Faculty Bibliography

EGFR ligands undergo complex processing during their maturation to active signaling proteins. Like its mammalian homologues, the predominant Drosophila EGFR ligand Spitz is produced as a transmembrane pro-protein. In the secretory pathway, Spitz is cleaved within its transmembrane domain to release the extracellular signaling domain. This domain is modified with an N-terminal palmitate group that tethers it to the plasma membrane. We found that the pro-protein can reach the cell surface in the absence of proteolysis, but it fails to activate the EGFR. To address why the transmembrane pro-protein is inactive, while membrane association through the palmitate group promotes activity, we generated a panel of chimeric constructs containing the Spitz extracellular region fused to exogenous transmembrane proteins. Although the orientation of the EGF domain and its distance from the plasma membrane varies in these chimeras, they are all active in vivo. Thus, tethering Spitz to the membrane via a transmembrane domain at either terminus does not prevent activity. Conversely, removing the N-terminal palmitate group from the C-terminally tethered pro-protein does not render it active. Furthermore, we show that the Spitz transmembrane pro-protein can activate the EGFR in a tissue culture assay, indicating that its failure to signal in vivo is not due to structural features. In polarized imaginal disc cells, unprocessed Spitz pro-protein localizes to apical puncta, whereas the active chimeric Spitz constructs are basolaterally localized. Together, our data support the model that localized trafficking of the pro-protein restricts its ability to activate the receptor in polarized tissues.

BACKGROUND: A multitude of different approaches have been proposed for achieving optimal aesthetic results after nipple reconstruction. In contrast, however, only a few studies focus on the morbidity associated with this procedure, particularly after implant-based breast reconstruction. METHODS: Using a cross-sectional study design all patients who underwent implant-based breast reconstruction with subsequent nipple reconstruction between 2000 and 2010 at Stanford University Medical Center were identified. The aim of the study was to analyze the impact of the following parameters on the occurrence of postoperative complications: age, final implant volume, time interval from placement of final implant to nipple reconstruction, and history of radiotherapy. RESULTS: A total of 139 patients with a mean age of 47.5 years (range, 29 to 75 years) underwent 189 nipple reconstructions. The overall complication rate was 13.2 percent (N = 25 nipple reconstructions). No association was observed between age (p = 0.43) or implant volume (p = 0.47) and the occurrence of complications. A trend towards higher complication rates in patients in whom the time interval between final implant placement and nipple reconstruction was greater than 8.5 months was seen (p = 0.07). Radiotherapy was the only parameter that was associated with a statistically significant increase in postoperative complication rate (51.7 percent vs. 6.25 percent; p < 0.00001). CONCLUSION: While nipple reconstruction is a safe procedure after implant-based breast reconstruction in patients without a history of radiotherapy, the presence of an irradiated field converts it to a high-risk one with a significant increase in postoperative complication rate. Patients with a history of radiotherapy should be informed about their risk profile and as a result may choose autologous reconstruction instead. LEVEL OF EVIDENCE: IV.

Forty two Candida albicans isolates were collected from clinical samples in Israel. Twenty strains were isolated from blood cultures and 22 from superficial candidiasis. Isolates were typed by MLST analysis. Thirty-seven Diploid Sequence Types (DSTs) were identified. Seventeen isolates (40.5%) displayed new DSTs; 34 (81%) clustered within previously described clades, while nine (19%) did not cluster in any known group. Clonal Complex (CC) 124 was the most prevalent in both candidemia and superficial candidiasis isolates, CC 918 was only found in candidemia strains. To the best of our knowledge, this is the first study analyzing C. albicans clinical isolates from Israel using MLST methodology, possibly pointing to geographic differences in strain distribution.

Abstract Purpose: To describe the location of the zygomatico-orbital foramen on the inferolateral orbital wall. Methods: This anatomic study examined 28 orbits of 14 dry human adult skulls. The zygomatico-orbital foramen was identified by passing a thin wire from the zygomatico-facial foramen to its orbital aspect and a thin flexible ruler was used to measure 1) the distance perpendicular to the closest point on the inferior orbital rim, 2) the distance from the inferior orbital fissure, and 3) the distance from the area used for retrobulbar injections. Results: The mean distance from the zygomatico-orbital foramen to the closest point on the inferior orbital rim was 4.7 mm (range from 1 to 7 mm). The mean distance from the inferior orbital fissure was 14.9 mm (range from 10 to 18 mm). The mean distance from the area of retrobulbar injection was 6.0 mm (range from 3 to 10 mm). Conclusions: The location of the zygomatico-orbital foramen within the inferolateral orbit is quite variable. This is the first study to attempt to quantify its proximity to the site of retrobulbar injection. We conclude that it is an important anatomical structure to consider when giving retrobulbar anesthesia, especially given the variability in technique among ophthalmologists.

Small molecules have been used since antiquity to regulate our sleep. Despite the explosion of diverse drugs to treat problems of too much or too little sleep, the detailed mechanisms of action and especially the neuronal targets by which these compounds alter human behavioural states are not well understood. Research efforts in model systems such as mouse, zebrafish and fruit fly are combining conditional genetics and optogenetics with pharmacology to map the effects of sleep-promoting drugs onto neural circuits. Recent studies raise the possibility that many small molecules alter sleep and wake via specific sets of critical neurons rather than through the global modulation of multiple brain targets. These findings also uncover novel brain areas as sleep/wake regulators and indicate that the development of circuit-selective drugs might alleviate sleep disorders with fewer side effects.

