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Risk factors for fibrocalcific aortic valve disease (FCAVD) are associated with systemic decreases in bioavailability of endothelium-derived nitric oxide (EDNO). In patients with bicuspid aortic valve (BAV), vascular expression of endothelial nitric oxide synthase (eNOS) is decreased, and eNOS−/− mice have increased prevalence of BAV. The goal of this study was to test the hypotheses that EDNO attenuates profibrotic actions of valve interstitial cells (VICs) in vitro and that EDNO deficiency accelerates development of FCAVD in vivo. As a result of the study, coculture of VICs with aortic valve endothelial cells (vlvECs) significantly decreased VIC activation, a critical early phase of FCAVD. Inhibition of VIC activation by vlvECs was attenuated by NG-nitro-l-arginine methyl ester or indomethacin. Coculture with vlvECs attenuated VIC expression of matrix metalloproteinase-9, which depended on stiffness of the culture matrix. Coculture with vlvECs preferentially inhibited collagen-3, compared with collagen-1, gene expression. BAV occurred in 30% of eNOS−/− mice. At age 6 mo, collagen was increased in both bicuspid and trileaflet eNOS−/− aortic valves, compared with wild-type valves. At 18 mo, total collagen was similar in eNOS−/− and wild-type mice, but collagen-3 was preferentially increased in eNOS−/− mice. Calcification and apoptosis were significantly increased in BAV of eNOS−/− mice at ages 6 and 18 mo. Remarkably, these histological changes were not accompanied by physiologically significant valve stenosis or regurgitation. In conclusion, coculture with vlvECs inhibits specific profibrotic VIC processes. In vivo, eNOS deficiency produces fibrosis in both trileaflet and BAVs but produces calcification only in BAVs.

Nuclear receptors (NRs) play a key role in endocrine signaling and metabolism and are important therapeutic targets in a number of hormone-dependent malignancies. Studies on the role of NRs in thyroid cancer are limited.The objective of the study was to examine systematically the expression of the 48 human NRs in a series of benign and malignant thyroid tissues. Within the papillary carcinoma cohort, we sought to determine if NR expression differed significantly by BRAF mutation status.RNA was isolated from multinodular goiter (MNG; n=6), papillary carcinoma (PTC, n=14), follicular carcinoma (FC; n=5), and Hürthle cell carcinoma (HCC; n=7). The 48 human NRs were profiled in this panel by quantitative real time polymerase chain reaction. Protein expression for selected NRs (Rev-erbα and LXR-β) was examined by immunohistochemistry (IHC) on tissue microarrays comprising benign and malignant thyroid tissues.Across all groups of benign and malignant thyroid tissue, there was prominent expression of LXR-β and ROR-γ. Key findings in PTC were marked overexpression of RXR-γ and Rev-erbα compared to MNG. Within the PTC cohort, when BRAF(V600E) tumors were compared with wild type BRAF, there was relative upregulation of RXR-γ and Rev-erbα and downregulation of AR, ERR-γ, and ROR-γ. In FC, EAR-2 was overexpressed, while PPAR-α and PPAR-δ were underexpressed compared to MNG. The NR expression profile of HCC was distinct, characterized by significant downregulation of a wide range of NRs. IHC for Rev-erbα and LXR-β localized protein expression to the tumor cells. Moderate to strong Rev-erbα immunostaining was seen in 22 out of 23 PTC, and, overall, staining was stronger than in the benign group.These results represent the first systematic examination of NR expression in thyroid cancer. Our finding of tumor-specific patterns of NR expression, as well as significant differences in NR expression between BRAF(V600E) and wild type BRAF PTC, provides a basis for further mechanistic studies and highlights potential novel therapeutic targets for this malignancy.

