Faculty Bibliography

The originally published version of this Article contained errors in Fig. 4 that were introduced during the production process. In panel c, the two uppermost labels 'IgE spleen' and 'IgE BM' incorrectly read 'IgG1 spleen' and 'IgE1 BM', respectively. These errors have now been corrected in both the PDF and HTML versions of the Article.

A method for specific quantification of hyaluronan (HA) concentration using AlphaScreen® (Amplified Luminescent Proximity Homogeneous Assay) technology is described. Two types of hydrogel-coated and chromophore-loaded latex nanobeads are employed. The proximity of the beads in solution is detected by excitation of the donor bead leading to the production of singlet oxygen, and chemiluminescence from the acceptor bead upon exposure to singlet oxygen. In the HA assay, the donor bead is modified with streptavidin, and binds biotin-labeled HA. The acceptor bead is modified with Ni(II), and is used to bind a specific recombinant HA-binding protein (such as HABP; aggrecan G1-IGD-G2) with a His-tag. Competitive inhibition of the HA-HABP interaction by free unlabeled HA in solution is used for quantification. The assay is specific for HA, and not dependent on HA molecular mass above the decasaccharide. HA can be quantified over a concentration range of approximately 30-1600 ng/mL using 2.5 μL of sample, for a detectable mass range of approximately 0.08-4 ng HA. This sensitivity of the AlphaScreen assay is greater than existing ELISA-like methods, due to the small volume requirements. HA can be detected in biological fluids using the AlphaScreen assay, after removal of bound proteins from HA and dilution or removal of other interfering proteins and lipids.

There is a great need for effective treatment options for post-traumatic stress disorder (PTSD). Neuropeptide Y (NPY) is associated with resilience to traumatic stress. MC4R antagonists, such as HS014, also reduce response to stress. Both regulate stress-responsive systems - the hypothalamic-pituitary-axis (HPA) and the noradrenergic nervous system and their associated behaviors. Therefore, we examined if their intranasal delivery to brain could attenuate development of PTSD-related symptoms in single prolonged stress (SPS) rodent PTSD model. Three regimens were used: (1) prophylactic treatment 30 min before SPS stressors, (2) early intervention right after SPS stressors, (3) therapeutic treatment when PTSD behaviors are manifested 1 wk or more after the traumatic stress. NPY delivered by regimen 1 or 2 prevented SPS-triggered elevation in anxiety, depressive-like behavior, and hyperarousal and reduced dysregulation of HPA axis. Hypothalamic CRH mRNA and GR in ventral hippocampus were significantly induced in vehicle- but not NPY-treated group. NPY also prevented hypersensitivity of LC/NE system to novel mild stressor and induction of CRH in amygdala. Some of these impairments were also reduced with HS014, alone or together with NPY. When given after symptoms were manifested (regiment 3), NPY attenuated anxiety and depressive behaviors. This demonstrates strong preclinical proof of concept for intranasal NPY, and perhaps MC4R antagonists, for non-invasive early pharmacological interventions for PTSD and comorbid disorders and possibly also as therapeutic strategy.

Major depressive disorder is a common psychiatric disorder that is thought to be triggered by both genetic and environmental factors. Depressive symptoms are an important public health problem and contribute to vulnerability to major depression. Although a substantial number of genetic and epigenetic studies have been performed to date, the detailed etiology of depression remains unclear and there are no validated biomarkers. DNA methylation is one of the major epigenetic modifications that play diverse roles in the etiology of complex diseases. In this study, we performed an epigenome-wide association study (EWAS) of DNA methylation on subjects with (N = 20) or without (N = 27) depressive symptoms in order to examine whether different levels of DNA methylation were associated with depressive tendencies. Employing methylation-array technology, a total of 363,887 methylation sites across the genomes were investigated and several candidate CpG sites associated with depressive symptoms were identified, especially annotated to genes linked to a G-protein coupled receptor protein signaling pathway. These data provide a strong impetus for validation studies using a larger cohort and support the possibility that G-protein coupled receptor protein signaling pathways are involved in the pathogenesis of depression.

