Faculty Bibliography

The crystal structure of the sensorless pore module of a voltage-gated K(+) (Kv) channel showed that lipids occupy a crevice between subunits. We asked if individual lipid monolayers of the bilayer embody independent modules linked to channel gating modulation. Functional studies using single channel current recordings of the sensorless pore module reconstituted in symmetric and asymmetric lipid bilayers allowed us to establish the deterministic role of lipid headgroup on gating. We discovered that individual monolayers with headgroups that coat the bilayer-aqueous interface with hydroxyls stabilize the channel open conformation. The hydroxyl need not be at a terminal position and the effect is not dependent on the presence of phosphate or net charge on the lipid headgroup. Asymmetric lipid bilayers allowed us to determine that phosphoglycerides with glycerol or inositol on the extracellular facing monolayer stabilize the open conformation of the channel. This indirect effect is attributed to a change in water structure at the membrane interface. By contrast, inclusion of the positively charged lysyl-dioleoyl-phosphatidylglycerol exclusively on the cytoplasmic facing monolayer of the bilayer increases drastically the probability of finding the channel open. Such modulation is mediated by a pi-cation interaction between Phe-19 of the pore module and the lysyl moiety anchored to the phosphatidylglycerol headgroup. The new findings imply that the specific chemistry of the lipid headgroup and its selective location in either monolayer of the bilayer dictate the stability of the open conformation of a Kv pore module in the absence of voltage-sensing modules.

In contrast to mammals, in the chicken major sites of lipoprotein synthesis and secretion are not only the liver and intestine, but also the kidney and the embryonic yolk sac. Two key components in the assembly of triglyceride-rich lipoproteins are the microsomal triglyceride transfer protein (MTP) and apolipoprotein B (apoB). We have analyzed the expression of MTP in the embryonic liver, small intestine, and kidney, and have studied the expression of MTP in, and the secretion of apoB from, the developing yolk sac (YS). Transcript and protein levels of MTP increase during embryogenesis in YS, liver, kidney, and small intestine, and decrease in YS, embryonic liver, and kidney after hatching. In small intestine, the MTP mRNA level rises sharply during the last trimester of embryo development (after day 15), while MTP protein is detectable only after hatching (day 21). In the YS of 15- and 20-day old embryos, apoB secretion was detected by pulse-chase metabolic radiolabeling experiments and subsequent immunoprecipitation. Taken together, our data reveal the importance of coordinated production of MTP and apoB in chicken tissues capable of secreting triglyceride-rich lipoproteins even before hatching.

Obesity is associated with infiltration of macrophages into adipose tissue (AT), contributing to insulin resistance and diabetes. However, relatively little is known regarding the origin of AT macrophages (ATMs). We discovered that murine models of obesity have prominent monocytosis and neutrophilia, associated with proliferation and expansion of bone marrow (BM) myeloid progenitors. AT transplantation conferred myeloid progenitor proliferation in lean recipients, while weight loss in both mice and humans (via gastric bypass) was associated with a reversal of monocytosis and neutrophilia. Adipose S100A8/A9 induced ATM TLR4/MyD88 and NLRP3 inflammasome-dependent IL-1beta production. IL-1beta interacted with the IL-1 receptor on BM myeloid progenitors to stimulate the production of monocytes and neutrophils. These studies uncover a positive feedback loop between ATMs and BM myeloid progenitors and suggest that inhibition of TLR4 ligands or the NLRP3-IL-1beta signaling axis could reduce AT inflammation and insulin resistance in obesity.

The principal function of an epithelium is to form a dynamic barrier that regulates movement between body compartments. Each epithelium is specialized with barrier functions that are specific for the tissues it serves. The apical surface commonly faces a lumen, but the retinal pigment epithelium (RPE) appears to be unique by a facing solid tissue, the sensory retina. Nonetheless, there exists a thin (subretinal) space that can become fluid filled during pathology. RPE separates the subretinal space from the blood supply of the outer retina, thereby forming the outer blood-retinal barrier. The intricate interaction between the RPE and sensory retina presents challenges for learning how accurately culture models reflect native behavior. The challenge is heightened by findings that detail the variation of RPE barrier proteins both among species and at different stages of the life cycle. Among the striking differences is the expression of claudin family members. Claudins are the tight junction proteins that regulate ion diffusion across the spaces that lie between the cells of a monolayer. Claudin expression by RPE varies with species and life-stage, which implies functional differences among commonly used animal models. Investigators have turned to transcriptomics to supplement functional studies when comparing native and cultured tissue. The most detailed studies of the outer blood-retinal barrier have focused on human RPE with transcriptome and functional studies reported for human fetal, adult, and stem-cell derived RPE.

Promoters and enhancers establish precise gene transcription patterns. The development of functional approaches for their identification in mammalian cells has been complicated by the size of these genomes. Here we report a high-throughput functional assay for directly identifying active promoter and enhancer elements called FIREWACh (Functional Identification of Regulatory Elements Within Accessible Chromatin), which we used to simultaneously assess over 80,000 DNA fragments derived from nucleosome-free regions within the chromatin of embryonic stem cells (ESCs) and identify 6,364 active regulatory elements. Many of these represent newly discovered ESC-specific enhancers, showing enriched binding-site motifs for ESC-specific transcription factors including SOX2, POU5F1 (OCT4) and KLF4. The application of FIREWACh to additional cultured cell types will facilitate functional annotation of the genome and expand our view of transcriptional network dynamics.

