharmacological or genetic interruption of CD40/CD40L interaction results in mitigation of AD-like pathology in vivo in transgenic AD mouse models, and in vitro. Recently, we showed that CD40L stimulation could increase Aβ levels, but the mechanism causing this phenomenon is not known. Here we show that CD40 ligation triggers internalization of APP and that internalization by endocytosis is associated with increased Aβ production. Furthermore, anthocyanins, which are known to impact trafficking to and from lipid rafts, impair the production of Aβ by CD40L stimulated CD40. However, anthocyanins have no effect on CD40L treatment of a neuroblastoma cell line over-expressing the C-99 APP fragment suggesting that CD40L internalization has no effect on γ-secretase. This finding is consistent with previous data suggesting that endocytosis increases BACE activity. In summary, these data suggest that a general mechanism of increased Aβ generation may be lipid raft mediated internalization of APP allowing increased BACE activity on its substrate.

Neurobehavioral profiles of two mouse models of Gulf War Illness

Laila Abdullah1, Alex Bishop1, John Phillips1, Benoit Mouzon1,2, Scott Ferguson1,2, Vani Ganapathi1, Myles Mullan1,2, Ghania Ait-Ghezala PhD1,2, Michael Mullan MD, PhD1,2 and Fiona Crawford PhD1,2

Affiliations: 1Roskamp Institute, 2040 Whitfield Ave, Sarasota, FL, 34243, 2James A. Haley VA Hospital, 13000 Bruce B. Downs Blvd, Tampa, FL, 33612.

Background: Gulf War Illness (GWI) is a multisymptom condition associated with service in the 1990-1991 Persian Gulf War conflict and affects around 250,000 US veterans. It is largely attributed to combined exposure to pyridostigmine bromide (PB) and overuse of pesticides and insect repellants.  After nearly two decades, there is still no treatment for GWI and the underlying pathologic factors associated with the observed central nervous system (CNS)-based symptoms in veterans remain unclear.  Current GWI animal models do not demonstrate the full spectrum of neurobehavioral features reported to be associated with GWI, which makes it particularly difficult to explore the efficacy of possible therapeutic options.  Therefore, we tested two different treatment paradigms in order to establish a mouse model of GWI, which exhibits motor, cognitive and anxiety-related symptoms that are observed in veterans with this illness.  Methods: For model A, a previously established treatment paradigm was used which showed pathological changes suggestive of neurodegeneration, however extensive neurobehavioral profiling was not performed. Treated C57BL6 mice received oral administration of 1.3mg/kg of PB in water, dermal application of 0.13mg/kg of permethrin (PER) and 40 mg/kg of N-N-diethyl m-toluamide 2 (DEET) in 70% ethanol and 5 minutes of restrained stress daily for 28 days, whereas control mice received vehicle for the same duration.  For model B, a new treatment paradigm was developed where CD1 mice in the treatment group were administered 2mg/kg of PB and 200 mg/kg of PER via i.p. in DMSO daily for 10 days and the control group received DMSO only.  Following treatment, neurobehavioral profiles were examined using the Rotarod test to assess motor deficits, the Open Field test for anxiety-related changes and the Morris Water Maze test to assess spatial memory.  Results: In model A, treatment was associated with significant impairment in sensorimotor function and presence of anxiety-related behavior, but there was no deficit in spatial memory.  In model B, a delayed adverse effect of treatment was observed on the outcome measures of anxiety and spatial memory, but there was no evidence of sensorimotor impairment.  Conclusion: These findings suggest that combined exposure to PB and pesticides/insect repellents may lead to CNS-based effects in mice that mimic some of the clinical symptoms observed in veterans with GWI.  Additional studies are required to determine whether all three neurobehavioral features can be produced in one mouse model and whether these changes correlate with pathological features associated with neurodegeneration.

Acknowledgment: Funding for this research is provided by a Congressionally Directed Medical Research award (GW080094) to Dr. Fiona Crawford.

