Faculty Bibliography

Differences in epidermal melanin levels result in the wide variation in color associated with ethnic skin diversity. Ethnic differences result from variance in melanogenesis, melanin transfer, and melanosome degradation in the epidermis, but the mechanisms underlying these differences, and their contribution to the regulation of skin color, are not fully understood. In this study, we explored proteins which were differentially expressed between Caucasian- and African American donor skin- derived melanocytes using twodimensional gel electrophoresis and mass spectroscopy. We consequently identified an important role for Heat Shock Protein 70 (Hsp70 encoded by HSPA1A) in skin color determination. In contrast to a previous report suggesting negative regulation of melanogenesis, Hsp70 was found to be more abundant in melanocytes from African American donors compared to those from Caucasian donors, which also reflected protein expression in the skin. In particular, inhibition of Hsp70, using either a specific inhibitor or siRNA targeting, significantly decreased melanin content of African American donor melanocytes, consistent with previous reports that Hsp70 is an Endoplasmic Reticulum (ER) chaperone involved in post-translational modification of tyrosinase. Indeed, inhibition of Hsp70 caused a decrease in tyrosinase protein levels by affecting its maturation. Taken together, our data reveal that Hsp70 plays an essential role in regulation of melanogenesis and thereby contributes to the ethnic diversity of skin color

Effective skin regeneration therapies require a successful interface between progenitor cells and biocompatible delivery systems. We previously demonstrated the efficiency of a biomimetic pullulan-collagen hydrogel scaffold for improving bone marrow-derived mesenchymal stem cell survival within ischemic skin wounds by creating a "stem cell niche" that enhances regenerative cytokine secretion. Adipose-derived mesenchymal stem cells (ASCs) represent an even more appealing source of stem cells because of their abundance and accessibility, and in this study we explored the utility of ASCs for hydrogel-based therapies. To optimize hydrogel cell seeding, a rapid, capillary force-based approach was developed and compared with previously established cell seeding methods. ASC viability and functionality following capillary hydrogel seeding were then analyzed in vitro and in vivo. In these experiments, ASCs were seeded more efficiently by capillary force than by traditional methods and remained viable and functional in this niche for up to 14 days. Additionally, hydrogel seeding of ASCs resulted in the enhanced expression of multiple stemness and angiogenesis-related genes, including Oct4, Vegf, Mcp-1, and Sdf-1. Moving in vivo, hydrogel delivery improved ASC survival, and application of both murine and human ASC-seeded hydrogels to splinted murine wounds resulted in accelerated wound closure and increased vascularity when compared with control wounds treated with unseeded hydrogels. In conclusion, capillary seeding of ASCs within a pullulan-collagen hydrogel bioscaffold provides a convenient and simple way to deliver therapeutic cells to wound environments. Moreover, ASC-seeded constructs display a significant potential to accelerate wound healing that can be easily translated to a clinical setting.

BACKGROUND:: Scarring represents a significant biomedical burden in clinical medicine. Mechanomodulation has been linked to scarring through inflammation, but until now a systematic approach to attenuate mechanical force and reduce scarring has not been possible. METHODS:: We conducted a twelve month, prospective, open label, randomized, multi-center clinical trial to evaluate abdominoplasty scar appearance following post-operative treatment with the embrace device to reduce mechanical forces on healing surgical incisions. Incisions from 65 healthy adult subjects were randomized to receive embrace treatment on one half of an abdominoplasty incision and control treatment (surgeon's optimal care methods) on the other half. The primary endpoint for this study was the difference between assessments of the scar appearance for the treated and control sides using the Visual Analogue Scale (VAS) scar score. RESULTS:: Final twelve month study photos were obtained from 36 subjects who completed at least five weeks of dressing application. The mean VAS score for embrace-treated scars (2.90) was significantly improved compared to control-treated scars (3.29) at 12 months (difference = 0.39, 95% confidence interval [0.14, 0.66], p = 0.027). Both subjects and investigators found that embrace-treated scars demonstrated significant improvements in overall appearance at 12 months using the Patient and Observer Scar Assessment Scale (POSAS) evaluation (p = 0.02 and p < 0.001, respectively). No serious adverse events were reported. CONCLUSION:: These results demonstrate that the embrace device significantly reduces scarring following abdominoplasty surgery. To our knowledge, this represents the first level one evidence for post-operative scar reduction.

The mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and metabolism. It controls many cell functions by integrating nutrient availability and growth factor signals. Amino acids, and in particular leucine, are among the main positive regulators of mTORC1 signaling. The current model for the regulation of mTORC1 by amino acids involves the movement of mTOR to the lysosome mediated by the Rag-GTPases. Here, we have examined the control of mTORC1 signaling and mTOR localization by amino acids and leucine in serum-fed cells, because both serum growth factors (or, e.g., insulin) and amino acids are required for full activation of mTORC1 signaling. We demonstrate that mTORC1 activity does not closely correlate with the lysosomal localization of mTOR. In particular, leucine controls mTORC1 activity without any detectable modification of the lysosomal localization of mTOR, indicating that the signal(s) exerted by leucine is likely distinct from those exerted by other amino acids. In addition, knock-down of the Rag-GTPases attenuated the inhibitory effect of amino acid- or leucine-starvation on the phosphorylation of mTORC1 targets. Furthermore, data from cells where Rag expression has been knocked down revealed that leucine can promote mTORC1 signaling independently of the lysosomal localization of mTOR. Our data complement existing models for the regulation of mTORC1 by amino acids and provide new insights into this important topic.

Alex Schier looks back at the life and research of his graduate mentor and friend Walter Gehring.

Chromatin structure determines DNA accessibility. We compare nucleosome occupancy in mouse and human embryonic stem cells (ESCs), induced-pluripotent stem cells (iPSCs) and differentiated cell types using MNase-seq. To address variability inherent in this technique, we developed a bioinformatic approach to identify regions of difference (RoD) in nucleosome occupancy between pluripotent and somatic cells. Surprisingly, most chromatin remains unchanged; a majority of rearrangements appear to affect a single nucleosome. RoDs are enriched at genes and regulatory elements, including enhancers associated with pluripotency and differentiation. RoDs co-localize with binding sites of key developmental regulators, including the reprogramming factors Klf4, Oct4/Sox2 and c-Myc. Nucleosomal landscapes in ESC enhancers are extensively altered, exhibiting lower nucleosome occupancy in pluripotent cells than in somatic cells. Most changes are reset during reprogramming. We conclude that changes in nucleosome occupancy are a hallmark of cell differentiation and reprogramming and likely identify regulatory regions essential for these processes.

The unfolded protein response (UPR) - the endoplasmic reticulum stress response - is found in various pathologies including ischemia-reperfusion injury (IRI). However, its role during IRI is still unclear. Here, by combining two different bioinformatical methods - a method based on ordinary differential equations (Time Series Network Inference) and an algebraic method (probabilistic polynomial dynamical systems) - we identified the IRE1alpha-XBP1 and the ATF6 pathways as the main UPR effectors involved in cell's adaptation to IRI. We validated these findings experimentally by assessing the impact of their knock-out and knock-down on cell survival during IRI.

The exon junction complex (EJC) is a highly conserved ribonucleoprotein complex that binds RNAs during splicing and remains associated with them following export to the cytoplasm. While the role of this complex in mRNA localization, translation, and degradation has been well characterized, its mechanism of action in splicing a subset of Drosophila and human transcripts remains to be elucidated. Here, we describe a novel function for the EJC and its splicing subunit, RnpS1, in preventing transposon accumulation in both Drosophila germline and surrounding somatic follicle cells. This function is mediated specifically through the control of piwi transcript splicing, where, in the absence of RnpS1, the fourth intron of piwi is retained. This intron contains a weak polypyrimidine tract that is sufficient to confer dependence on RnpS1. Finally, we demonstrate that RnpS1-dependent removal of this intron requires splicing of the flanking introns, suggesting a model in which the EJC facilitates the splicing of weak introns following its initial deposition at adjacent exon junctions. These data demonstrate a novel role for the EJC in regulating piwi intron excision and provide a mechanism for its function during splicing.

Neural stem cells in the adult brain exist primarily in a quiescent state but are reactivated in response to changing physiological conditions. How do stem cells sense and respond to metabolic changes? In the Drosophila CNS, quiescent neural stem cells are reactivated synchronously in response to a nutritional stimulus. Feeding triggers insulin production by blood-brain barrier glial cells, activating the insulin/insulin-like growth factor pathway in underlying neural stem cells and stimulating their growth and proliferation. Here we show that gap junctions in the blood-brain barrier glia mediate the influence of metabolic changes on stem cell behavior, enabling glia to respond to nutritional signals and reactivate quiescent stem cells. We propose that gap junctions in the blood-brain barrier are required to translate metabolic signals into synchronized calcium pulses and insulin secretion.