Faculty Bibliography

The organization of nucleosomes influences transcriptional activity by controlling accessibility of DNA binding proteins to the genome. Genome-wide nucleosome binding profiles have identified a canonical nucleosome organization at gene promoters, where arrays of well-positioned nucleosomes emanate from nucleosome-depleted regions. The mechanisms of formation and the function of canonical promoter nucleosome organization remain unclear. Here we analyze the genome-wide location of nucleosomes during zebrafish embryogenesis and show that well-positioned nucleosome arrays appear on thousands of promoters during the activation of the zygotic genome. The formation of canonical promoter nucleosome organization is independent of DNA sequence preference, transcriptional elongation, and robust RNA polymerase II (Pol II) binding. Instead, canonical promoter nucleosome organization correlates with the presence of histone H3 lysine 4 trimethylation (H3K4me3) and affects future transcriptional activation. These findings reveal that genome activation is central to the organization of nucleosome arrays during early embryogenesis.

Magnetic resonance imaging (MRI) of autism populations is confounded by the inherent heterogeneity in the individuals' genetics and environment, two factors difficult to control for. Imaging genetic animal models that recapitulate a mutation associated with autism quantify the impact of genetics on brain morphology and mitigate the confounding factors in human studies. Here, we used MRI to image three genetic mouse models with single mutations implicated in autism: Neuroligin-3 R451C knock-in, Methyl-CpG binding protein-2 (MECP2) 308-truncation and integrin beta3 homozygous knockout. This study identified the morphological differences specific to the cerebellum, a structure repeatedly linked to autism in human neuroimaging and postmortem studies. To accomplish a comparative analysis, a segmented cerebellum template was created and used to segment each study image. This template delineated 39 different cerebellar structures. For Neuroligin-3 R451C male mutants, the gray (effect size (ES) = 1.94, FDR q = 0.03) and white (ES = 1.84, q = 0.037) matter of crus II lobule and the gray matter of the paraflocculus (ES = 1.45, q = 0.045) were larger in volume. The MECP2 mutant mice had cerebellar volume changes that increased in scope depending on the genotype: hemizygous males to homozygous females. The integrin beta3 mutant mouse had a drastically smaller cerebellum than controls with 28 out of 39 cerebellar structures smaller. These imaging results are discussed in relation to repetitive behaviors, sociability, and learning in the context of autism. This work further illuminates the cerebellum's role in autism. Autism Res 2013, : -. (c) 2013 International Society for Autism Research, Wiley Periodicals, Inc.

The Cancer/Testis Antigen (CTA), Prostate-associated Gene 4 (PAGE4), is a stress-response protein that is upregulated in prostate cancer (PCa) especially in precursor lesions that result from inflammatory stress. In cells under stress, translocation of PAGE4 to mitochondria increases while production of reactive oxygen species decreases. Furthermore, PAGE4 is also upregulated in human fetal prostate, underscoring its potential role in development. However, the proteins that interact with PAGE4 and the mechanisms underlying its pleiotropic functions in prostatic development and disease remain unknown. Here, we identified c-Jun as a PAGE4 interacting partner. We show that both PAGE4 and c-Jun are overexpressed in the human fetal prostate; and in cell-based assays, PAGE4 robustly potentiates c-Jun transactivation. Single-molecule Forster resonance energy transfer experiments indicate that upon binding to c-Jun, PAGE4 undergoes conformational changes. However, no interaction is observed in presence of BSA or unilamellar vesicles containing the mitochondrial inner membrane diphosphatidylglycerol lipid marker cardiolipin. Together, our data indicate that PAGE4 specifically interacts with c-Jun and that, conformational dynamics may account for its observed pleiotropic functions. To our knowledge, this is the first report demonstrating crosstalk between a CTA and a proto-oncogene. Disrupting PAGE4/c-Jun interactions using small molecules may represent a novel therapeutic strategy for PCa.

