Faculty Bibliography

Cryo-electron tomography (cryoET) has become a powerful tool for direct visualization of 3D structures of native biological specimens at molecular resolution, but its application is limited to thin specimens (<300 nm). Recently, vitreous sectioning and cryoFIB milling technologies were developed to physically reduce the specimen thickness; however, cryoET analysis of membrane protein complexes within native cell membranes remains a great challenge. Here, we use phage ΦX174 lysis gene E to rapidly produce native, intact, bacterial cell membranes for high resolution cryoET. We characterized E gene-induced cell lysis using FIB/SEM and cryoEM and showed that the bacteria cytoplasm was largely depleted through spot lesion, producing ghosts with the cell membranes intact. We further demonstrated the utility of E-gene-induced lysis for cryoET using the bacterial chemotaxis receptor signaling complex array. The described method should have a broad application for structural and functional studies of native, intact cell membranes and membrane protein complexes.

Age and physiological status, like menopause, are risk factors for breast cancer. Less clear is what factors influence the diversity of breast cancer. In this study, we investigated the effect of host age on the distribution of tumor subtypes in mouse mammary chimera consisting of wild-type hosts and Trp53 nullizygous epithelium, which undergoes a high rate of neoplastic transformation. Wild-type mammary glands cleared of endogenous epithelium at 3 weeks of age were subsequently implanted during puberty (5 weeks) or at maturation (10 weeks) with syngeneic Trp53 null mammary tissue fragments and monitored for 1 year. Tumors arose sooner from adult hosts (AH) compared to juvenile hosts (JH). However, compared to AH tumors, JH tumors grew several times faster, were more perfused, exhibited a 2-fold higher mitotic index and were more highly positive for insulin-like growth factor receptor phosphorylation. Most tumors in each setting were ER positive (80% JH vs 70% AH) but JH tumors were significantly more ER immunoreactive (p=0.0001) than AH tumors. A differential expression signature (JvA) of juvenile versus adult tumors revealed a luminal transcriptional program. Centroids of the human homologs of JvA genes showed that JH tumors were more like luminal A tumors and AH tumors were more like luminal B tumors. Hierarchical clustering with the JvA human ortholog gene list segregated luminal A and luminal B breast cancers across data sets. These data support the notion that age-associated host physiology greatly influences the intrinsic subtype of breast cancer.

Densely ionizing radiation, which is present in the space radiation environment and used in radiation oncology, has potentially greater carcinogenic effect compared to sparsely ionizing radiation that is prevalent on earth. Here we used a radiation chimera in which mice were exposed to densely ionizing 350 MeV/amu Si-particles, gamma-radiation, or sham-irradiated and transplanted 3 days later with syngeneic Trp53 null mammary fragments. Trp53 null tumors arising in mice densely irradiated had a shorter median time to appearance and grew faster once detected compared to those in sham-irradiated or gamma-irradiated mice. Tumors were further classified by markers keratin 8/18 (K18, KRT18), keratin 14 (K14, KRT18) and estrogen receptor (ER, ESR1) and expression profiling. Most tumors arising in sham irradiated hosts were comprised of both K18 and K14 positive cells (K14/18) while those tumors arising in irradiated hosts expressed mostly K18. Keratin staining was significantly associated with ER status. K14/18 tumors were predominantly ER positive while K18 tumors were predominantly ER negative. Genes differentially expressed in K18 tumors compared to K14/18 tumor were associated with ERBB2 and KRAS, metastasis and loss of E-cadherin. Although K18 tumors tended to grow faster and be more metastatic than K14/18 tumors, K18 tumors in particle-irradiated mice grew significantly larger compared to controls and were more metastatic compared to sham-irradiated mice. An expression profile that distinguished K18 tumors arising in particle-irradiated compared sham-irradiated mice was enriched in mammary stem cell, stroma, and Notch signaling genes. These data suggest that the carcinogenic effects of densely ionizing radiation is mediated by the microenvironment, which elicits more aggressive tumors compared to similar tumors arising in sham-irradiated hosts.

We investigated H2S attenuation by dissimilatory perchlorate-reducing bacteria (DPRB). All DPRB tested oxidized H2S coupled to (per)chlorate reduction without sustaining growth. H2S was preferentially utilized over organic electron donors resulting in an enriched (34S)-elemental sulfur product. Electron microscopy revealed elemental sulfur production in the cytoplasm and on the cell surface of the DPRB Azospira suillum. Based on our results, we propose a novel hybrid enzymatic-abiotic mechanism for H2S oxidation similar to that recently proposed for nitrate-dependent Fe(II) oxidation. The results of this study have implications for the control of biosouring and biocorrosion in a range of industrial environments.

