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FARE2014 WINNERS Sorted by Institute/Center FARE2014 WINNERS Sorted By Institute/Center CC Xu, Ziyue Postdoctoral Fellow Radiology/Imaging/PET and Neuroimaging Computerized Inspection of Airways and Lungs (CIAL): Introducing a novel, robust, accurate, and fast software tool for analyzing CT scans Pulmonary tuberculosis (TB) is a common infection with high mortality and morbidity. CT is often used for spatial information analysis, visualization, and volume quantification of airways and cavities. Characterization of airways and cavitation are typically assessed by radiologists via visual analysis, which can be time consuming and provide limited accuracy with high inter-observer variance. In this study, we designed and tested a novel computer aided detection and quantification tool to detect and quantify airways and cavities. Airway lumen and wall thickness are located and measured with a novel graph- based algorithm that worked quickly and reliably. Cavities were then detected and delineated with a proposed automatic algorithm. With our methodology, multiple advanced imaging markers like airway radius, wall thickness, cavity volume and shape dispersion, minimum distance from cavities to airway, and the evolution of the cavity over time were extracted in high accuracy and efficiency. This can provide an analytical tool for improving radiologic research and patient-care diagnosis in the clinical and preclinical settings. Airway segmentation is developed based on a novel hybrid multi-scale approach, wall thickness estimation is based on novel spatially constrained random walk segmentation, and cavity detection uses both intensity and shape information within an efficient fuzzy connectedness delineation framework. The integration of these methods for our algorithm can reliably identify both airway structure and cavitation from CT scans, enabling automatic extraction of features relative to clinical applications. We retrospectively tested our computer-aided system on 40 human CT scans and a longitudinal study of TB with 12 rabbits for 7 weeks. The airway identification algorithm was found to be among the best of state-of-the-art methods, in accuracy and time-cost. For airway wall estimation, dice similarity coefficients (DSC) were found to be 73.87% and 81.42%, with respect to the surrogate truths (defined by radiologist visual analysis); inter-observer agreement was found to be 77.61%. For cavity detection and delineation, DSC was found to be over 95%. Our experimental results confirm that cavities are formed within the vicinity of the airway structure, with a mean distance of 1.59 mm; volume change could be statistically integrated with clinical variables to improve mathematical modeling of the disease when identifying potentially important biomarkers. FDA/CBER Gupta, Charu Postdoctoral Fellow Metabolomics/Proteomics Identification of biomarkers of Trypanosoma cruzi infection in blood using aptamer technology and proteomics Most individuals infected with the blood borne protozoan parasite Trypanosoma cruzi (T. cruzi), causing Chagas disease, are not aware of their chronic infection. This causes a threat to blood supplies because T. cruzi can be transmitted by blood transfusion. Current antibody-based tests have issues of cross reactivity and sensitivity, suggesting a need for an alternative and sensitive non-antibody based assay for T. cruzi diagnostic and blood donors screening. We have developed an aptamer-based assay for the detection of T. cruzi biomarkers in blood. Aptamers are small single stranded nucleic acid molecules which bind to their target based on shape recognition. We selected RNA aptamers against T. cruzi excreted/secreted antigens (TESA) from a library of 10 superscript 12 (10^12) sequences (170 nucleotide long) using a SELEX (Systematic Evolution of Ligands by Exponential Enrichment) strategy. Aptamers were used as T. cruzi specific ligands in an Enzyme Linked Aptamer (ELA) assay and were selected for their ability to bind TESA and react with T. cruzi infected mice plasma. Aptamer L44 showing high binding affinity and specificity to TESA also reacted specifically with T. cruzi infected mice plasma, in the ELA assay. We used a proteomics based approach to identify the target (biomarker) of aptamer L44. Whole cell extracts of T. cruzi (Tulahuen strain) trypomastigotes, were fractionated using chromatofocusing and the fractions were tested using Apt-L44 ELA assay. Positive and negative fractions were selected and digested with trypsin. The peptides generated were analyzed by 2D nano-LCMS/MS. A comparison of top hits, having zero false positive rates, from ELA positive and negative fractions, resulted in 13 proteins as possible targets of Apt-L44. Of those, 6 were reported to be secreted by some trypanosomes and other