The emergence of transcriptomics, fuelled by high-throughput sequencing technologies, has changed the nature of cancer research and resulted in a massive accumulation of data. Computational analysis, integration, and data visualization are now major bottlenecks in cancer biology and translational research. Although many tools have been brought to bear on these problems, their use remains unnecessarily restricted to computational biologists, as many tools require scripting skills, data infrastructure, and powerful computational facilities. New user-friendly, integrative, and automated analytical approaches are required to make computational methods more generally useful to the research community. Here we present INsPeCT (INtegrative Platform for Cancer Transcriptomics), which allows users with basic computer skills to perform comprehensive in-silico analyses of microarray, ChlPseq, and RNA-seq data. INsPeCT supports the selection of interesting genes for advanced functional analysis. Included in its automated workflows are (i) a novel analytical framework, RMaNI (regulatory module network inference), which supports the inference of cancer subtype-specific transcriptional module networks and the analysis of modules; and (ii) WGCNA (weighted gene co-expression network analysis), which infers modules of highly correlated genes across microarray samples, associated with sample traits, eg survival time. INsPeCT is available free of cost from Bioinformatics Resource Australia-EMBL and can be accessed at http://inspect.braembl.org.au .

Previous research from our lab has shown that when using a rodent model of ischemic stroke (permanent middle cerebral artery occlusion), mild sensory stimulation, when delivered within two hours of ischemic onset, completely protects the cortex from impending ischemic stroke damage when assessed 24 hours post-occlusion. However, the long-term stability of this protection remains unclear. Using intrinsic signal optical imaging for assessment of cortical function, laser speckle imaging for assessment of blood flow, a battery of behavioral tests and cresyl violet for histological assessment, the present study examined whether this protection was long-lasting. When assessed 4 months post-occlusion (this length of time being equivalent to 10-15 years in humans), rats receiving sensory stimulation treatment immediately after ischemic onset exhibit normal neuronal and vascular function, and they are behaviorally and histologically equivalent to healthy controls (surgical shams). Thus, the complete neuroprotection due to cortical activation via sensory stimulation remains stable with time. These findings add support to the translational potential of this sensory stimulation-based treatment.

The Sleeping Beauty (SB) transposon mutagenesis screen is a powerful tool to facilitate the discovery of cancer genes that drive tumorigenesis in mouse models. In this study, we sought to identify genes that functionally cooperate with sonic hedgehog signaling to initiate medulloblastoma (MB), a tumor of the cerebellum. By combining SB mutagenesis with Patched1 heterozygous mice (Ptch1(lacZ/+)), we observed an increased frequency of MB and decreased tumor-free survival compared with Ptch1(lacZ/+) controls. From an analysis of 85 tumors, we identified 77 common insertion sites that map to 56 genes potentially driving increased tumorigenesis. The common insertion site genes identified in the mutagenesis screen were mapped to human orthologs, which were used to select probes and corresponding expression data from an independent set of previously described human MB samples, and surprisingly were capable of accurately clustering known molecular subgroups of MB, thereby defining common regulatory networks underlying all forms of MB irrespective of subgroup. We performed a network analysis to discover the likely mechanisms of action of subnetworks and used an in vivo model to confirm a role for a highly ranked candidate gene, Nfia, in promoting MB formation. Our analysis implicates candidate cancer genes in the deregulation of apoptosis and translational elongation, and reveals a strong signature of transcriptional regulation that will have broad impact on expression programs in MB. These networks provide functional insights into the complex biology of human MB and identify potential avenues for intervention common to all clinical subgroups.

Objective To reduce young children's distress and increase coping behavior among children undergoing a voiding cystourethrogram (VCU). Methods Three- to seven-year-old children were stratified based on prior VCU experience and randomly assigned to an intervention (n = 20) or a standard care (n = 20) condition. The intervention included provision of information, coping skills training, and parent coaching. We hypothesized that the intervention would reduce children's distress as assessed by child report, parent and technician ratings, and behavioral observations. Results Children in the intervention displayed fewer distress behaviors and greater coping behaviors and were rated as more cooperative than children receiving standard care. Children's fear and pain ratings did not differ significantly between groups. Conclusions A cognitive-behavioral treatment package effectively reduced children's distress, increased coping, and increased cooperation during voiding cystourethrogram procedures. This type of an intervention should be integrated into routine pediatric radiological procedures.

