Faculty Bibliography

Induced pluripotent stem (iPS) cells can be generated using retroviral vectors expressing Oct4, Klf4, Sox2, and cMyc. Most prior studies have required multiple retroviral vectors for reprogramming, resulting in high numbers of genomic integrations in iPS cells and limiting their use for therapeutic applications. Here we describe the use of a single lentiviral vector expressing a 'stem cell cassette' composed of the four transcription factors and a combination of 2A peptide and internal ribosome entry site technology, generating iPS cells from postnatal fibroblasts. iPS cells generated in this manner display embryonic stem cell-like morphology, express stem cell markers, and exhibit in vivo pluripotency, as evidenced by their ability to differentiate in teratoma assays and their robust contribution to mouse chimeras. Combining all factors into a single transcript achieves the most efficient reprogramming system to date and allows derivation of iPS cells with a single viral integration. The use of a single lentiviral vector for reprogramming represents a powerful laboratory tool and a significant step toward the application of iPS technology for clinical purposes

Atherosclerosis is the leading cause of death worldwide. To date, the use of statins to lower LDL levels has been the major intervention used to delay or halt disease progression. These drugs have an incomplete impact on plaque burden and risk, however, as evidenced by the substantial rates of myocardial infarctions that occur in large-scale clinical trials of statins. Thus, it is hoped that by understanding the factors that lead to plaque regression, better approaches to treating atherosclerosis may be developed. A transplantation-based mouse model of atherosclerosis regression has been developed by allowing plaques to form in a model of human atherosclerosis, the apoE-deficient mouse, and then placing these plaques into recipient mice with a normolipidemic plasma environment. Under these conditions, the depletion of foam cells occurs. Interestingly, the disappearance of foam cells was primarily due to migration in a CCR7-dependent manner to regional and systemic lymph nodes after 3 days in the normolipidemic (regression) environment. Further studies using this transplant model demonstrated that liver X receptor and HDL are other factors likely to be involved in plaque regression. In conclusion, through the use of this transplant model, the process of uncovering the pathways regulating atherosclerosis regression has begun, which will ultimately lead to the identification of new therapeutic targets

OBJECTIVE: To develop a rapid, high-throughput screening method of gene suspension array technique to simultaneously detect five bioterrorism bacteria: Bacillus anthracis, Francisella tularensis, Yersinia pestis, Brucella spp. and Burkholderia pseudomallei. METHODS: Highly validated specific primers were used to amplify diagnostic regions unique to each pathogen. Biotin labelled PCR products were hybridized to corresponding probes coupling on the unique sets of fluorescent beads. The hybridized beads were processed through the Bio-plex, which identified the presence of PCR products. RESULTS: Multiplex PCR-suspension array can detect five bioterrorism bacteria correctly with high specificity and high sensitivity, the results suggest the utility of suspension array system for high-throughput screening of bioterrorism samples. CONCLUSION: A multiplex PCR-suspension array for rapid detection of five bioterrorism bacteria was established.

Caspofungin is used for the treatment of acute invasive candidiasis and as salvage treatment for invasive aspergillosis. We report characteristics of isolates of Candida albicans and Aspergillus fumigatus detected in a patient with breakthrough infection complicating severe gastrointestinal surgery and evaluate the capability of susceptibility methods to identify candin resistance. The susceptibility of C. albicans to caspofungin and anidulafungin was investigated by Etest, microdilution (European Committee on Antibiotic Susceptibility Testing [EUCAST] and CLSI), disk diffusion, agar dilution, and FKS1 sequencing and in a mouse model. Tissue was examined by immunohistochemistry, PCR, and sequencing for the presence of A. fumigatus and resistance mutations. The MICs for the C. albicans isolate were as follows: >32 microg/ml caspofungin and 0.5 microg/ml anidulafungin by Etest, 2 microg/ml caspofungin and 0.125 microg/ml anidulafungin by EUCAST methods, and 1 microg/ml caspofungin and 0.5 microg/ml anidulafungin by CLSI methods. Sequencing of the FKS1 gene revealed a mutation leading to an S645P substitution. Caspofungin and anidulafungin failed to reduce kidney CFU counts in animals inoculated with this isolate (P > 0.05 compared to untreated control animals), while both candins completely sterilized the kidneys in animals infected with a control isolate. Disk diffusion and agar dilution methods clearly separated the two isolates. Immunohistochemistry and sequencing confirmed the presence of A. fumigatus without FSK1 resistance mutations in liver and lung tissues. Breakthrough disseminated aspergillosis and candidiasis developed despite an absence of characteristic FKS1 resistance mutations in the Aspergillus isolates. EUCAST and CLSI methodology did not separate the candin-resistant clinical isolate from the sensitive control isolate as well as did the Etest and agar methods.

