Faculty Bibliography

Human infants, like non-human primates, can breathe and swallow simultaneously, due to similarities in the anatomy of the aerodigestive tract (ADT). Morphometric studies lack standardized methods to investigate the descent of the hyolaryngeal apparatus in relation to the skull base and bony Eustachian tube - a process taking place throughout the first three years of postnatal life. This study tests the hypothesis that 3D landmark coordinates taken on sagittal CT scans can be reliably used to assess ADT and craniofacial structures via 3D geometric morphometrics. We used the computer program Osirix to identify 14 landmarks on head and neck sagittal CT scans obtained from 22 monkeys (Macaca mulatta) as a model to test accuracy. After four training sessions on images apart from the study sample, two observers assessed the study sample. Each observer performed three assessments per specimen and no specimen was evaluated twice in the same day. We used Generalized Procrustes Analysis, involving optimal superimposition and scaling by Centroid Size, and measured the procrustes distances to interpret data. Inter-observer errors were significantly high (> 5%), indicating that plotting landmarks on sagittal CTs allows for low reproducibility of measurements via 3D analysis. ANOVA of inter-observer distances per landmark yielded a significant result (p<0.0001), indicating that some landmarks are less reliable, and a series of t-tests with Bonferroni-Dunn adjustment revealed that the craniometric point Prosthion was the least accurate (p<0.05). Tracking the descent of the hyolaryngeal complex relative to craniofacial structures in early postnatal life requires a careful methodological approach. This study indicates that using 3D landmarks obtained from CT sagittal slices to ascertain measurements of the hyoid bone and craniofacial structures situated in different planes yields intrinsic errors of measurements that may jeopardize reproducibility of geometric morphometric analysis. Next step of this project is to use volume rendered 3D CT scans and collect coordinates for the same landmarks to determine interobserver variability, assess accuracy and validate reproducibility

Cardiolipin is a specific mitochondrial phospholipid that has a high affinity for proteins and that stabilizes the assembly of supercomplexes involved in oxidative phosphorylation. We found that sequestration of cardiolipin in protein complexes is critical to protect it from degradation. The turnover of cardiolipin is slower by almost an order of magnitude than the turnover of other phospholipids. However, in subjects with Barth syndrome, cardiolipin is rapidly degraded via the intermediate monolyso-cardiolipin. Treatments that induce supercomplex assembly decrease the turnover of cardiolipin and the concentration of monolyso-cardiolipin, whereas dissociation of supercomplexes has the opposite effect. Our data suggest that cardiolipin is uniquely protected from normal lipid turnover by its association with proteins, but this association is compromised in subjects with Barth syndrome, leading cardiolipin to become unstable, which in turn causes the accumulation of monolyso-cardiolipin.

OBJECTIVES: Cartilage is a highly mechano-responsive tissue. Chondrocytes undergo a series of complex changes, including proliferation and metabolic alteration as the target of external biomechanical and biochemical stimuli. IL-1beta is known to regulate chondrocyte metabolism and play an important role in the pathogenesis of osteoarthritis. The objective of this study was to employ low-intensity pulsed ultrasound (LIPUS) as a localized mechanical stimulus and assess its effects on chondrocyte migration, proliferation, metabolism, and differentiation, as well as its ability to suppress IL-1beta mediated catabolism in cartilage. METHODS: Human cartilage explants and chondrocytes were stimulated by LIPUS in presence and absence of of IL-1beta to asses cartilage degradation, chondrocytes metabolism, migration and proliferation. Western blot analyses were conducted to study IL-1beta the associated NFkappaB pathway in chondrocytes. RESULTS: LIPUS stimulation increased the proteoglycan content in human cartilage explants and inhibited IL-1beta induced loss of proteoglycans. LIPUS stimulation increased rates of chondrocyte migration and proliferation, and promoted chondrogenesis in mesenchymal stem cells. Further, LIPUS suppressed IL-1beta induced activation of phosphorylation of NFkappaB-p65 and IkBalpha leading to reduced expression of MMP13 and ADAMT5 in chondrocytes. CONCLUSIONS: Collectively, these data demonstrate the potential therapeutic effects of LIPUS in preventing cartilage degradation and treating osteoarthritis via a mechanical stimulation that inhibits the catabolic action of IL-1beta and stimulates chondrocyte migration, proliferation, and differentiation.