The effects of interleukin-1 alpha (IL-1alpha) on chromium-51 absorption, tissue retention, and urinary excretion were studied in adult male Sprague-Dawley rats. Ten rats were deprived of food for 12 h, injected intraperitoneally with mouse recombinant IL-1alpha (1 microg/kg of body weight in phosphate-buffered saline [PBS]) or control (0.1% bovine serum albumin [BSA] in PBS). Two hours after dosing with the IL-1alpha, rats were fed 50 microL (200 microCi, 0.36 microg Cr) of 51CrCl3 by micropipet. Blood was collected from the tail at 0.25, 0.5, 1, 2, and 4 h. Six hours after dosing with 51CrCl3, rats were exsanguinated and blood and tissues were sampled. The IL-1alpha significantly decreased chromium-51 in blood, urine, and some tissues compared to the control. The decreased absorption, retention, and urinary excretion of chromium-51 from 51CrCl3 in this study may be due to IL-1alpha-mediated increases in the production of prostaglandins and/or decreased production of gastric acid.

A cDNA encoding pinto bean α-D-galactosidase [E.C. 3.2.1.22] was obtained by amplification of cDNA using highly conserved sequences found in eucaryotic α-D-galactosidases. Subsequently a full length Phaseolus cDNA clone was obtained that is 1537 nt long and contains untranslated 5′ and 3′ sequences. The nucleotide sequence of the cDNA has a high degree of homology with other eucaryotic (α-D-galactosidase genes. The recombinant α-D-galactosidase (rGal) was expressed in Escherichia coli and purified by ion exchange and affinity chromatography. Purified rGal was homogeneous by SDS-PAGE and had relative masses of 40.1 and 45.4 kDa under nonreducing and reducing conditions, respectively. The N-terminal sequence of the expressed protein contained the sequence GNGLGQTPPMG corresponding to that deduced from the cDNA sequence. The native molecular weight for rGal was determined to be 32.18 kDa by Sephacryl S-200 chromatography. The specific activity of the rGal was 349 μmoles of PNP-α-D-galactopyranoside hydrolyzed per mg of pure rGal per min. rGal was highly specific for α-D-galactosyl residues and degraded B oligosaccharide. No detectable hemagglutinin or protease activity was present in the preparations. Furthermore, rGal was active against the blood group B antigen on native human erythrocytes in cell suspension assays. The only detectable RBC phenotypic change was loss of the B and P1 epitopes. Recombinant Phaseolus vulgaris α-D-galactosidase may have useful biotechnical applications in the potential mass production of enzymatically converted, universally transfusable type O RBCs. α-D-galactosidase [E.C. 3.2.1.22] has been purified from a variety of procaryotic and eucaryotic species (1,2,3,4). Most α-D-galactosidases have similar low molecular weight substrate specificities, but activity against high molecular weight substrates is variable (5,6). Terminal α-D-galactoside residues are present in glycoproteins and glycolipids (7). Some α-D-galactosidases have activity against α-D-galactosyl residues on cell membrane glycoconjugates. Glycosidases with this property are useful for carbohydrate structural studies and biotechnical applications (8,9). Enzymes free of other glycosidase activities with activity near neutral pH are particularly useful for membrane modification studies on native cells. Complex sugar chains in glycolipids and glycoproteins have often been implicated in the growth and development of eucaryotes (10). In particular, complex sugar chains play an important role in the recognition of self in the immune system (11). Some α-D-galactosidases can modify certain carbohydrate membrane epitopes, thereby modulating the immune response. For example, the blood group B epitope expressed on erythrocytes contains a terminal α-D-galactosyl residue. Individuals lacking this antigen produce naturally occurring complement fixing antibodies to the B epitope (12). Hydrolysis of this terminal saccharide destroys the antigenic activity of the B determinant producing H antigen (blood type O) on erythrocytes (13). Only rare individuals produce clinically significant antibodies to the H antigen, and therefore, type O red blood cells are “universally” compatible and in great demand (14). Dhar purified α-D-galactosidase isozymes from Phaseolus vulgaris and characterized their activity (15). To our knowledge, our laboratory, in a brief report, is the first to describe the cloning of the gene and the use of recombinant enzyme for seroconverting blood type B to O cells (16). This paper describes the cloning, sequence, expression, purification, and characterization of recombinant α-D-galactosidase. Activity of the recombinant enzyme on the native human erythrocyte blood group B epitope is shown.