A substantial proportion of fractures can present with nonunion, and the management of nonunion continues to present a challenge for orthopaedic surgeons. A variety of biological, mechanical, patient, and injury factors can contribute to the occurrence of nonunion, and often the cause of nonunion may be multifactorial. Successful management often requires assessment and treatment of more than one of these factors. This article reviews common factors that may contribute to nonunion including infection, impaired biology, and metabolic disorders. In addition, new and evolving strategies for diagnosing the cause and effectively treating nonunion including the diagnosis of infection, metabolic workup, bone grafting, cell-based therapies, and biological adjuvants are reviewed and discussed.

ATP-sensitive K+ (KATP) channels uniquely link cellular energy metabolism to membrane excitability and are expressed in diverse cell types that range from the endocrine pancreas to neurons and smooth, skeletal, and cardiac muscle. A decrease in the surface expression of KATP channels has been linked to various disorders, including dysregulated insulin secretion, abnormal blood pressure, and impaired resistance to cardiac injury. In contrast, up-regulation of KATP channel surface expression may be protective, for example, by mediating the beneficial effect of ischemic preconditioning. Molecular mechanisms that regulate KATP channel trafficking are poorly understood. Here, we used cellular assays with immunofluorescence, surface biotinylation, and patch clamping to demonstrate that Eps15 homology domain-containing protein 2 (EHD2) is a novel positive regulator of KATP channel trafficking to increase surface KATP channel density. EHD2 had no effect on cardiac Na+ channels (Nav1.5). The effect is specific to EHD2 as other members of the EHD family-EHD1, EHD3, and EHD4-had no effect on KATP channel surface expression. EHD2 did not directly affect KATP channel properties as unitary conductance and ATP sensitivity were unchanged. Instead, we observed that the mechanism by which EHD2 increases surface expression is by stabilizing KATP channel-containing caveolar structures, which results in a reduced rate of endocytosis. EHD2 also regulated KATP channel trafficking in isolated cardiomyocytes, which validated the physiologic relevance of these observations. Pathophysiologically, EHD2 may be cardioprotective as a dominant-negative EHD2 mutant sensitized cardiomyocytes to ischemic damage. Our findings highlight EHD2 as a potential pharmacologic target in the treatment of diseases with KATP channel trafficking defects.-Yang, H. Q., Jana, K., Rindler, M. J., Coetzee, W. A. The trafficking protein, EHD2, positively regulates cardiac sarcolemmal KATP channel surface expression: role in cardioprotection.

BACKGROUND:Radiation therapy is a mainstay in the treatment of many malignancies, but collateral damage to surrounding tissue, with resultant hypovascularity, fibrosis, and atrophy, can be difficult to reconstruct. Fat grafting has been shown to improve the quality of irradiated skin, but volume retention of the graft is significantly decreased. Deferoxamine is a U.S. Food and Drug Administration-approved iron-chelating medication for acute iron intoxication and chronic iron overload that has also been shown to increase angiogenesis. The present study evaluates the effects of deferoxamine treatment on irradiated skin and subsequent fat graft volume retention. METHODS:Mice underwent irradiation to the scalp followed by treatment with deferoxamine or saline and perfusion and were analyzed using laser Doppler analysis. Human fat grafts were then placed beneath the scalp and retention was also followed up to 8 weeks radiographically. Finally, histologic evaluation of overlying skin was performed to evaluate the effects of deferoxamine preconditioning. RESULTS:Treatment with deferoxamine resulted in significantly increased perfusion, as demonstrated by laser Doppler analysis and CD31 immunofluorescent staining (p < 0.05). Increased dermal thickness and collagen content secondary to irradiation, however, were not affected by deferoxamine (p > 0.05). Importantly, fat graft volume retention was significantly increased when the irradiated recipient site was preconditioned with deferoxamine (p < 0.05). CONCLUSIONS:The authors' results demonstrated increased perfusion with deferoxamine treatment, which was also associated with improved fat graft volume retention. Preconditioning with deferoxamine may thus enhance fat graft outcomes for soft-tissue reconstruction following radiation therapy.