Advances in financial engineering are radically reshaping the biomedical marketplace. For instance, new methods of pooling diversified drug development programs by placing them in a special purpose vehicle (SPV) have been proposed to create a securitized cancer megafund allowing for debt and equity participation. In this study, we perform theoretical and numerical simulations that highlight the role of empirical validation of the projects comprising a cancer megafund. We quantify the degree to which the deliberately designed structure of derivatives and investments is key to its liquidity. Research megafunds with comprehensive in silico and laboratory validation protocols and ability to issue both debt, and equity as well as hybrid financial products may enable conservative investors including pension funds and sovereign government funds to profit from unique securitization opportunities. Thus, while hedging investor's longevity risk, such well-validated megafunds will contribute to health, well being and longevity of the global population.

In the endeavor to associate genetic variation with complex traits, closely related taxa are particularly fruitful for understanding the neurophysiological and genetic underpinnings of species-specific attributes. Similarity to humans has motivated research into nonhuman primate models, yet few studies of wild primates have investigated immediate causal factors of evolutionarily diverged social behaviors. Neurotransmitter differences have been invoked to explain the distinct behavioral suites of two baboon species in Awash, Ethiopia, which differ markedly in social behavior despite evolutionary propinquity. With this natural experiment, we test the hypothesis that genomic regions associated with monoamine neurotransmitters would be highly differentiated, and we identify a dopamine pathway as an outlier, highlighting the system as a potential cause of species-specific social behaviors. Dopamine levels and resultant variation in impulsivity were likely under differential selection in the species due to social system structure differences, with either brash or circumspect social behavior advantageous to secure mating opportunities depending on the social backdrop. Such comparative studies into the causes of the behavioral agendas that create and interact with social systems are of particular interest, and differences in temperament related to boldness and associated with dopamine variation likely played important roles in the evolution of all social, behaviorally complex animals, including baboons and humans.

Background: Immune cells, particularly myeloid-derived ones, play a pivotal role in the microenvironment of breast cancer. Because of the high diagnostic and therapeutic values of these immune cells, they have been extensively investigated, mostly invasively. Therefore, non-invasive breast cancer immune cell imaging methods can have great impact on diagnosis, disease management, and evaluation of therapy. Here, we describe the development of a high-density lipoprotein (HDL) -based positron emission tomography (PET) nano-tracer to noninvasively image immune cells in a breast cancer model. Methods: Radiolabeled HDL-based nano-tracers were developed by using two different approaches that incorporated the long-lived positron-emitting nuclide ⁸⁹Zr into HDL. The nano-tracers are composed of the phospholipid DMPC and apolipoprotein A-I (apoA-I) in a 2.5 : 1 weight ratio. DFO chelators, conjugated to either phospholipids or apoA-I proteins, were used to complex with ⁸⁹Zr to generate ⁸⁹Zr-PL-HDL (phospholipid-labeled) or ⁸⁹Zr-AI-HDL (apoA-1- labeled). In vivo evaluation was carried out in an orthotropic mouse model of breast cancer and included pharmacokinetic analysis, biodistribution studies, and PET imaging. Ex vivo radioautography and histology analyses of tumor tissues were performed to assess regional distribution of the nano-tracers. Fluorescent analogs of the nanotracers were used to determine cell-targeting specificity by using flow cytometry. Results: ⁸⁹Zr-PL-HDL (phospholipid-labeled) was produced in 79 +/- 13% (n = 6) radiochemical yield; ⁸⁹Zr-AI-HDL (apoA-I-labeled), 94 +/- 6% (n = 6). Both nano-tracers had at least 99% radiochemical purity. Intravenous administration of both nano-tracers resulted in high tumor radioactivity accumulation (16.5 +/- 2.8 %ID/g for ⁸⁹Zr-PL-HDL and 8.6 +/- 1.3 %ID/g for ⁸⁹Zr-AI-HDL) at 24 hours post injection. Radioautography and histology analyses showed high colocalization of radioactivity with macrophage-rich areas in tumors. Flow cytometry revealed high accumulation of the nano-tracers in myeloid-derived immune cells (preferentially in tumor-associated macrophages and monocytes, followed by dendritic cells and neutrophils), whereas low uptake was observed in endothelial cells and tumor cells (n = 4). Conclusions: Based on natural HDL particles, we have developed immune cell-targeting PET nano-tracers. In an orthotropic mouse model of breast cancer, we have demonstrated their specificity for myeloid-derived immune cells. Quantitative immune cell PET imaging with our ⁸⁹Zr-PET nano-tracers could be valuable for non-invasive diagnosis of breast cancer and evaluation of immunotherapy response. (Figure Presented)