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow (ischemia). Middle cerebral artery (MCA) delivers a great majority of blood to the lateral surface of the cortex, is the most common site of human stroke, and ischemia within its territory can result in extensive dysfunction or death. Survivors of ischemic stroke often suffer loss or disruption of motor capabilities, sensory deficits, and infarct. In an effort to capture these key characteristics of stroke, and thereby develop effective treatment, a great deal of emphasis is placed upon animal models of ischemia in MCA. Here we present a method of permanently occluding a cortical surface blood vessel. We will present this method using an example of a relevant vessel occlusion that models the most common type, location, and outcome of human stroke, permanent middle cerebral artery occlusion (pMCAO). In this model, we surgically expose MCA in the adult rat and subsequently occlude via double ligature and transection of the vessel. This pMCAO blocks the proximal cortical branch of MCA, causing ischemia in all of MCA cortical territory, a large portion of the cortex. This method of occlusion can also be used to occlude more distal portions of cortical vessels in order to achieve more focal ischemia targeting a smaller region of cortex. The primary disadvantages of pMCAO are that the surgical procedure is somewhat invasive as a small craniotomy is required to access MCA, though this results in minimal tissue damage. The primary advantages of this model, however, are: the site of occlusion is well defined, the degree of blood flow reduction is consistent, functional and neurological impairment occurs rapidly, infarct size is consistent, and the high rate of survival allows for long-term chronic assessment.

Membrane microdomains such as lipid rafts and caveolae regulate a myriad of cellular functions including cell signalling, protein trafficking, cell viability, and cell movement. They have been implicated in diseases such as cancer, diabetes and Alzheimer's disease, highlighting the essential role they play in cell processes. Despite much research and debate on the size, composition and dynamics of membrane microdomains, the molecular mechanism(s) of their action remain poorly understood. Most studies have dealt solely with the content and properties of the membrane microdomain as an entity in itself. However, recent work shows that membrane microdomain disruption has wide ranging effects on other subcellular compartments, and the cell as a whole. Hence we propose that a systems approach incorporating many cellular attributes such as subcellular localisation is required in order to understand the global impact of microdomains on cell function. Although analysis of sub-proteome changes already provides additional insight, we further propose biological network analysis of functional proteomics data to capture effects at the systems level. In this review, we highlight the use of protein-protein interactions networks and mixed networks to portray and visualize the relationships between proteins within and between subcellular fractions. Such a systems analysis will be required to improve our understanding of the full cellular function of membrane microdomains.

Inference of gene regulatory network from expression data is a challenging task. Many methods have been developed to this purpose but a comprehensive evaluation that covers unsupervised, semi-supervised and supervised methods, and provides guidelines for their practical application, is lacking. We performed an extensive evaluation of inference methods on simulated and experimental expression data. The results reveal low prediction accuracies for unsupervised techniques with the notable exception of the Z-SCORE method on knockout data. In all other cases, the supervised approach achieved the highest accuracies and even in a semi-supervised setting with small numbers of only positive samples, outperformed the unsupervised techniques.

