Faculty Bibliography

Affinity grids (AG) are specialized EM grids that bind macromolecular complexes containing tagged proteins to obtain maximum occupancy for structural analysis through single-particle EM. In this study, utilizing AG, we show that His-tagged activated PKC betaII binds to the small ribosomal subunit (40S). We reconstructed a cryo-EM map which shows that PKC betaII interacts with RACK1, a seven-bladed beta-propeller protein present on the 40S and binds in two different regions close to blades 3 and 4 of RACK1. This study is a first step in understanding the molecular framework of PKC betaII/RACK1 interaction and its role in translation.

Angiogenesis is essential to wound repair, and vascular endothelial growth factor (VEGF) is a potent factor to stimulate angiogenesis. Here, we examine the potential of VEGF-overexpressing adipose-derived stromal cells (ASCs) for accelerating wound healing using nonviral, biodegradable polymeric vectors. Mouse ASCs were transfected with DNA plasmid encoding VEGF or green fluorescent protein (GFP) using biodegradable poly (beta-amino) esters (PBAE). Cells transfected using Lipofectamine 2000, a commercially available transfection reagent, were included as controls. ASCs transfected using PBAEs showed enhanced transfection efficiency and 12-15-fold higher VEGF production compared with cells transfected using Lipofectamine 2000 (*P < 0.05). When transplanted into a mouse wild-type excisional wound model, VEGF-overexpressing ASCs led to significantly accelerated wound healing, with full wound closure observed at 8 days compared to 10-12 days in groups treated with ASCs alone or saline control (*P < 0.05). Histology and polarized microscopy showed increased collagen deposition and more mature collagen fibers in the dermis of wound beds treated using PBAE/VEGF-modified ASCs than ASCs alone. Our results demonstrate the efficacy of using nonviral-engineered ASCs to accelerate wound healing, which may provide an alternative therapy for treating many diseases in which wound healing is impaired.

The transcription factor Nrf2 is responsible for regulating a battery of antioxidant and cellular protective genes, primarily in response to oxidative stress. A member of the cap 'n' collar family of transcription factors, Nrf2 activation is tightly controlled by a series of signaling events. These events can be separated into the basal state, a preinduction response, gene induction, and finally a postinduction response, culminating in the restoration of redox homeostasis. However, despite the immensely intricate level of control the cellular environment imposes on Nrf2 activity, there are many opportunities for perturbations to arise in the signaling events that favor carcinogenesis and, therefore, implicate Nrf2 as both a tumor suppressor and a protooncogene. Herein, we highlight the ways in which Nrf2 is regulated, and discuss some of the Nrf2-inducible antioxidant (NQO1, NQO2, HO-1, GCLC), antiapoptotic (Bcl-2), metabolic (G6PD, TKT, PPARgamma), and drug efflux transporter (ABCG2, MRP3, MRP4) genes. In addition, we focus on how Nrf2 functions as a tumor suppressor under normal conditions and how its ability to detoxify the cellular environment makes it an attractive target for other oncogenes either via stabilization or degradation of the transcription factor. Finally, we discuss some of the ways in which Nrf2 is being considered as a therapeutic target for cancer treatment.

