Faculty Bibliography

Following skeletal muscle injury, muscle stem cells (satellite cells) are activated, proliferate, and differentiate to form myofibers. We show that mRNA-decay protein AUF1 regulates satellite cell function through targeted degradation of specific mRNAs containing 3' AU-rich elements (AREs). auf1-/- mice undergo accelerated skeletal muscle wasting with age and impaired skeletal muscle repair following injury. Satellite cell mRNA analysis and regeneration studies demonstrate that auf1-/- satellite cell self-renewal is impaired due to increased stability and overexpression of ARE-mRNAs, including cell-autonomous overexpression of matrix metalloprotease MMP9. Secreted MMP9 degrades the skeletal muscle matrix, preventing satellite-cell-mediated regeneration and return to quiescence. Blocking MMP9 activity in auf1-/- mice restores skeletal muscle repair and maintenance of the satellite cell population. Control of ARE-mRNA decay by AUF1 represents a mechanism for adult stem cell regulation and is implicated in human skeletal muscle wasting diseases.

Aging leads to a proinflammatory state within the vasculature without disease, yet whether this inflammatory state occurs during atherogenesis remains unclear. Here, we examined how aging impacts atherosclerosis using Ldlr(-/-) mice, an established murine model of atherosclerosis. We found that aged atherosclerotic Ldlr(-/-) mice exhibited enhanced atherogenesis within the aorta. Aging also led to increased LDL levels, elevated blood pressure on a low-fat diet, and insulin resistance after a high-fat diet (HFD). On a HFD, aging increased a monocytosis in the peripheral blood and enhanced macrophage accumulation within the aorta. When we conducted bone marrow transplant experiments, we found that stromal factors contributed to age-enhanced atherosclerosis. To delineate these stromal factors, we determined that the vasculature exhibited an age-enhanced inflammatory response consisting of elevated production of CCL-2, osteopontin, and IL-6 during atherogenesis. In addition, in vitro cultures showed that aging enhanced the production of osteopontin by vascular smooth muscle cells. Functionally, aged atherosclerotic aortas displayed higher monocyte chemotaxis than young aortas. Hence, our study has revealed that aging induces metabolic dysfunction and enhances vascular inflammation to promote a peripheral monocytosis and macrophage accumulation within the atherosclerotic aorta.

BACKGROUND:Nipple reconstruction is the last stage in cosmetic reconstruction of the breast after mastectomy, but no method produces reliable and consistent aesthetic results. This study examined the use of the Biodesign Nipple Reconstruction Cylinder (NRC) during reconstruction of the nipple after mastectomy. METHODS:Patients with a history of breast cancer and mastectomy desiring nipple reconstruction were invited to participate. After obtaining consent, unilateral or bilateral nipple reconstruction was performed. Skin flaps were raised, the NRC was placed beneath the flaps as a stent, and the site was protected for up to 4 weeks with a nipple shield. Nipple projection was measured for 12 months after surgery. Patient satisfaction was measured and adverse events were recorded. Follow-up examinations were performed at 1 week, and then at 1, 3, 6, and 12 months after surgery. RESULTS:Eighty-two nipple reconstructions were performed in 50 patients. Related postoperative adverse events were minor, but reported in 8 reconstructions (9.8%) representing 7 patients (14.0%). Average projection at 6 and 12 months was 4.1 ± 1.6 mm and 3.8 ± 1.5 mm, respectively, compared with 10.5 ± 2.2 mm 1 week after surgery. Of patients completing the satisfaction questionnaire at 12 months, 70/75 (93.3%) of reconstructions were rated "pleased" or "very pleased" with the overall outcome. Overall, 45/46 (97.8%) patients would recommend nipple reconstruction to other women. CONCLUSIONS:The Biodesign NRC offers a safe alternative to nipple reconstruction, resulting in stable projection and a high level of patient satisfaction for 12 months after placement.

Helical reconstruction represents a convenient and powerful approach for structure determination of macromolecules that assemble into helical arrays. In the case of membrane proteins, formation of tubular crystals with helical symmetry represents an attractive alternative, especially when their small size precludes the use of single-particle analysis. An essential first step for helical reconstruction is to characterize the helical symmetry. This process is often daunting, due to the complexity of helical diffraction and to the low signal-to-noise ratio in images of individual assemblies. Furthermore, the large diameters of the tubular crystals produced by membrane proteins exacerbates the innate ambiguities that, if not resolved, will produce incorrect structures. In this report, we describe a set of tools that can be used to eliminate ambiguities and to validate the choice of symmetry. The first approach increases the signal-to-noise ratio along layer lines by incoherently summing data from multiple helical assemblies, thus producing several candidate indexing schemes. The second approach compares the layer lines from images with those from synthetic models built with the various candidate schemes. The third approach uses unit cell dimensions measured from collapsed tubes to distinguish between these candidate schemes. These approaches are illustrated with tubular crystals from a boron transporter from yeast, Bor1p, and a beta-barrel channel from the outer membrane of E. coli, OmpF.