OBJECTIVE:Sustained hemodynamic stress mediated by high blood flow promotes arteriogenesis, the outward remodeling of existing arteries. Here, we examined whether Ca²⁺/calmodulin-dependent kinase II (CaMKII) regulates arteriogenesis. METHODS AND RESULTS:Ligation of the left common carotid led to an increase in vessel diameter and perimeter of internal and external elastic lamina in the contralateral, right common carotid. Deletion of CaMKIIδ (CaMKIIδ-/-) abolished this outward remodeling. Carotid ligation increased CaMKII expression and was associated with oxidative activation of CaMKII in the adventitia and endothelium. Remodeling was abrogated in a knock-in model in which oxidative activation of CaMKII is abolished. Early after ligation, matrix metalloproteinase 9 (MMP9) was robustly expressed in the adventitia of right carotid arteries of WT but not CaMKIIδ-/- mice. MMP9 mainly colocalized with adventitial macrophages. In contrast, we did not observe an effect of CaMKIIδ deficiency on other proposed mediators of arteriogenesis such as expression of adhesion molecules or smooth muscle proliferation. Transplantation of WT bone marrow into CaMKIIδ-/- mice normalized flow-mediated remodeling. CONCLUSION:CaMKIIδ is activated by oxidation under high blood flow conditions and is required for flow-mediated remodeling through a mechanism that includes increased MMP9 expression in bone marrow-derived cells invading the arterial wall.

51 Chromium excretion was studied in adult female Sprague Dawley rats following bile-duct cannulation. The cannulation technique allowed the separation of bile from pancreatic secretions while enterohepatic circulation remained intact. Three days post-surgery, animals were deprived of food overnight and intubated with a 1 ml solution of aspirin (20 mg/kg body weight) in 20% ethanol or 1 ml of 20% ethanol as a control. Intubation was followed immediately by an oral dose of 25 μCi of 51 CrCl 3 (0.18 μg of chromium) by micropipette. Weighed samples of bile and blood were collected at 0, 15 and 30 minutes, and 1, 2 and 4 hours after dosing. Six days post-surgery, the same bile-duct cannulated rats were dosed with either aspirin in 20% ethanol or 20% ethanol alone in a crossover design. Aspirin increased 51 Cr in blood compared to control but did not affect the concentration of 51 Cr in bile significantly. Concentrations of 51 Cr in bile were lower than in blood at all time periods. While bile appears to be a major avenue for the elimination of managese and copper, our data indicate that the biliary excretion of chromium during the first 4 hours after dosing is minimal and that biliary excretion is not an important homeostatic mechanism for chromium.

Background Cancer outlier profile analysis (COPA) has proven to be an effective approach to analyzing cancer expression data, leading to the discovery of the TMPRSS2 and ETS family gene fusion events in prostate cancer. However, the original COPA algorithm did not identify down-regulated outliers, and the currently available R package implementing the method is similarly restricted to the analysis of over-expressed outliers. Here we present a modified outlier detection method, mCOPA, which contains refinements to the outlier-detection algorithm, identifies both over- and under-expressed outliers, is freely available, and can be applied to any expression dataset. Results We compare our method to other feature-selection approaches, and demonstrate that mCOPA frequently selects more-informative features than do differential expression or variance-based feature selection approaches, and is able to recover observed clinical subtypes more consistently. We demonstrate the application of mCOPA to prostate cancer expression data, and explore the use of outliers in clustering, pathway analysis, and the identification of tumour suppressors. We analyse the under-expressed outliers to identify known and novel prostate cancer tumour suppressor genes, validating these against data in Oncomine and the Cancer Gene Index. We also demonstrate how a combination of outlier analysis and pathway analysis can identify molecular mechanisms disrupted in individual tumours. Conclusions We demonstrate that mCOPA offers advantages, compared to differential expression or variance, in selecting outlier features, and that the features so selected are better able to assign samples to clinically annotated subtypes. Further, we show that the biology explored by outlier analysis differs from that uncovered in differential expression or variance analysis. mCOPA is an important new tool for the exploration of cancer datasets and the discovery of new cancer subtypes, and can be combined with pathway and functional analysis approaches to discover mechanisms underpinning heterogeneity in cancers