In 1943, Luria and Delbruck used a phage-resistance assay to establish spontaneous mutation as a driving force of microbial diversity. Mutation rates are still studied using such assays, but these can only be used to examine the small minority of mutations conferring survival in a particular condition. Newer approaches, such as long-term evolution followed by whole-genome sequencing, may be skewed by mutational 'hot' or 'cold' spots. Both approaches are affected by numerous caveats. Here we devise a method, maximum-depth sequencing (MDS), to detect extremely rare variants in a population of cells through error-corrected, high-throughput sequencing. We directly measure locus-specific mutation rates in Escherichia coli and show that they vary across the genome by at least an order of magnitude. Our data suggest that certain types of nucleotide misincorporation occur 104-fold more frequently than the basal rate of mutations, but are repaired in vivo. Our data also suggest specific mechanisms of antibiotic-induced mutagenesis, including downregulation of mismatch repair via oxidative stress, transcription-replication conflicts, and, in the case of fluoroquinolones, direct damage to DNA.

Background: Chronic traumatic encephalopathy (CTE) is a neurodegenerative disorder resulting from repetitive mild brain trauma. Currently, the definite diagnosis of CTE is established postmortem, and diagnosis in life is complicated by symptom overlap with Alzheimer's disease (AD) and the increased risk of developing AD after repeated head injuries. We aim to determine whether neuropsychological testing and neuroimaging can provide biomarkers for diagnosing CTE in vivo. Methods: This is the clinical case of a 39-year-old retired National Football League player with a history of 22 concussions and cognitive complaints. Evaluation included neurologic and neuropsychological assessment, structural MRI, [18F]florbetapir amyloid positron emission tomography (PET) imaging, and experimental tau PET imaging with [18F]T807. Additional neuropsychological data from 2010 and a structural MRI from 2011 enabled longitudinal analysis of neuropsychological performance, cortical thickness, and subcortical volumes. Results: Cognitive performance declined from 2010-2015, especially in the domains of executive functioning, verbal fluency, and fine motor skills. Performance was below average on a naming task but was average or higher in other memory and language tests. In longitudinal structural analysis, left Broca's area and medial orbitofrontal cortex, left lateral temporal areas, and the left basal ganglia showed greatest volume losses (more than 2%), with apparent sparing of medial temporal lobe structures. PET imaging was negative for amyloid but revealed possible multifocal [18F]T807 retention, consistent with postmortem patterns of tau deposition in CTE at the junction of cortical grey matter and white matter. Conclusions: Although the definitive identification of the neuropathological retention of [18F]T807 requires postmortem correlation, our data suggest that [18F]T807 may inform future diagnostic criteria for CTE in living patients and help develop predictive biomarkers for specifying CTE from AD

The basolateral recycling and transcytotic pathways of epithelial cells were previously defined using markers such as transferrin (TfR) and polymeric IgA (pIgR) receptors. In contrast, our knowledge of the apical recycling pathway remains fragmentary. Here we utilize quantitative live-imaging and mathematical modelling to outline the recycling pathway of Megalin (LRP-2), an apical receptor with key developmental and renal functions, in MDCK cells. We show that, like TfR, Megalin is a long-lived and fast-recycling receptor. Megalin enters polarized MDCK cells through segregated apical sorting endosomes and subsequently intersects the TfR and pIgR pathways at a perinuclear Rab11-negative compartment termed common recycling endosomes (CRE). Whereas TfR recycles to the basolateral membrane from CRE, Megalin, like pIgR, traffics to subapical Rab11-positive apical recycling endosomes (ARE) and reaches the apical membrane in a microtubule- and Rab11-dependent manner. Hence, Megalin defines the apical recycling pathway of epithelia, with CRE as its apical sorting station.

DNA polymerase theta (Poltheta) promotes insertion mutations during alternative end-joining (alt-EJ) by an unknown mechanism. Here, we discover that mammalian Poltheta transfers nucleotides to the 3' terminus of DNA during alt-EJ in vitro and in vivo by oscillating between three different modes of terminal transferase activity: non-templated extension, templated extension in cis, and templated extension in trans. This switching mechanism requires manganese as a co-factor for Poltheta template-independent activity and allows for random combinations of templated and non-templated nucleotide insertions. We further find that Poltheta terminal transferase activity is most efficient on DNA containing 3' overhangs, is facilitated by an insertion loop and conserved residues that hold the 3' primer terminus, and is surprisingly more proficient than terminal deoxynucleotidyl transferase. In summary, this report identifies an unprecedented switching mechanism used by Poltheta to generate genetic diversity during alt-EJ and characterizes Poltheta as among the most proficient terminal transferases known.