BACKGROUND:Indications for nipple-sparing mastectomy (NSM) are expanding; however, high-risk patients have more ischemic complications. Surgical devascularization of the nipple-areolar complex (NAC) prior to NSM can reduce complications. This study reports perfusion patterns and complications in high-risk patients undergoing 2-stage NSM. METHODS:Surgical devascularization of the NAC was performed 3-6 weeks prior to NSM in 28 women. Risk factors included ptosis, obesity, smoking, prior breast surgery, and radiation. Using indocyanine green (ICG)-based fluorescence and an infrared camera, blood inflow was visualized intraoperatively. NAC perfusion patterns were classified as: V1, underlying breast; V2, surrounding skin; V3 = V1 + V2, or V4, capillary fill following devascularization. Ischemic complications were analyzed. RESULTS:Baseline perfusion for 54 breasts was 35 % V1, 32 % V2, and 33 % V3. Increasing ptosis was associated with V1 pattern: 86 % for grade 3, 31 % for grade 2, and 18 % for grade 1. Postdevascularization epidermolysis was observed in 63 % of V1 baseline, 41 % of V2, and 22 % of V3 (P = .042) and after NSM in 26 % for V1, 7 % for V2, and 6 % for V3 (P = .131). Ptosis was significantly associated with epidermolysis postdevascularization (P = .002) and NSM (P = .002). Smoking and BMI ≥30 were related to increased ischemic complications. Two or more risk factors were associated with postdevascularization ischemic changes (P = .026), but were not significant after NSM. Nipple loss was not observed, but 2 patients underwent partial areolar resection. CONCLUSION/CONCLUSIONS:Adaptive circulatory changes after devascularization allow tissues to tolerate the additional ischemic challenge of mastectomy. Our findings support extending 2-staged operations to high-risk women previously considered unsuitable for NSM.

The misfolding and accumulation of the protein fragment beta-amyloid (Abeta) is an early and essential event in the pathogenesis of Alzheimer's disease (AD). Despite close biological similarities among primates, humans appear to be uniquely susceptible to the profound neurodegeneration and dementia that characterize AD, even though nonhuman primates deposit copious Abeta in senile plaques and cerebral amyloid-beta angiopathy as they grow old. Because the amino acid sequence of Abeta is identical in all primates studied to date, we asked whether differences in the properties of aggregated Abeta might underlie the vulnerability of humans and the resistance of other primates to AD. In a comparison of aged squirrel monkeys (Saimiri sciureus) and humans with AD, immunochemical and mass spectrometric analyses indicate that the populations of Abeta fragments are largely similar in the 2 species. In addition, Abeta-rich brain extracts from the brains of aged squirrel monkeys and AD patients similarly seed the deposition of Abeta in a transgenic mouse model. However, the epitope exposure of aggregated Abeta differs in sodium dodecyl sulfate-stable oligomeric Abeta from the 2 species. In addition, the high-affinity binding of 3H Pittsburgh Compound B to Abeta is significantly diminished in tissue extracts from squirrel monkeys compared with AD patients. These findings support the hypothesis that differences in the pathobiology of aggregated Abeta among primates are linked to post-translational attributes of the misfolded protein, such as molecular conformation and/or the involvement of species-specific cofactors.

INTRODUCTION:Epidemiologic studies have demonstrated an association between diabetes and dementia. Insulin signaling within the brain, in particular within the hypothalamus regulates carbohydrate, lipid, and branched chain amino acid (BCAA) metabolism in peripheral organs such as the liver and adipose tissue. We hypothesized that cerebral amyloidosis impairs central nervous system control of metabolism through disruption of insulin signaling in the hypothalamus, which dysregulates glucose and BCAA homeostasis resulting in increased susceptibility to diabetes. METHODS:We examined whether APP/PS1 mice exhibit increased susceptibility to aging or high-fat diet (HFD)-induced metabolic impairment using metabolic phenotyping and insulin-signaling studies. RESULTS:APP/PS1 mice were more susceptible to high-fat feeding and aging-induced metabolic dysregulation including disrupted BCAA homeostasis and exhibited impaired hypothalamic insulin signaling. DISCUSSION:Our data suggest that AD pathology increases susceptibility to diabetes due to impaired hypothalamic insulin signaling, and that plasma BCAA levels could serve as a biomarker of hypothalamic insulin action in patients with AD.