Objective Sustained hemodynamic stress mediated by high blood flow promotes arteriogenesis, the outward remodeling of existing arteries. Here, we examined whether Ca2+/calmodulin-dependent kinase II (CaMKII) regulates arteriogenesis. Methods and Results Ligation of the left common carotid led to an increase in vessel diameter and perimeter of internal and external elastic lamina in the contralateral, right common carotid. Deletion of CaMKIIδ (CaMKIIδ−/−) abolished this outward remodeling. Carotid ligation increased CaMKII expression and was associated with oxidative activation of CaMKII in the adventitia and endothelium. Remodeling was abrogated in a knock-in model in which oxidative activation of CaMKII is abolished. Early after ligation, matrix metalloproteinase 9 (MMP9) was robustly expressed in the adventitia of right carotid arteries of WT but not CaMKIIδ−/− mice. MMP9 mainly colocalized with adventitial macrophages. In contrast, we did not observe an effect of CaMKIIδ deficiency on other proposed mediators of arteriogenesis such as expression of adhesion molecules or smooth muscle proliferation. Transplantation of WT bone marrow into CaMKIIδ−/− mice normalized flow-mediated remodeling. Conclusion CaMKIIδ is activated by oxidation under high blood flow conditions and is required for flow-mediated remodeling through a mechanism that includes increased MMP9 expression in bone marrow-derived cells invading the arterial wall.

Effects of five over-the-counter drugs on retention and urinary excretion of 51 chromium from 51 chromium chloride ( 51 CrCl 3 ) were evaluated using adult female rats. In addition to the control group dosed with distilled water (pH 7.89), groups were dosed with vitamin C (5 mg, pH 2.76), Tums® (40 mg, pH 9.72), Maalox Plus® (40 mg, pH 8.39), aspirin (40 mg, pH 2.74) or Bufferin® (40 mg, pH 8.73). After a 12 h fast, the drugs were administered by gastric intubation (0.5 ml) followed immediately by an oral dose of 51 CrCl 3 (55 uCi) in 0.14 M HCl (50 ul). Blood was sampled from the tail at 0.75, 1.5, 3.0 and 6.0 h. Urine samples were composited at 3, 6, and I2 h and tissues were sampled at 12 h. 51 Chromium in blood, urine and tissues was higher (p 51 Cr in blood, urine and tissues than the other groups. Increased 51 Cr absorption in the aspirin group and decreased 51 Cr absorption in the Maalox group did not appear to be mediated solely by pH. Potential effects of long-term use of aspirin or antacids on Cr status need evaluation because over-the-counter drugs altered Cr absorption in this study.

Background Accumulated research has shown that the older adult brain is significantly more vulnerable to stroke than the young adult brain. Although recent evidence in young adult rats demonstrates that single‐whisker stimulation can result in complete protection from ischemic damage after permanent middle cerebral artery occlusion ( pMCAO ), it remains unclear whether the same treatment would be effective in older animals. Methods and Results Aged rats (21 to 24 months of age) underwent pMCAO and subsequently were divided into “treated” and “untreated” groups. Treated aged rats received intermittent single‐whisker stimulation during a 120‐minute period immediately after pMCAO , whereas untreated aged rats did not. These animals were assessed using a battery of behavioral tests 1 week before and 1 week after pMCAO , after which their brains were stained for infarct. An additional treated aged group and a treated young adult group also were imaged with functional imaging. Results demonstrated that the recovery of treated aged animals was indistinguishable from that of the treated young adult animals. Treated aged rats had fully intact sensorimotor behavior and no infarct, whereas untreated aged rats were impaired and sustained cortical infarct. Conclusions Taken together, our results confirm that single‐whisker stimulation is protective in an aged rodent pMCAO model, despite age‐associated stroke vulnerability. These findings further suggest potential for translation to the more clinically relevant older adult human population.