Mouse models have increased our understanding of the pathogenesis of medulloblastoma (MB), the most common malignant pediatric brain tumor that often forms in the cerebellum. A major goal of ongoing research is to better understand the early stages of tumorigenesis and to establish the genetic and environmental changes that underlie MB initiation and growth. However, studies of MB progression in mouse models are difficult due to the heterogeneity of tumor onset times and growth patterns and the lack of clinical symptoms at early stages. Magnetic resonance imaging (MRI) is critical for noninvasive, longitudinal, three-dimensional (3D) brain tumor imaging in the clinic but is limited in resolution and sensitivity for imaging early MBs in mice. In this study, high-resolution (100 mum in 2 hours) and high-throughput (150 mum in 15 minutes) manganese-enhanced MRI (MEMRI) protocols were optimized for early detection and monitoring of MBs in a Patched-1 (Ptch1) conditional knockout (CKO) model. The high tissue contrast obtained with MEMRI revealed detailed cerebellar morphology and enabled detection of MBs over a wide range of stages including pretumoral lesions as early as 2 to 3 weeks postnatal with volumes close to 0.1 mm(3). Furthermore, longitudinal MEMRI allowed noninvasive monitoring of tumors and demonstrated that lesions within and between individuals have different tumorigenic potentials. 3D volumetric studies allowed quantitative analysis of MB tumor morphology and growth rates in individual Ptch1-CKO mice. These results show that MEMRI provides a powerful method for early in vivo detection and longitudinal imaging of MB progression in the mouse brain.

We report the first systems biology investigation of regulators controlling arterial plaque macrophage transcriptional changes in response to lipid lowering in vivo in two distinct mouse models of atherosclerosis regression. Transcriptome measurements from plaque macrophages from the Reversa mouse were integrated with measurements from an aortic transplant-based mouse model of plaque regression. Functional relevance of the genes detected as differentially expressed in plaque macrophages in response to lipid lowering in vivo was assessed through analysis of gene functional annotations, overlap with in vitro foam cell studies, and overlap of associated eQTLs with human atherosclerosis/CAD risk SNPs. To identify transcription factors that control plaque macrophage responses to lipid lowering in vivo, we used an integrative strategy--leveraging macrophage epigenomic measurements--to detect enrichment of transcription factor binding sites upstream of genes that are differentially expressed in plaque macrophages during regression. The integrated analysis uncovered eight transcription factor binding site elements that were statistically overrepresented within the 5' regulatory regions of genes that were upregulated in plaque macrophages in the Reversa model under maximal regression conditions and within the 5' regulatory regions of genes that were upregulated in the aortic transplant model during regression. Of these, the TCF/LEF binding site was present in promoters of upregulated genes related to cell motility, suggesting that the canonical Wnt signaling pathway may be activated in plaque macrophages during regression. We validated this network-based prediction by demonstrating that beta-catenin expression is higher in regressing (vs. control group) plaques in both regression models, and we further demonstrated that stimulation of canonical Wnt signaling increases macrophage migration in vitro. These results suggest involvement of canonical Wnt signaling in macrophage emigration from the plaque during lipid lowering-induced regression, and they illustrate the discovery potential of an epigenome-guided, systems approach to understanding atherosclerosis regression.

OBJECTIVE: To investigate how epithelial mechanotransduction pathways impact wound repair. BACKGROUND: Mechanical forces are increasingly recognized to influence tissue repair, but their role in chronic wound pathophysiology remains unknown. Studies have shown that chronic wounds exhibit high levels of matrix metalloproteinase 9 (MMP9), a key proteolytic enzyme that regulates wound remodeling. We hypothesized that epithelial mechanosensory pathways regulated by keratinocyte-specific focal adhesion kinase (FAK) control dermal remodeling via MMP9. METHODS: A standard wound model was applied to keratinocyte-specific FAK knockout (KO) and control mice. Rates of wound healing were measured and tissue was obtained for histologic and molecular analyses. Transcriptional and immunoblot assays were used to assess the activation of FAK, intracellular kinases, and MMP9 in vitro. A cell suspension model was designed to validate the importance of FAK mechanosensing, p38, and MMP9 secretion in human cells. Biomechanical testing was utilized to evaluate matrix tensile properties in FAK KO and control wounds. RESULTS: Wound healing in FAK KO mice was significantly delayed compared with controls (closure at 15 days compared with 20 days, P = 0.0003). FAK KO wounds demonstrated decreased dermal thickness and collagen density. FAK KO keratinocytes exhibited overactive p38 and MMP9 signaling in vitro, findings recapitulated in human keratinocytes via the deactivation of FAK in the cell suspension model. Functionally, FAK KO wounds were significantly weaker and more brittle than control wounds, results consistent with the histologic and molecular analyses. CONCLUSIONS: Keratinocyte FAK is highly responsive to mechanical cues and may play a critical role in matrix remodeling via regulation of p38 and MMP9. These findings suggest that aberrant epithelial mechanosensory pathways may contribute to pathologic dermal proteolysis and wound chronicity.

Abstract Metabolic syndrome is a complex metabolic condition caused by abnormal adipose deposition and function, dyslipidemia and hyperglycemia, which affects >47 million American adults and approximately 1 million children. Individuals with the metabolic syndrome have essentially twice the risk for developing cardiovascular disease (CVD) and Type 2 diabetes mellitus (T2D), compared to those without the syndrome. In the search for improved and novel therapeutic strategies, microRNAs (miRNA) have been shown to be interesting targets due to their regulatory role on gene networks controlling different crucial aspects of metabolism, including lipid and glucose homeostasis. More recently, the discovery of circulating miRNAs suggest that miRNAs may be involved in facilitating metabolic crosstalk between organs as well as serving as novel biomarkers of diseases, including T2D and atherosclerosis. These findings highlight the importance of miRNAs for regulating pathways that underlie metabolic diseases, and their potential as therapeutic targets for the development of novel treatments.