Proteomic identification of plasma TBI biomarkers

Benoit Mouzon1,2, Alex Bishop1, Gogce Kayihan1,2, Ben Katz1, Scott Ferguson1,2, Jon Reed1, Venkatarajan Mathura1, Michael Mullan1,2 and Fiona Crawford1,2

1Roskamp Institute, Sarasota, Florida

2James A. Haley Veterans’ Hospital, Tampa, Florida

Traumatic Brain Injury (TBI) is a major cause of mortality and morbidity in both military and civilian populations. The current lack of prognostic biomarkers for TBI confounds treatment and management of patients and is of increasing concern as the TBI population grows.  As part of our TBI research program we are generating brain and plasma proteomic profiles from APOE3 and APOE4 transgenic mice which demonstrate relatively favorable and unfavorable outcomes respectively, following TBI.  In this study we used proteomic approaches to identify the changes in plasma protein profiles in APOE3 and APOE4 mice following severe TBI, in order to determine peripheral biomarkers associated with a poor outcome after TBI.

Using a quantitative proteomics approach (isobaric tagging for relative and absolute quantitation – iTRAQ) we have identified proteins that are significantly modulated as a function of APOE genotype, injury and the interactive term of “genotype*injury”. Analysis of modulated plasma proteins revealed significant differences in proteomic response at 24 hours, 1 month and 3 months post injury across genotypes.  From these proteomic datasets we have identified 83 proteins at the 24 hour timepoint, 170 at 1 month and 129 at 3 months post TBI. For each timepoint, the identified proteins included those whose response was dependent on injury or the injury*genotype interaction, suggesting them as potential biomarkers of injury or outcome following injury.

In pilot validation studies, using antibody-based approaches in the original plasma from these mice, we have demonstrated the validity of our approach.  These preliminary data clearly demonstrate plasma protein changes that are not only injury-dependent but also interaction-dependent. The identified proteins include biomarkers that have been previously implicated in human TBI, and their time course and relationship to neurobehavior and pathology are now to be examined in these mouse models.  Importantly these results demonstrate the presence of TBI-dependent and interaction-dependent plasma proteins at a 3 months time point, which is a considerable time post-injury in the mouse model and will potentially be of significance for combat veterans receiving assessment at extended periods post-injury.  Furthermore, our identification of clusters of related proteins indicates disturbance of particular biological modules which increases their value beyond that of solitary biomarkers.  Clinical assays for many of these proteins are already established, which will facilitate translation of our findings from mouse to human.

The biomarker panels developed from this work will aid clinicians in the determination of diagnosis, prognosis, appropriate treatment and monitoring response to treatment, all of which are urgently needed in TBI management. The next step will be to investigate these potential biomarkers in human TBI patients and those studies will begin this year with the VA patient population and our clinical collaborators.

Acknowledgement:  This research was funded by a Department of Defense award (W81XWH-07-1-0700) to Dr. Fiona Crawford.

The Roskamp Institute is devoted to understanding causes and finding cures for neuropsychiatric and neurodegenerative disorders and addictions. The Institute utilizes a broad range of scientific approaches to understanding the causes of and potential therapies for these disorders with an emphasis on Alzheimer’s disease. For more information, please call (941)752-2949


Advertisements

Handling Unstructured Data in Biological Research and Clinical Trials

Venkatarajan S. Mathura

Complex information that are available in a crude format make computational modeling and processing
a difficult objective in Biological and Health care research. Protocol revisions, process flexibility,
user adoptability should be considered in mind to develop an user friendly information management solution.
There is a great need to integrate data from several fields of research in a common platform to increase
process efficieny and reduce human errors. Realizing cost savings, application of knowledge mining tools on existing data and the need for data organization due to regulatory compliance, research groups and drug-discovery related industries are adopting electronic data capture and management solutions (EDC or EDM). Small scale setups still require an enterprise wide system that is both efficient, light weight, cost effective information management solutions that will be flexible to accomodate growth in the future. With the availability of field specific ontologies, controlled keywords/vocabularies, meta data management and language mapping tools, organization of unstructured data is becoming a feasible task. At the Roskamp Institute the Bioinformatics Group has developed several information management software to aid: Genomics, Proteomics, Animal Colony (Vivarium) and Clinical data management.