S SELECTED FOR PRESENTATION 14.4 Inhibition of sympathetic activity by rilmenidine reverses angiotensin II-induced hypertension, blood pressure variability and cardiac remodeling R. Sabharwal, R.N. El Accaoui, R.M. Weiss, M.K. Davis, F.M. Abboud, M.W. Chapleau Department of Internal Medicine, University of Iowa Veterans Affairs Medical Center, USA The relative contribution of increased sympathetic activity to angiotensin II (Ang II)-induced hypertension and end-organ damage is controversial. To determine the importance of the sympathetic nervous system, we examined whether sustained inhibition of sympathetic activity induced by intracerebroventricular infusion of rilmenidine (RIL) attenuates Ang II-induced hypertension, blood pressure variability and left ventricular (LV) remodeling. Blood pressure (BP) and heart rate (HR) were measured by telemetry in C57BL/6 mice (n= 7) and mice deficient in the antioxidant enzyme methionine sulfoxide reductase A (MsrA) (n= 8), before and during four weeks of Ang II infusion (1000 ng/kg/min, SC). LV end-diastolic volume (EDV), ejection fraction and mass were measured by echocardiography. Subgroups of mice were administered RIL (2.5 mg/kg/day, ICV, n = 4) over the last 2 weeks of Ang II infusion. Ang II infusion increased mean BP, BP variability, cardiac and vasomotor sympathetic tone, and LVEDV/mass ratio; and decreased cardiac vagal tone and spontaneous baroreflex sensitivity (sequence technique) (P b 0.05). All measures of dysfunction were exacerbated in MsrA−/− mice. As expected, RIL decreased sympathetic tone in all mice. Remarkably, RIL completely reversed Ang II-induced hypertension, BP variability, autonomic dysregulation, and LV remodeling in C57BL/6 andMsrA−/− mice (P b 0.05). RIL also increased (normalized) LV ejection fraction and locomotor activity in Ang II-infused MsrA−/− mice (P b 0.05). We conclude that Ang II-induced hypertension, BP variability and LV remodeling are critically dependent on increased sympathetic activity. The results strongly support therapeutic targeting of the brain to inhibit sympathetic activity in hypertension. (HL14388, VA). doi:10.1016/j.autneu.2015.07.320 14.5 Brain and renal alterations following denervation of ischemic kidney in renovascular hypertension E.E. Nishi, C.T. Bergamaschi, G.N. Gomes, J.C. Perry, G.S. Lincevicius, R.R. Campos Physiology Dpt., Universidade Federal de Sao Paulo, Brazil Psychobiology Dpt., Universidade Federal de Sao Paulo, Brazil Our aim was to investigate whether renal nerves play a role in the renal injury process and modulation of AngII receptors expression in the central nuclei and kidneys in renovascular hypertension. Denervation of clipped kidney (RD: visible bundles were dissected + 10% phenol) was performed in Wistar male rats (n = 5/group) 4–5 weeks after clip implantation (gap width: 0.2 mm). Ten days after RD, blood pressure (BP) was significantly reduced in the 2 K-1C rats. AT1R and AT2R upregulation within the rostral ventrolateral medulla (RVLM) and paraventricular nucleus of the hypothalamus (PVN) in the 2 K-1C group was normalized after RD. No change in the expression of either AngII receptor was found within the nucleus of the tractus solitarius (NTS). Following RD, there was no change in plasma renin activity in control and 2 K-1C rats. In order to distinguish the effects of RD from the reduction in BP, 2 K-1C rats were treated orally with hydralazine (25 mg/kg/day for seven consecutive days). Hydralazine significantly reduced BP and AT1R within the PVN in the 2 K-1C rats, but there were no changes in AT1R or AT2R within the RVLM and NTS. Following RD, renal injury was attenuated in both ischemic and contralateral kidneys of hypertensive rats, although hydralazine decreased injury only in the contralateral kidney. These results demonstrate that the renal nerves may modulate AngII receptors within the RVLM and renal injury in the ischemic kidney independent of BP and plasma renin activity in the 2 K-1C model of arterial hypertension. Supported by FAPESP and CNPq. doi:10.1016/j.autneu.2015.07.321 Symposium 15: Sleep and autonomic nervous system: At the crossroad between health and disease 15.1 Cardiovascular alterations induced by acute paradoxical sleep deprivation in rats