Classic tissue engineering paradigms are limited by the incorporation of a functional vasculature and a reliable means for reimplantation into the host circulation. We have developed a novel approach to overcome these obstacles using autologous explanted microcirculatory beds (EMBs) as bioscaffolds for engineering complex three-dimensional constructs. In this study, EMBs consisting of an afferent artery, capillary beds, efferent vein, and surrounding parenchymal tissue are explanted and maintained for 24 h ex vivo in a bioreactor that preserves EMB viability and function. Given the rapidly advancing field of stem cell biology, EMBs were subsequently seeded with three distinct stem cell populations, multipotent adult progenitor cells (MAPCs), and bone marrow and adipose tissue-derived mesenchymal stem cells (MSCs). We demonstrate MAPCs, as well as MSCs, are able to egress from the microcirculation into the parenchymal space, forming proliferative clusters. Likewise, human adipose tissue-derived MSCs were also found to egress from the vasculature and seed into the EMBs, suggesting feasibility of this technology for clinical applications. We further demonstrate that MSCs can be transfected to express a luciferase protein and continue to remain viable and maintain luciferase expression in vivo. By using the vascular network of EMBs, EMBs can be perfused ex vivo and seeded with stem cells, which can potentially be directed to differentiate into neo-organs or transfected to replace failing organs and deficient proteins

Excessive transforming growth factor-beta (TGF-beta) signaling characterizes the progression of aortic aneurysm in mouse models of Marfan syndrome, a systemic disorder of the connective tissue that is caused by mutations in the gene encoding the extracellular matrix protein fibrillin-1. Fibrillin-1 mutations are believed to promote abnormal Smad2/3 signaling by impairing the sequestration of latent TGF-beta complexes into the extracellular matrix. Here we report that promiscuous Smad2/3 signaling is the cell-autonomous phenotype of primary cultures of vascular smooth muscle cells (VSMC) explanted from the thoracic aortas of Fbn1 mutant mice with either neonatal onset or progressively severe aortic aneurysm. This cellular phenotype was characterized in VSMC isolated from Fbn1-null (mgN/mgN) mice, which recapitulate the most severe form of Marfan syndrome. We found that loss of fibrillin-1 deposition promotes the production of intracellular reactive oxygen species and abnormal accumulation of phosphorylated TGF-beta-activated kinase 1 and p38 MAPK, in addition to increasing the levels of endogenous phospho-Smad2. We showed that improper Smad2/3 signaling in Fbn1-null VSMC is in part stimulated by phospho-p38 MAPK, which is in turn activated in response to signals other than those mediated by the kinase activity of the ALK5 receptor. Consistent with these cell culture data, in vivo analyses documented that phospho-p38 MAPK accumulates earlier than phospho-Smad2 in the aortic wall of mgN/mgN mice and that systemic inhibition of phospho-p38 MAPK activity lowers the levels of phospho-Smad2 in this tissue. Collectively, these findings indicate that improper activation of p38 MAPK is a precursor of constitutive Smad2/3 signaling in the aortic wall of a mouse model of neonatal lethal Marfan syndrome

Posttranscriptional regulation and RNA metabolism have become central topics in the understanding of mammalian gene expression and cell signalling, with the 3' untranslated region emerging as the coordinating unit. The 3' untranslated region trans-acting factor Hu protein R (HuR) forms a central posttranscriptional pathway node bridging between AU-rich element-mediated processes and microRNA regulation. While (m)RNA control by HuR has been extensively characterized, the molecular mode of action still remains elusive. Here we describe the identification of the first RRM3 (RNA recognition motif 3) targeted low molecular weight HuR inhibitors from a one-bead-one-compound library screen using confocal nanoscanning. A further compound characterization revealed the presence of an ATP-binding pocket within HuR RRM3, associated with enzymatic activity. Centered around a metal-ion-coordinating DxD motif, the catalytic site mediates 3'-terminal adenosyl modification of non-polyadenylated RNA substrates by HuR. These findings suggest that HuR actively contributes to RNA modification and maturation and thereby shed an entirely new light on the role of HuR in RNA metabolism.

Quantitative and qualitative alterations of mitochondrial cardiolipin have been implicated in the pathogenesis of Barth syndrome, an X-linked cardioskeletal myopathy caused by a deficiency in tafazzin, an enzyme in the cardiolipin remodeling pathway. We have generated and previously reported a tafazzin-deficient Drosophila model of Barth syndrome that is characterized by low cardiolipin concentration, abnormal cardiolipin fatty acyl composition, abnormal mitochondria, and poor motor function. Here, we first show that tafazzin deficiency in Drosophila disrupts the final stage of spermatogenesis, spermatid individualization, and causes male sterility. This phenotype can be genetically suppressed by inactivation of the gene encoding a calcium-independent phospholipase A(2), iPLA2-VIA, which also prevents cardiolipin depletion/monolysocardiolipin accumulation, although in wild-type flies inactivation of the iPLA2-VIA does not affect the molecular composition of cardiolipin. Furthermore, we show that treatment of Barth syndrome patients' lymphoblasts in tissue culture with the iPLA(2) inhibitor, bromoenol lactone, partially restores their cardiolipin homeostasis. Taken together, these findings establish a causal role of cardiolipin deficiency in the pathogenesis of Barth syndrome and identify iPLA2-VIA as an important enzyme in cardiolipin deacylation, and as a potential target for therapeutic intervention