“Back of the Bakaletz Lab Video Box”

Lauren O. Bakaletz, Ph.D.

Centers for Microbial Pathogenesis & Battelle Center for Mathematical Medicine Joint Center Meeting

September 13, 2011

What types of questions are you asking?

Nature of protein-protein interactions and protein structure determinations for the type IV pilus of NTHI (in collaboration with the Munson Lab)

Predicting nature of interactions between bacterial or viral pathogens and antibody that targets them via either naturally occurring immune response or one directed by immunization

Predicting/mapping interactions of a specific class of bacterial proteins (or peptides derived from them) with dsDNA

Studying the kinetics and mechanisms by which several unique ‘treatments’ can dissociate an existing bacterial biofilm

Studying when and where in the context of the disease course, specific bacterial genes are expressed

Protein folding and structure determinations for host defense peptides

Host responses (i.e. inflammation, edema etc.) to bacterial or viral pathogens as assessed by small animal imaging (ie. Micro CT)

What kinds of data are generated? Biosensor-derived protein-protein interaction data based on surface

plasmon resonance (think equilibrium curves)

Epitope mapping data – quantitative data sets based on interaction between linear (overlapping) peptides and antibodies of interest

Time lapse video microscopy image files

Immunohistochemistry data, TEM and SEM image files

Promoter activity data generated using Xenogen imaging/kinetic plate reader

3D (soon to be 4D) imaging of bacterial biofilms

COMSTAT analyses of z-stack images of biofilms with and without host cells (from in vitro systems as well as in vivo models)

Otoscopy and tympanometry data

Two or three dimensional representations of proteins

Two or three dimensional representation of anatomical structures in animals

How are the data captured?

Typically via dedicated software associated with instrument being used although many standard image files

Transfer and renaming of files by hand so that they can be re-analyzed Examples:- use Zeiss software to capture biofilm images on confocal scope then convert images by hand for COMSTAT analysis

- capture biofilm resolution images with camera then hand assemble the sequentially captured images to create a video

- do kinetic analyses of bacteria grown under various conditions on the 96-well plate reader, generating data that then have to be downloaded and assembled in graphing software by hand

Otoscopy image files

How much data do you generate?

Otoscopy & biomass image files from the TCIM study (a 35-animal study) are already ~150 Mb

GSK2010 otoscopy image files: 700 Mb (20MB in images/day x 35 days)

Time-lapse imaging of biofilms in chamber slide experiments generates 18720 images, totaling over 52 Gb of data for a single run

Two dimensional images have low memory requirements (70-100 Kb)

Three dimensional representations (~1000 – 2000 Kb/image often requiring 60 images per study) and movies (1000 – 4000 Kb)

SEM image files (100 Kb per image)

What do your data tell you?

Why certain vaccines work and others don’t – and ultimately how to design better vaccine candidates

The molecular mechanisms by which vaccines are effective

The molecular mechanisms by which specific treatments are better than others, as well as the relative kinetics of these interactions

How bacterial gene products interact with each other physically as well as with antibodies directed against them