CliniProteus:
Clinical trials involve multi-site heterogeneous data generation with complex data input-formats and forms. The data should be captured and queried in an integrated fashion to facilitate further analysis. Electronic case-report forms (eCRF) are gaining popularity since it allows capture of clinical information in a rapid manner. We have designed and developed an XML based flexible clinical trials data management framework in .NET environment that can be used for efficient design and deployment of eCRFs to efficiently collate data and analyze information from multi-site clinical trials. The main components of our system include an XML form designer, a Patient registration eForm, reusable eForms, multiple-visit data capture and consolidated reports. A unique id is used for tracking the trial, site of occurrence, the patient and the year of recruitment.
Availability: http://www.rfdn.org/bioinfo/CTMS/ctms.html.

PWIMS 1.0: Proteomics Workflow and Information Management System

PWIMS is a software package that can systematically manage data in a proteomic laboratory setup. It is implemented in the LAMPP (Linux-Apache-MySQL-Perl/PHP) environment as a three-tier architecture. The client-tier is a web-browser that uses a thin-client HTTP to request resources and display responses to the user. The middle tier consists of an Apache web server, PHP scripting language, the Zend (PHP script) engine. The database tier uses MySQL RDBMS. Data Models and Entity-Relationships have been defined for handling data at various levels.

Some of the features of PWIMS are:

User Management: Multiple user settings, password authentication and access restriction depending on user role

Project Management: Project tracking, online availability of experiment status, experiment results, coordinators information, timeline and date records of the project.

Scheduling & Workflow Control: Tracks the workflow using unique codes for gels and target plates, controls workflow step-skipping and erroneous data entry, systematically schedules next step in the process, lists pending jobs

Data Capture: Form-based data entry, automatic mapping and transfer of large project files using FTP protocols, data can also be entered in a simple EXCEL sheet and uploaded

Data Integration & Analysis: Mass spec results are integrated to gels and projects. Protein hits can be filtered and exported for future reference or any other software, e.g., PDQuest. External links to UniProt, PUBMED, etc. are automatically provided

Data Mining & Presentation: Sequence motif search, functional keyword search and advanced queries can be specified. Results are made available for presentation and sharing.

GEMAT Genomics Experiment Management and Analysis Tool . GEMAT is an information management system designed as client-server tool for handling Affymetrix GeneChip information. It has builtin analytical tools for performing data mining and posting microarry data to endusers.

Tech Tips:

By Our Mass Spectrometry Guru Jon Reed

This will be the first in an ongoing series of technical tips for those interested in biochemistry and mass spectrometry.  Anyone involved with these fields knows there can be considerable headache and heartache that goes hand-in-hand with even the simplest of experiments.  Along the way, I’ve picked up quite a few tricks that I feel should be shared with others.  Most of these are pretty simple, and are intended to do one or more of the following: save time, save money, and improve your experiments.  I’d like to keep these segments informal.  After all, this is a blog and not a review article or method journal.

One recurring theme you will find will be (hopefully) the destruction of the “because we’ve always done it that way” mentality that pervades many laboratories.  This way of thinking runs counter-intuitive to everything the progressive nature of science strives to achieve, and stems from laziness and ego- neither of which have any place in a well-functioning lab.  Let’s face it though,  it’s going to be hard to get rid of lab egos.  We’re all nerds.  Some nerds think they’re not nerds.  Deal with it.

OK, now on to this week’s topic….