Identification and characterization of molecular mechanisms that connect genetic risk factors to initiation and evolution of disease pathophysiology represent major goals and opportunities for improving therapeutic and diagnostic outcomes in Alzheimer's disease (AD). Integrative genomic analysis of the human AD brain transcriptome holds potential for revealing novel mechanisms of dysfunction that underlie the onset and/or progression of the disease. We performed an integrative genomic analysis of brain tissue-derived transcriptomes measured from two lines of mice expressing distinct mutant AD-related proteins. The first line expresses oligomerogenic mutant APP(E693Q) inside neurons, leading to the accumulation of amyloid beta (Abeta) oligomers and behavioral impairment, but never develops parenchymal fibrillar amyloid deposits. The second line expresses APP(KM670/671NL)/PSEN1(Deltaexon9) in neurons and accumulates fibrillar Abeta amyloid and amyloid plaques accompanied by neuritic dystrophy and behavioral impairment. We performed RNA sequencing analyses of the dentate gyrus and entorhinal cortex from each line and from wild-type mice. We then performed an integrative genomic analysis to identify dysregulated molecules and pathways, comparing transgenic mice with wild-type controls as well as to each other. We also compared these results with datasets derived from human AD brain. Differential gene and exon expression analysis revealed pervasive alterations in APP/Abeta metabolism, epigenetic control of neurogenesis, cytoskeletal organization and extracellular matrix (ECM) regulation. Comparative molecular analysis converged on FMR1 (Fragile X Mental Retardation 1), an important negative regulator of APP translation and oligomerogenesis in the post-synaptic space. Integration of these transcriptomic results with human postmortem AD gene networks, differential expression and differential splicing signatures identified significant similarities in pathway dysregulation, including ECM regulation and neurogenesis, as well as strong overlap with AD-associated co-expression network structures. The strong overlap in molecular systems features supports the relevance of these findings from the AD mouse models to human AD.

The wide range in human skin color results from varying levels of the pigment melanin. Genetic mechanisms underlying coloration differences have been explored, but identified genes do not account for all variation seen in the skin color spectrum. Post-transcriptional and post-translational regulation of factors that determine skin color, including melanin synthesis in epidermal melanocytes, melanosome transfer to keratinocytes and melanosome degradation, is also critical for pigmentation. We therefore investigated proteins that are differentially expressed in melanocytes derived from either White or African American (AA) skin. Two dimensional gel electrophoresis (2-DGE) and mass spectrometry demonstrated that Heat Shock Protein 70-1A (Hsp70-1A) protein levels were significantly higher in AA melanocytes compared to White melanocytes. Hsp70-1A expression significantly correlated with levels of tyrosinase, the rate-limiting melanogenic enzyme, consistent with a proposed role for Hsp70-family members in tyrosinase post-translational modification. Additionally, pharmacologic inhibition and siRNA-mediated down-regulation of Hsp70-1A correlated with pigmentation changes in cultured melanocytes, modified human skin substitutes and ex vivo skin. Furthermore, Hsp70-1A inhibition led to increased autophagy-mediated melanosome degradation in keratinocytes. Our data thus reveal that epidermal Hsp70-1A contributes to the diversity of skin color by regulating the amount of melanin synthesized in melanocytes and modulating autophagic melanosome degradation in keratinocytes.

In visceral leishmaniasis (VL), it is well-known that a patient in clinical remission of VL remains immune to reinfection, which provides a rationale for the feasibility of a vaccine against this deadly disease. In earlier studies, observation of significant cellular responses in treated Leishmania patients as well as in hamsters against leishmanial antigens from different fractions led to its further proteomic characterization, wherein S-Adenosyl-L-Homocysteine Hydrolase (AdoHcy) was identified as Th1 stimulatory protein. The present study includes immunological characterization of this protein, its cellular responses (lymphoproliferation, NO production and cytokine responses) in treated Leishmania infected hamsters and patients as well as prophylactic efficacy against Leishmania challenge in hamsters and the immune responses generated thereof. Significantly higher cellular responses were noticed against recombinant L. donovani S-Adenosyl-L-Homocysteine Hydrolase (rLdAdoHcy) as compared to soluble L. donovani antigen in treated samples. Moreover, stimulation of peripheral blood mononuclear cells with rLdAdoHcy up-regulated the levels of IFN-gamma, IL-12 and down-regulated IL-10. Furthermore, vaccination with rLdAdoHcy generated perceptible delayed type hypersensitivity response and exerted considerably good prophylactic efficacy ( approximately 70% inhibition) against L. donovani challenge. The efficacy was confirmed by the increased expression levels of inducible NO synthase and Th1-type cytokines, IFN-gamma and IL-12 and down-regulation of IL-4, IL-10 and TGF-beta. The results indicate towards the potentiality of rLdAdoHcy protein as a suitable vaccine candidate against VL