Prior studies of clay-virus interactions have focused on the stability and infectivity of nonenveloped viruses, yielding contradictory results. We hypothesize that the surface charge distribution of the clay and virus envelope dictates how the components react and affect aggregation, viral stability, and infectivity. The bacteriophage Cystoviridae species φ6 used in this study is a good model for enveloped pathogens. The interaction between φ6 and montmorillonite (MMT) clay (the primary component of bentonite) is explored by transmission electron microscopy. The analyses show that MMT-φ6 mixtures undergo heteroaggregation, forming structures in which virtually all the virions are either sequestered between MMT platelet layers or attached to platelet edges. The virions swell and undergo disassembly resulting in partial or total envelope loss. Edge-attached viral envelopes distort to increase contact area with the positively charged platelet edges indicating that the virion surface is negatively charged. The nucleocapsid (NCs) remaining after envelope removal also exhibit distortion, in contrast to detergent-produced NCs which exhibit no distortion. This visually discernible disassembly is a mechanism for loss of infectivity previously unreported by studies of nonenveloped viruses. The MMT-mediated sequestration and disassembly result in reduced infectivity, suggesting that clays may reduce infectivity of enveloped pathogenic viruses in soils and sediments.

Zebularine is a cytidine analogue incorporated into DNA during replication, inhibiting DNA methyltransferase 1 (DNMT1), resulting in demethylation and changes in gene expression. Such modification may improve radiosensitivity in resistant lymphoma cells. The hypothesis of this study was that zebularine and radiation would synergistically inhibit cell growth and viability. Human MEC1 malignant B cells were incubated with 0-200 microM zebularine for 48 h. Media containing zebularine was removed, and the cells were irradiated with 0-2 Gy of either external beam irradiation or (177) Lu-DOTA-TATE, a radiolabelled somatostatin analogue. Concentration and viability were measured over 48-72 h. The proportion of apoptotic cells was identified using an active Caspase 3/7 assay. Zebularine inhibited growth of cells in a dose-dependent manner during exposure. No residual growth inhibition occurred following removal of the drug. Zebularine and external irradiation inhibited cell proliferation in a dose-dependent, synergistic interaction, but the effect on viability was additive. Treatment with zebularine and (177) Lu-DOTA-TATE resulted in less inhibition of proliferation (P = 0.0135), but a synergistic decrease in viability. Apoptotic fraction was much higher in cells irradiated with (177) Lu-DOTA-TATE than external irradiation. External irradiation induces growth arrest rather than apoptosis. Apoptosis is the primary effect of radiopharmaceutical therapy on tumour cells. Treatment with the methylation inhibitor, zebularine, appears to synergistically augment these natural effects in vitro, which could be exploited clinically.

OBJECTIVE: One of the major risk factors for atherosclerosis is the plasma level of low-density lipoprotein (LDL), which is a product of very-low-density lipoprotein (VLDL). Hepatic apolipoprotein B100 (apoB100) is the essential component that provides structural stability to VLDL particles. Newly translated apoB100 is partially lipidated in the endoplasmic reticulum (ER), forming nascent apoB100-VLDL particles. These particles are further modified to form fully mature VLDLs in the Golgi apparatus. Therefore, the transport of nascent VLDL from the ER to the Golgi represents a critical step during VLDL maturation and secretion and in regulating serum LDL cholesterol levels. Our previous studies showed that apoB100 exits the ER in coat complex II vesicles (COPII), but the cohort of related factors that control trafficking is poorly defined. APPROACH AND RESULTS: Expression levels of Kelch-like protein 12 (KLHL12), an adaptor protein known to assist COPII-dependent transport of procollagen, were manipulated by using a KLHL12-specific small interfering RNA and a KLHL12 expression plasmid in the rat hepatoma cell line, McArdle RH7777. KLHL12 knockdown decreased the secreted and intracellular pools of apoB100, an effect that was attenuated in the presence of an autophagy inhibitor. KLHL12 knockdown also significantly reduced secretion of the most lipidated apoB100-VLDL species and led to the accumulation of apoB100 in the ER. Consistent with these data, KLHL12 overexpression increased apoB100 recovery and apoB100-VLDL secretion. Images obtained from confocal microscopy revealed colocalization of apoB100 and KLHL12, further supporting a direct link between KLHL12 function and VLDL trafficking from the ER. CONCLUSIONS: KLHL12 plays a critical role in facilitating the ER exit and secretion of apoB100-VLDL particles, suggesting that KLHL12 modulation would influence plasma lipid levels.