Western Blotting: Is methanol the be-all, end-all solvent for Western blotting buffers?

NO.  Not by a long shot.  It’s pricey and toxic.  You can find cheaper substitutes on the shelves of Walmart and Sam’s Club (if you live in the U.S., that is) that work just as well, if not better than MeOH – and you’re not paying the high prices charged by chemical companies.  It’s also ACS grade when sold in stores like that, so you don’t have to worry about it being junk.

So why is MeOH used almost exclusively for blots?

Oh wait…

“BECAUSE WE’VE ALWAYS DONE IT THAT WAY!”

I’m certainly not the first person to think of this, but I figure it warrants repeating.  Simply substitute isopropanol (IPA) for MeOH when preparing your Towbin’s buffer recipe.  Remember that most stores sell it at a 70% or 90% concentration, so be sure to adjust your volumes!   You can also use IPA to wet your PVDF membranes.

I’ll post some western blots run in IPA vs. MeOH, as well as images of the stained PVDF membranes so you can see the results for yourself.  What’s the point in changing things if they’re not as good or better than the original, right?

Other solvent sins: No matter what solvent you’re using for westerns, at no point should you use HPLC-grade solvents for blotting.  Good grief!  They certainly won’t ruin your experiments, but they will definitely ruin your budget, and I can’t tell you how many times I’ve seen labs doing this as a matter of routine.   Electrophoresis-grade buffers make perfect sense, but HPLC-grade solvents?  They make perfect nonsense.  Find a lesser-grade solvent in your catalog or simply head to the local megamart and compare prices (that’s right, you have to do some of the work here too).

So, if you’re running mass spectrometry experiments, now is not the time to be cheap with your solvents.  If you’re western blotting, that’s a different story.  Be bold, be cheap, be happy.

Next week: preparing frits for nano-LC columns.


Roskamp Institute is participating in VA Research Week Activities

Veterans Affairs is celebrating 85th year in research this week (April 26-30, 2010). One of the events is a poster session where scientists will be presenting their most recent work. We are proud to say that most of our PhD students and research assistants will be in the auditorium of the James A Haley VA Hospital in Tampa, FL to present their posters tomorrow (April 29th 2010). For more information about the VA Research Week please visit this site.

The Roskamp Institute is devoted to understanding causes and finding cures for neuropsychiatric and neurodegenerative disorders and addictions. The Institute utilizes a broad range of scientific approaches to understanding the causes of and potential therapies for these disorders with an emphasis on Alzheimer’s disease. For more information, please call (941)752-2949


Roskamp Institute-LECOM collaboration

Dr. Corbin Bachmeier of the Roskamp Institute is currently investigating the drug efflux transport interactions of various antidepressants in collaboration with Dr. Gary Levin of the Lake Erie College of Osteopathic Medicine (LECOM) in Bradenton, FL.  Dr. Bachmeier and Dr. Levin are co-investigators of a grant, provided by Wyeth Pharmaceuticals, which examines the interactions of two antidepressant medications currently on the market, venlafaxine (Effexor) and desvenlafaxine (Pristiq), with the drug efflux transport proteins P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP).  Previous efforts by this group found that treatment with venlafaxine in caco-2 cells, a model of intestinal absorption, induced the mRNA expression levels for P-gp.  This work is reported in the scientific journal Human Psychopharmacology: Clinical and Experimental (Ehret et al., 2007).  Their existing research aims to expound upon these findings by not only examining the impact of treatment with venlafaxine and its metabolite, desvenlafaxine, on the expression of P-gp at the protein level, but also determine their effect on BCRP protein expression levels.  In addition to testing in the caco-2 cells, this research will also evaluate the impact of these drugs on transporter expression in brain endothelial cells, which constitute the blood-brain barrier (BBB).  This research will examine the potential for drug-drug interactions with these therapies and may influence the manner in which these drugs are currently prescribed.