Transforming growth factor beta (TGFbeta) plays critical roles in regulating a plethora of physiological processes in normal organs, including morphogenesis, embryonic development, stem cell differentiation, immune regulation, and wound healing. Though considered a tumor suppressor, TGFbeta is a critical mediator of tumor microenvironment, in which it likewise mediates tumor and stromal cell phenotype, recruitment, inflammation, immune function, and angiogenesis. The fact that activation of TGFbeta is an early and persistent event in irradiated tissues and that TGFbeta signaling controls effective DNA damage response provides a new means to manipulate tumor response to radiation. Here we discuss preclinical studies unraveling TGFbeta effects in cancer treatment and review TGFbeta biology in lung cancer as an example of the opportunities for TGFbeta pathway inhibition as a pharmaceutical approach to augment radiation therapy.

Bradykinin, acting via the bradykinin B2 receptor (B2R), is a potent stimulator of adrenomedullary catecholamine biosynthesis and release and likely plays an important role in the adrenomedullary stress response. However, the effects of stress on the expression of this receptor in the adrenal medulla are currently unclear. Here, we examined the changes in adrenomedullary B2R gene expression in male rats in response to single (1 time) and repeated (6 times) exposure to 2 hours immobilization stress (IMO). Immediately after 1 or 6 times IMO, B2R mRNA levels were increased by 9-fold and 7-fold, respectively, and returned to unstressed control levels 3 hours later. This large, but transient, increase in mRNA elicited a doubling of protein levels 3 hours after the stress exposure. Next, the role of the hypothalamic-pituitary-adrenocortical axis in the stress-induced upregulation of B2R gene expression was examined. Treatment with endogenous (corticosterone) and synthetic (dexamethasone) glucocorticoids dose-dependently increased B2R mRNA levels in adrenomedullary-derived PC12 cells. Furthermore, cortisol supplementation at levels mimicking stress exposure elevated B2R mRNA levels in the adrenal medulla of hypophysectomized rats. In response to 1 exposure to IMO, the stress-triggered rise in plasma corticosterone and adrenomedullary B2R mRNA levels was attenuated in CRH-knockout mice and absent in pharmacologically adrenalectomized rats, indicating a requirement for glucocorticoids in the upregulation of B2R gene expression with stress. Overall, the increase in B2R gene expression in response to the stress-triggered rise in glucocorticoids likely enhances catecholamine biosynthesis and release and may serve as an adaptive response of the adrenomedullary catecholaminergic system to stress.

Atherosclerosis is a chronic inflammatory disease that arises from an imbalance in lipid metabolism and a maladaptive immune response driven by the accumulation of cholesterol-laden macrophages in the artery wall. Through the analysis of the progression and regression of atherosclerosis in animal models, there is a growing understanding that the balance of macrophages in the plaque is dynamic and that both macrophage numbers and the inflammatory phenotype influence plaque fate. In this Review, we summarize recently identified pro- and anti-inflammatory pathways that link lipid and inflammation biology with the retention of macrophages in plaques, as well as factors that have the potential to promote their egress from these sites.

Establishment of specific characteristics of each embryonic cardiac chamber is crucial for development of a fully functional adult heart. Despite the importance of defining and maintaining unique features in ventricular and atrial cardiomyocytes, the regulatory mechanisms guiding these processes are poorly understood. Here, we show that the homeodomain transcription factors Nkx2.5 and Nkx2.7 are necessary to sustain ventricular chamber attributes through repression of atrial chamber identity. Mutation of nkx2.5 in zebrafish yields embryos with diminutive ventricular and bulbous atrial chambers. These chamber deformities emerge gradually during development, with a severe collapse in the number of ventricular cardiomyocytes and an accumulation of excess atrial cardiomyocytes as the heart matures. Removal of nkx2.7 function from nkx2.5 mutants exacerbates the loss of ventricular cells and the gain of atrial cells. Moreover, in these Nkx-deficient embryos, expression of vmhc, a ventricular gene, fades, whereas expression of amhc, an atrial gene, expands. Cell-labeling experiments suggest that ventricular cardiomyocytes can transform into atrial cardiomyocytes in the absence of Nkx gene function. Through suggestion of transdifferentiation from ventricular to atrial fate, our data reveal a pivotal role for Nkx genes in maintaining ventricular identity and highlight remarkable plasticity in differentiated myocardium. Thus, our results are relevant to the etiologies of fetal and neonatal cardiac pathology and could direct future innovations in cardiac regenerative medicine.

Harvesting adipose-derived stromal cells (ASCs) for tissue engineering is frequently done through liposuction. However, several different techniques exist. Although third-generation ultrasound-assisted liposuction has been shown to not have a negative effect on ASCs, the impact of laser-assisted liposuction on the quality and differentiation potential of ASCs has not been studied. Therefore, ASCs were harvested from laser-assisted lipoaspirate and suction-assisted lipoaspirate. Next, in vitro parameters of cell yield, cell viability and proliferation, surface marker phenotype, osteogenic differentiation, and adipogenic differentiation were performed. Finally, in vivo bone formation was assessed using a critical-sized cranial defect in athymic nude mice. Although ASCs isolated from suction-assisted lipoaspirate and laser-assisted lipoaspirate both successfully underwent osteogenic and adipogenic differentiation, the cell yield, viability, proliferation, and frequency of ASCs (CD34(+)CD31(-)CD45(-)) in the stromal vascular fraction were all significantly less with laser-assisted liposuction in vitro (p < .05). In vivo, quantification of osseous healing by micro-computed tomography revealed significantly more healing with ASCs isolated from suction-assisted lipoaspirate relative to laser-assisted lipoaspirate at the 4-, 6-, and 8-week time points (p < .05). Therefore, as laser-assisted liposuction appears to negatively impact the biology of ASCs, cell harvest using suction-assisted liposuction is preferable for tissue-engineering purposes.