Introduction: Portal/mesenteric vein resection (PVR) is technically challenging and adds potential morbidity to a pancreaticoduodenectomy (PD). We reviewed our experience with PD for pancreatic adenocarcinoma to evaluate both short and long term outcomes following PVR. Methods: From our institutional pancreatic adenocarcinoma database, we identified 223 patients who underwent pancreaticoduodenectomy (PD) with (Group I n= 20) or without (Group II n= 203) PVR during the period 1990-2011. The study end-points were overall morbidity, 30-day mortality, length of post-operative stay (LOS), overall survival (OS). Differences between groups were evaluated using t-test or chi-squared test. OS for each group was estimated with Kaplan-Meier method and compared using the log-rank statistics. Results: The two groups were similar in terms of gender, age, ethnicity, underlying comorbidities and performance status (see table 1). One patient in Group I and 8 in Group II were deemed borderline resectable (5.0% vs. 3.9%, p = 0.8) and underwent neo-adjuvant treatment. Duration of surgery was longer in Group I (532 vs. 456 min, p = 0.04), but there were no differences in operative blood losses (1047 vs. 991 ml, p = 0.8), length of stay (13.9 vs. 14.4 days, p = 0.8), overall morbidity (55% vs. 38%, p = 0.14). There were only 2 post-operative deaths, both in the Group II (p = 0.7). Pathology revealed similar TNM stage and rates of resections with negative margins (85% vs. 75%, p = 0.8). At median follow-up of 14 months there was no significant difference in OS (20.5 vs. 15.8 months, p = 0.6) Conclusions: In our experience, post-operative and long-term outcomes were not adversely affected by PVR. PVR should be offered to patients with pancreatic cancer involving portal or mesenteric veins. (Table Presented)

A key event in prion diseases is the conversion of the prion protein (PrP) from its native alpha-helical conformation to a misfolded, beta-sheet rich conformation. Thus, preventing or reversing PrP misfolding could provide a means to disrupt prion disease progression and transmission. However, determining the structure of misfolded PrP has been notoriously difficult due to its inherent heterogeneity and aggregation behavior. For these reasons, simplified peptide fragments have been used as models that recapitulate characteristics of full-length PrP, such as amyloid-like aggregation and fibril formation, and in vitro toxicity. We provide a biochemical and structural comparison of PrP(127-147) peptides from elk, bovine and hamster using electrophysiology, electron microscopy and fluorescence. Our results demonstrate that the PrP(127-147) peptides adopt distinct populations of fibril structures. In addition, the elk PrP(127-147) peptide is unique in its ability to enhance Thioflavin T fluorescence and its ability to modulate neuronal ion channel conductances.

Myelinated axons are organized into specialized domains critical to their function in saltatory conduction, i.e., nodes, paranodes, juxtaparanodes, and internodes. Here, we describe the distribution and role of the 4.1B protein in this organization. 4.1B is expressed by neurons, and at lower levels by Schwann cells, which also robustly express 4.1G. Immunofluorescence and immuno-EM demonstrates 4.1B is expressed subjacent to the axon membrane in all domains except the nodes. Mice deficient in 4.1B have preserved paranodes, based on marker staining and EM in contrast to the juxtaparanodes, which are substantially affected in both the PNS and CNS. The juxtaparanodal defect is evident in developing and adult nerves and is neuron-autonomous based on myelinating cocultures in which wt Schwann cells were grown with 4.1B-deficient neurons. Despite the juxtaparanodal defect, nerve conduction velocity is unaffected. Preservation of paranodal markers in 4.1B deficient mice is associated with, but not dependent on an increase of 4.1R at the axonal paranodes. Loss of 4.1B in the axon is also associated with reduced levels of the internodal proteins, Necl-1 and Necl-2, and of alpha-2 spectrin. Mutant nerves are modestly hypermyelinated and have increased numbers of Schmidt-Lanterman incisures, increased expression of 4.1G, and express a residual, truncated isoform of 4.1B. These results demonstrate that 4.1B is a key cytoskeletal scaffold for axonal adhesion molecules expressed in the juxtaparanodal and internodal domains that unexpectedly regulates myelin sheath thickness. (c) 2012 Wiley Periodicals, Inc.

Alterations in the metabolic control of lipid and glucose homeostasis predispose an individual to develop cardiometabolic diseases, such as type 2-diabetes mellitus and atherosclerosis. Work over the last years has suggested that microRNAs (miRNAs) play an important role in regulating these physiological processes. The contribution of miRNAs in regulating metabolism is exemplified by miR-33, an intronic miRNA encoded in the Srebp genes. miR-33 controls cellular cholesterol export and fatty acid degradation, whereas its host genes stimulate cholesterol and fatty acid synthesis. Other miRNAs, such as miR-122, also play a critical role in regulating lipid homeostasis by controlling cholesterol synthesis and lipoprotein secretion in the liver. This review article summarizes the recent findings in the field, highlighting the contribution of miRNAs in regulating lipid and glucose metabolism. We will also discuss how the modulation of specific miRNAs may be a promising strategy to treat metabolic diseases.