The Roskamp Institute is devoted to understanding causes and finding cures for neuropsychiatric and neurodegenerative disorders and addictions. The Institute utilizes a broad range of scientific approaches to understanding the causes of and potential therapies for these disorders with an emphasis on Alzheimer’s disease. For more information, please call (941)752-2949

High serum Abeta and vascular risk factors in first-degree relatives of Alzheimer's disease patients

Alzheimer’s disease is clinically characterized by progressive cognitive decline accompanied by the presence of amyloid plaques and neurofibrillary tangles in the brain of Alzheimer’s patients. A small protein fragment beta-amyloid (Abeta) with 42 amino acids is shown to deposit earlier in the disease process than the slightly shorter form (40 amino acid fragment).  Both species of this protein fragment are considered toxic to the brain and are shown to have an important role in causing Alzheimer’s disease.  Current research suggests that the disease process in Alzheimer’s begins long before the presence of palpable symptoms and widespread damage in the brain. Therefore, use of beta-amyloid seems promising in identification of individuals at-risk of developing Alzheimer’s disease.  Clinical studies have previously shown that blood and cerebrospinal fluid levels of Abeta may be helpful in diagnosis of Alzheimer’s disease but are influenced by factors such as presence of family history and other risk factors. The main objective of a recent study published by the scientists at the Roskamp Institute was to determine whether elevated blood Abeta levels among the first-degree relatives of patients with Alzheimer’s disease are associated with certain risk factors of cardiovascular disease that are also risk factors Alzheimer’s disease, such as hypertension.  Blood Abeta was measured in disease-free first-degree relatives of patients with Alzheimer-like dementia. Study participants were recruited as part of an ancillary study of the Alzheimer’s Disease Anti-inflammatory Prevention Trial (ADAPT subpopulation) which was funded by the National Institutes of Health (UO1AG15477).  Examination of Abeta in this group of individuals showed that Abeta(1-40) fragment was positively associated with age and use of anti-hypertensive medications, but a negative relationship was observed in those individuals who experienced some increase in systolic blood pressure, despite being on anti-hypertensive medication.  On the other hand, the more toxic Abeta(1-42) was associated with statin use (medications used for lowering cholesterol) and with high-density lipoproteins was observed among statin nonusers. These findings suggest that high Abeta in blood samples of family history-enriched individuals may be due to enrichment of vascular risk factors and may reflect presymptomatic stage of Alzheimer’s disease.  As anti-hypertensive medications and statins are considered to be protective against Alzheimer’s disease onset, it remains to be determined whether their association with Abeta reflects mitigation of Abeta-related toxicity in the brain. Longitudinal evaluation of blood Abeta in this cohort will provide a better understanding of the significance of this association in Alzheimer’s disease etiology.

By Abdullah L, Luis C, Paris D, Ait-ghezala G, Mouzon B, Allen E, Parrish J, Mullan MA, Ferguson S, Wood M, Crawford F, Mullan M. These findings were published in Molecular Medicine 2009 Mar-Apr;15(3-4):95-100.

The Roskamp Institute is devoted to understanding causes and finding cures for neuropsychiatric and neurodegenerative disorders and addictions. The Institute utilizes a broad range of scientific approaches to understanding the causes of and potential therapies for these disorders with an emphasis on Alzheimer’s disease. For more information, please call (941)752-2949

Journal Club: The presence of sodium dodecyl sulphate-stable Aβ dimers is strongly associated with Alzheimer-type dementia

JC Nowell Ganey will present “The presence of sodium dodecyl sulphate-stable Aβ dimers is strongly associated with Alzheimer-type dementia” on Thursday (29th April 2010) at 4:00pm in Roskamp Institute. You can find the article in the following link.

The Roskamp Institute provides a full range of services for individuals with Alzheimer’s disease including diagnostic work-up and follow-up treatment, neuropsychological examination, clinical trial opportunities, and memory screening.  For more information, please call (941)752-2949