parasites. To identify the true target from these candidates, recombinant proteins will be made and assessed for their Apt-L44 binding activity. This biomarker will be used further to generate additional aptamers that will be used to modify the current ELA assay into a quantitative sandwich aptamer-based diagnostic test. FDA/CBER Fortes de Araujo, Fernanda Postdoctoral Fellow Molecular Biology - Eukaryotic Deletion of endonuclease G in Leishmania mexicana leads to disruption in mitochondrial homeostasis and abolition of immunopathology in Balb/C mice Cutaneous Leishmaniasis is a disease caused by the blood borne human protozoan parasite Leishmania. There are no effective vaccines against cutaneous leishmaniasis which is a serious public health problem. Previous vaccination approaches using heat-killed parasites, recombinant protein(s) and DNA vaccines have not been successful in humans. In contrast, genetically attenuated parasites (GAP) allow for interaction between a broad repertoire of parasitic antigens and the host immune system that is essential for the development of protective immunity. An ideal GAP vaccine would allow limited replication of the parasites in the host thus facilitating development of robust immunity and importantly cause no pathology. Endonuclease G (endoG) is a mitochondrial nuclease implicated in apoptosis. Recent reports showed endoG function as a determinant of the mitochondrial functions in humans. Since mitochondrial activities are essential for amastigote (the stage of Leishmania parasites in humans) survival, we investigated if deletion of endoG from Leishmania mexicana results in impaired mitochondrial activities and thus strong attenuation in parasite virulence. Results showed that deletion of endoG by homologous recombination (endoG-/-) leads to a significant growth reduction of amastigotes in vitro. This reduction was accompanied by impaired mitochondrial functions including reduced mitochondrial respiration, impaired lipid metabolism and reduced ATP production. Furthermore, deletion of endoG led to altered karyotype, such as a high proportion of abnormal, multinucleated amastigotes indicating defects in the cell cycle progression. Importantly, EM studies revealed the presence of large lipid bodies in endoG-/- mutants indicating impairment in macro- autophagy which regulates lipid metabolism. On the other hand, infection experiments with sand flies, arthropod vectors of Leishmania parasites, showed poor differentiation of endoG-/- mutants into infective metacyclic parasites. Autophagy is known to be essential for metacyclogenesis and our sand fly infection results further supports that autophagy is impaired in endoG-/- mutants. Balb/C infection experiments showed a 6-log fold difference in the parasite burden (endoG-/- versus wild type) and no pathology 10 weeks after the infection. These results demonstrate that deletion of endoG leads to a strong attenuation of virulence and endoG-/- mutants can be tested as a novel live attenuated Leishmania vaccine. FDA/CBER Dharmasena, Madushini Postdoctoral Fellow Molecular Biology - Prokaryotic Chromosomal Super-recombineering in Development of a Combined Oral Vaccine for Simultaneous Protection Against Both Typhoid Fever and Shigellosis Typhoid fever and shigellosis cause high morbidity and mortality worldwide, yet no anti-Shigella vaccine is currently available. A live, attenuated bacterial oral vaccine for typhoid fever, strain Ty21a, has been safely administered to > 200 million travelers/military worldwide and affords long-term protection (> 8 yrs) after 3 doses given over a 5-day period. We have utilized Ty21a as a safe, broad-based oral vaccine vector for expression of multiple foreign antigens to protect against shigellosis, anthrax, and plague. Shigella LPS is a potent vaccine antigen for serotype-specific protection against shigellosis. Previously, we separately cloned/expressed S. sonnei and S. dysenteriae 1 LPS’s from a low copy plasmid in Ty21a. The pGB-2 plasmid carrying Shigella LPS genes was stable during nonselective growth, but inexplicably lost stability upon removal of a selectable antibiotic-resistance gene. In this study, we have manipulated exisitng recombineering methods to insert 10-15 kb blocks of Shigella O-antigen-encoding genes into a targeted site within the Ty21a chromosome. The chromosomally-inserted Shigella LPS genes are 100% stable during subsequent growth for > 75 generations. Ty21a containing chromosomally-inserted S. sonnei O-antigen genes (i.e. Ty21a-Ss) simultaneously expressed both S.Typhi and S. sonnei O-antigens, elicited high serum antibody titers against both LPS’s in mice and protected mice against a virulent S. sonnei challenge. Separately, the S. dysenteriae 1 O-antigen genes were inserted into the Ty21a chromosome.
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