BACKGROUND: Mechanically offloading or shielding an incision significantly reduces scarring in both animal and first-in-human studies. Whether or not this strategy would be effective following scar revision surgery was previously unknown. In this article, the authors report that the embrace device, which uses principles of mechanomodulation, significantly improves aesthetic outcomes following scar revision surgery. METHODS: A prospective, open-label, randomized, single-center study was conducted to evaluate the appearance of scars following revision and embrace treatment. Revision surgery was performed on 12 patients, each acting as his or her own control, and outcomes were assessed at 6 months. A visual analogue scale was used to evaluate each scar, rated by four independent surgeons who were not involved in the study. RESULTS: Evaluation of 6-month scar images by four independent surgeons using the visual analogue scale demonstrated a highly significant improvement in scar appearance following embrace treatment (p < 0.005). CONCLUSION: The embrace device represents a powerful new technology for significantly improving scar appearance following revision surgery. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, II.

INTRODUCTION: Adipose-derived stem cells (ASCs) have shown potential for cell-based therapy in the field of plastic surgery. However, the fate of ASCs after transplantation and the mechanism(s) of their biologic capabilities remain unclear. METHODS: We isolated and cultured ASCs from transgenic mice that express both luciferase and green fluorescent protein and injected the cells into the inguinal fat pads of wild-type mice. We tested 4 experimental groups, namely, ischemic fat pads with/without ASCs and control fat pads with/without ASCs. RESULTS: Transplanted ASCs were tracked with bioluminescence imaging. The luminescence gradually decreased over 28 days, indicating cell death after transplantation. More ASCs were retained in ischemic fat pads on day 7 compared to control fat pads. On day 14, adipose tissue vascular density was higher in the ASC transplantation groups compared to those without ASCs. On day 28, there was decreased atrophy of adipose tissue in ASC-treated ischemic fat pads. Transplanted ASCs were detected as nonproliferating green fluorescent protein-positive cells, whereas native endothelial cells adjacent to the transplanted ASCs were proliferative. Protein analysis demonstrated higher expression of hepatocyte growth factor and vascular endothelial growth factor in the ASC transplantation groups, suggesting a paracrine mechanism, which was confirmed by in vitro experiments with conditioned media from ASCs. CONCLUSIONS: Transplanted ASCs are preferentially retained in ischemic adipose tissue, although most of the cells eventually undergo cell death. They exert an angiogenic effect on adipose tissue mainly through a paracrine mechanism. Increased understanding of these effects will help develop ASCs as a tool for cell-based therapy.

Remyelination occurs in multiple sclerosis (MS) lesions but is generally considered to be insufficient. One of the major challenges in MS research is to understand the causes of remyelination failure and to identify therapeutic targets that promote remyelination. Activation of pancreatic endoplasmic reticulum kinase (PERK) signaling in response to endoplasmic reticulum stress modulates cell viability and function under stressful conditions. There is evidence that PERK is activated in remyelinating oligodendrocytes in demyelinated lesions in both MS and its animal model, experimental autoimmune encephalomyelitis (EAE). In this study, we sought to determine the role of PERK signaling in remyelinating oligodendrocytes in MS and EAE using transgenic mice that allow temporally controlled activation of PERK signaling specifically in oligodendrocytes. We demonstrated that persistent PERK activation was not deleterious to myelinating oligodendrocytes in young, developing mice or to remyelinating oligodendrocytes in cuprizone-induced demyelinated lesions. We found that enhancing PERK activation, specifically in (re)myelinating oligodendrocytes, protected the cells and myelin against the detrimental effects of interferon-gamma, a key proinflammatory cytokine in MS and EAE. More important, we showed that enhancing PERK activation in remyelinating oligodendrocytes at the recovery stage of EAE promoted cell survival and remyelination in EAE demyelinated lesions. Thus, our data provide direct evidence that PERK activation cell-autonomously enhances the survival and preserves function of remyelinating oligodendrocytes in immune-mediated demyelinating diseases.

In this issue, guest editors Kathy Green and Mario Delmar, who are leaders in the fields of epidermal desmosomes and heart intercalated discs respectively, have joined forces to collate a two-part series of reviews focused on junctional proteins and genes that are targets of skin and heart diseases.