Significance: Poor wound healing remains a significant health issue for a large number of patients in the United States. The physiologic response to local wound hypoxia plays a critical role in determining the success of the normal healing process. Hypoxia-inducible factor-1 (HIF-1), as the master regulator of oxygen homeostasis, is an important determinant of healing outcomes. HIF-1 contributes to all stages of wound healing through its role in cell migration, cell survival under hypoxic conditions, cell division, growth factor release, and matrix synthesis throughout the healing process. Recent Advances: Positive regulators of HIF-1, such as prolyl-4-hydroxylase inhibitors, have been shown to be beneficial in enhancing diabetic ischemic wound closure and are currently undergoing clinical trials for treatment of several human-ischemia-based conditions. Critical Issues: HIF-1 deficiency and subsequent failure to respond to hypoxic stimuli leads to chronic hypoxia, which has been shown to contribute to the formation of nonhealing ulcers. In contrast, overexpression of HIF-1 has been implicated in fibrotic disease through its role in increasing myofibroblast differentiation leading to excessive matrix production and deposition. Both positive and negative regulators of HIF-1 therefore provide important therapeutic targets that can be used to manipulate HIF-1 expression where an excess or deficiency in HIF-1 is known to correlate with pathogenesis. Future Directions: Targeting HIF-1 during wound healing has many important clinical implications for tissue repair. Counteracting the detrimental effects of excessive or deficient HIF-1 signaling by modulating HIF-1 expression may improve future management of poorly healing wounds.

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.

Inactivation of the Rb tumor suppressor can lead to increased cell proliferation or cell death depending on specific cellular context. Therefore, identification of the interacting pathways that modulate the effect of Rb loss will provide novel insights into the roles of Rb in cancer development and promote new therapeutic strategies. Here, we identify a novel synthetic lethal interaction between Rb inactivation and deregulated Wg/Wnt signaling through unbiased genetic screens. We show that a weak allele of axin, which deregulates Wg signaling and increases cell proliferation without obvious effects on cell fate specification, significantly alters metabolic gene expression, causes hypersensitivity to metabolic stress induced by fasting, and induces synergistic apoptosis with mutation of fly Rb ortholog, rbf. Furthermore, hyperactivation of Wg signaling by other components of the Wg pathway also induces synergistic apoptosis with rbf. We show that hyperactivated Wg signaling significantly increases TORC1 activity and induces excessive energy stress with rbf mutation. Inhibition of TORC1 activity significantly suppressed synergistic cell death induced by hyperactivated Wg signaling and rbf inactivation, which is correlated with decreased energy stress and decreased induction of apoptotic regulator expression. Finally the synthetic lethality between Rb and deregulated Wnt signaling is conserved in mammalian cells and that inactivation of Rb and APC induces synergistic cell death through a similar mechanism. These results suggest that elevated TORC1 activity and metabolic stress underpin the evolutionarily conserved synthetic lethal interaction between hyperactivated Wnt signaling and inactivated Rb tumor suppressor.

BACKGROUND: & Aims: Subsets of leukocytes synergize with regenerative growth factors to promote hepatic regeneration. gammadeltaTau cells are early responders to inflammation-induced injury in a number of contexts. We investigated the role of gammadeltaTau cells in hepatic regeneration using mice with disruptions in Tcrd (encodes the T cell receptor delta chain) and Clec7a (encodes C-type lectin domain family 7 member a, also known as DECTIN1). METHODS: We performed partial hepatectomies on wild-type C57BL/6, CD45.1, Tcrd-/-, or Clec7a-/- mice. Cells were isolated from livers of patients and mice via mechanical and enzymatic digestion. gammadeltaTau cells were purified by fluorescence-activated cell sorting. RESULTS: In mice, partial hepatectomy upregulated expression of CCL20 and ligands of Dectin-1, associated with recruitment and activation of gammadeltaTau cells and their increased production of interleukin (IL)17 family cytokines. Recruited gammadeltaTau cells induced production of IL6 by antigen-presenting cells and suppressed expression of interferon gamma by natural killer T cells, promoting hepatocyte proliferation. Absence of IL17-producing gammadeltaTau cells or deletion of Dectin-1 prevented development of regenerative phenotypes in subsets of innate immune cells. This slowed liver regeneration and was associated with reduced expression of regenerative growth factors and cell cycle regulators. Conversely, exogenous administration of IL17 family cytokines or Dectin-1 ligands promoted regeneration. More broadly, we found that gammadeltaTau cells are required for inflammatory responses mediated by IL17 and Dectin-1. CONCLUSIONS: gammadeltaT cells regulate hepatic regeneration by producing IL22 and IL17, which have direct mitogenic effects on hepatocytes and promote a regenerative phenotype in hepatic leukocytes, respectively. Dectin-1 ligation is required for gammadeltaT cells to promote hepatic regeneration.