BACKGROUND: Seventy-five million people are estimated to be hypertensive in sub-Saharan Africa. This translates in high morbidity and mortality, as hypertension is now considered to be the number one single risk factor for death worldwide. Accurate data from countries lacking national disease surveillance is needed to guide future evidence-driven health policies. The authors aimed to estimate the prevalence, awareness, management and control of hypertension and associated factors in an adult population of Angola. METHODS: A community-based survey of 1,464 adults, following the World Health Organization's Stepwise Approach to Chronic Disease Risk Factor Surveillance, was conducted to estimate the prevalence of hypertension, awareness, treatment and control in Dande, Northern Angola. Using a demographic surveillance system database, a representative sample of subjects, stratified by sex and age (18-40 and 41-64 years old), was selected. RESULTS: Prevalence of hypertension (systolic blood pressure >/=140 mmHg and/or diastolic blood pressure >/=90 mmHg and/or hypertensive therapy) was of 23% (95% CI: 21% to 25.2%). A follow-up consultation confirmed the hypertensive status in 82% of the subjects who had a second measurement on average 23 days after the first. Amongst hypertensive individuals, 21.6% (95% CI: 17.0% to 26.9%) were aware of their status. Only 13.9% (95% CI: 5.9% to 29.1%) of the subjects aware of their condition were under pharmacological treatment, of which approximately one-third were controlled. Older age, lower level of education, higher body mass index and abdominal obesity were found to be significantly (p<0.01) associated with hypertension. CONCLUSIONS: Our survey is the first to provide insightful data on hypertension prevalence in Angola. There is an urgent need for strategies to improve prevention, diagnosis and access to adequate treatment in this country, where a massive economic growth and consequent potential impact on lifestyle risk factors could lead to an increase in the prevalence of hypertension and cardiovascular disease.

The mechanistic target of rapamycin complex 1 (mTORC1) pathway regulates organismal growth in response to many environmental cues, including nutrients and growth factors. Cell-based studies showed that mTORC1 senses amino acids through the RagA-D family of GTPases (also known as RRAGA, B, C and D), but their importance in mammalian physiology is unknown. Here we generate knock-in mice that express a constitutively active form of RagA (RagA(GTP)) from its endogenous promoter. RagA(GTP/GTP) mice develop normally, but fail to survive postnatal day 1. When delivered by Caesarean section, fasted RagA(GTP/GTP) neonates die almost twice as rapidly as wild-type littermates. Within an hour of birth, wild-type neonates strongly inhibit mTORC1, which coincides with profound hypoglycaemia and a decrease in plasma amino-acid concentrations. In contrast, mTORC1 inhibition does not occur in RagA(GTP/GTP) neonates, despite identical reductions in blood nutrient amounts. With prolonged fasting, wild-type neonates recover their plasma glucose concentrations, but RagA(GTP/GTP) mice remain hypoglycaemic until death, despite using glycogen at a faster rate. The glucose homeostasis defect correlates with the inability of fasted RagA(GTP/GTP) neonates to trigger autophagy and produce amino acids for de novo glucose production. Because profound hypoglycaemia does not inhibit mTORC1 in RagA(GTP/GTP) neonates, we considered the possibility that the Rag pathway signals glucose as well as amino-acid sufficiency to mTORC1. Indeed, mTORC1 is resistant to glucose deprivation in RagA(GTP/GTP) fibroblasts, and glucose, like amino acids, controls its recruitment to the lysosomal surface, the site of mTORC1 activation. Thus, the Rag GTPases signal glucose and amino-acid concentrations to mTORC1, and have an unexpectedly key role in neonates in autophagy induction and thus nutrient homeostasis and viability.