Faculty Bibliography

The synthesis of proteins in the endoplasmic reticulum (ER) that exceeds the protein folding capacity of this organelle is a frequent cause of cellular dysfunction and disease. An example of such a disease is alpha-1-antitrypsin (A1AT) deficiency, caused by destabilizing mutations in this glycoprotein. It is considered that the mutant proteins are recognized in the ER by lectins and are subsequently degraded through the proteasome, leading to a deficiency in this enzyme in the afflicted patients. We previously established a Drosophila model of this disease by overexpressing the null Hong Kong (NHK) allele of this gene and found that the Drosophila lectin, ER degradation-enhancing alpha-mannosidase-like protein 2 (EDEM2), can accelerate the degradation of A1AT when overexpressed. NHK is a rare allele, and in this study, we investigated in depth the mechanisms through which Drosophila EDEMs affect the degradation of the Z variant, which is the predominant disease allele. Specifically, we report that the Z allele does not activate ER stress signaling as prominently as the NHK allele, but similarly requires both Drosophila EDEM1 and EDEM2 for the degradation of the protein. We demonstrate that EDEMs are required for their ubiquitination, and without EDEMs, glycosylated A1AT mutants accumulate in cells. These results support the role of the EDEM-mediated ubiquitination of the alpha-1-antitrypsin Z (ATZ) allele, and establish a Drosophila model for the study of this protein and disease.

Dalbavancin is an intravenous lipoglycopeptide with activity against Gram-positive pathogens and an MIC90 for Staphylococcus aureus of 0.06 mug/ml. With a terminal half-life of >14 days, dosing regimens with infrequent parenteral administration become available to treat infectious diseases such as osteomyelitis and endocarditis that otherwise require daily dosing for many weeks. In order to support a rationale for these novel regimens, the pharmacokinetics over an extended dosing interval and the distribution of dalbavancin into bone and articular tissue were studied in two phase I trials and pharmacokinetic modeling was performed. Intravenous administration of 1,000 mg of dalbavancin on day 1 followed by 500 mg weekly for seven additional weeks was well tolerated and did not demonstrate evidence of drug accumulation. In a separate study, dalbavancin concentrations in cortical bone 12 h after infusion of a single 1,000-mg intravenous infusion were 6.3 mug/g and 2 weeks later were 4.1 mug/g. A two-dose, once-weekly regimen that would provide tissue exposure over the dalbavancin MIC for Staphylococcus aureus for 8 weeks, maximizing the initial exposure to treatment while minimizing the frequency of intravenous therapy, is proposed.

BACKGROUND:: Fat graft volume retention remains highly unpredictable, but addition of adipose-derived stromal cells (ASCs) to fat grafts has been shown to improve retention. The present study aimed to investigate the mechanisms involved in ASC enhancement of fat grafting. METHODS:: ASCs isolated from human lipoaspirate were labeled with green fluorescent protein (GFP) and luciferase. Fat grafts enhanced with ASCs were injected into the scalp and bioluminescent imaging was performed to follow retention of ASCs within the fat graft. Fat grafts were also explanted at days 1, 5, and 10 post-grafting for ASC extraction and single-cell gene analysis. Finally, CD31 immunohistochemical staining was performed on fat grafts enriched with ASCs. RESULTS:: Bioluminescent imaging demonstrated significant reduction in luciferase+ ASCs within fat grafts at five days post-grafting. A similar reduction in viable GFP+ ASCs retrieved from explanted grafts was also noted. Single cell analysis revealed expression of multiple genes/markers related to cell survival and angiogenesis including BMPR2, CD90, CD105, FGF2, CD248, TGFss1, and VEGFA. Genes involved in adipogenesis were not expressed by ASCs. Finally, CD31 staining revealed significantly higher vascular density in fat grafts explanted at day 10 post-grafting. CONCLUSIONS:: Although ASC survival in the hypoxic graft environment decreases significantly over time, these cells provide multiple angiogenic growth factors. Therefore, improved fat graft volume retention with ASC enrichment may be due to improved graft vascularization.

Inflammation drives atherosclerotic plaque progression and rupture, and is a compelling therapeutic target. Consequently, attenuating inflammation by reducing local macrophage accumulation is an appealing approach. This can potentially be accomplished by either blocking blood monocyte recruitment to the plaque or increasing macrophage apoptosis and emigration. Because macrophage proliferation was recently shown to dominate macrophage accumulation in advanced plaques, locally inhibiting macrophage proliferation may reduce plaque inflammation and produce long-term therapeutic benefits. To test this hypothesis, we used nanoparticle-based delivery of simvastatin to inhibit plaque macrophage proliferation in apolipoprotein E deficient mice (Apoe-/- ) with advanced atherosclerotic plaques. This resulted in rapid reduction of plaque inflammation and favorable phenotype remodeling. We then combined this short-term nanoparticle intervention with an eight-week oral statin treatment, and this regimen rapidly reduced and continuously suppressed plaque inflammation. Our results demonstrate that pharmacologically inhibiting local macrophage proliferation can effectively treat inflammation in atherosclerosis.

Analysis of single molecules in living cells has provided quantitative insights into the kinetics of fundamental biological processes; however, the dynamics of messenger RNA (mRNA) translation have yet to be addressed. We have developed a fluorescence microscopy technique that reports on the first translation events of individual mRNA molecules. This allowed us to examine the spatiotemporal regulation of translation during normal growth and stress and during Drosophila oocyte development. We have shown that mRNAs are not translated in the nucleus but translate within minutes after export, that sequestration within P-bodies regulates translation, and that oskar mRNA is not translated until it reaches the posterior pole of the oocyte. This methodology provides a framework for studying initiation of protein synthesis on single mRNAs in living cells.