Background Altered networks of gene regulation underlie many complex conditions, including cancer. Inferring gene regulatory networks from high-throughput microarray expression data is a fundamental but challenging task in computational systems biology and its translation to genomic medicine. Although diverse computational and statistical approaches have been brought to bear on the gene regulatory network inference problem, their relative strengths and disadvantages remain poorly understood, largely because comparative analyses usually consider only small subsets of methods, use only synthetic data, and/or fail to adopt a common measure of inference quality. Methods We report a comprehensive comparative evaluation of nine state-of-the art gene regulatory network inference methods encompassing the main algorithmic approaches (mutual information, correlation, partial correlation, random forests, support vector machines) using 38 simulated datasets and empirical serous papillary ovarian adenocarcinoma expression-microarray data. We then apply the best-performing method to infer normal and cancer networks. We assess the druggability of the proteins encoded by our predicted target genes using the CancerResource and PharmGKB webtools and databases. Results We observe large differences in the accuracy with which these methods predict the underlying gene regulatory network depending on features of the data, network size, topology, experiment type, and parameter settings. Applying the best-performing method (the supervised method SIRENE) to the serous papillary ovarian adenocarcinoma dataset, we infer and rank regulatory interactions, some previously reported and others novel. For selected novel interactions we propose testable mechanistic models linking gene regulation to cancer. Using network analysis and visualization, we uncover cross-regulation of angiogenesis-specific genes through three key transcription factors in normal and cancer conditions. Druggabilty analysis of proteins encoded by the 10 highest-confidence target genes, and by 15 genes with differential regulation in normal and cancer conditions, reveals 75% to be potential drug targets. Conclusions Our study represents a concrete application of gene regulatory network inference to ovarian cancer, demonstrating the complete cycle of computational systems biology research, from genome-scale data analysis via network inference, evaluation of methods, to the generation of novel testable hypotheses, their prioritization for experimental validation, and discovery of potential drug targets.

Imaging of pregnant patients with non-obstetric abdominal pain is reviewed, with an accompanying pictorial essay of cases with concentration on magnetic resonance imaging. Non-obstetric causes of abdominal pain during pregnancy are similar to those of non-pregnant patients. The most common causes are appendicitis and cholecystitis. Other causes are myriad and include biliary, gastrointestinal, infectious, inflammatory, and malignant etiologies, among others. The approach to imaging in pregnant patient is unique, as it is imperative to minimize potentially harmful radiation exposures to the fetus. Ultrasound and MRI are the primary modalities for evaluation of the pregnant patient with abdominal pain. The use of intravenous contrast is discouraged, except in highly-selected patients where there is no other way to obtain vital diagnostic information. CT is still used as the mainstay of evaluation of blunt abdominal trauma and is commonly used for diagnosis of small bowel obstruction, stone disease, and work-up of malignancy during pregnancy. A discussion of test selection and underlying rationale is presented.

Motivation: Protein–protein interactions (PPIs) are pivotal for many biological processes and similarity in Gene Ontology (GO) annotation has been found to be one of the strongest indicators for PPI. Most GO-driven algorithms for PPI inference combine machine learning and semantic similarity techniques. We introduce the concept of inducers as a method to integrate both approaches more effectively, leading to superior prediction accuracies. Results: An inducer (ULCA) in combination with a Random Forest classifier compares favorably to several sequence-based methods, semantic similarity measures and multi-kernel approaches. On a newly created set of high-quality interaction data, the proposed method achieves high cross-species prediction accuracies (Area under the ROC curve ≤ 0.88), rendering it a valuable companion to sequence-based methods. Availability: Software and datasets are available at http://bioinformatics.org.au/go2ppi/ Contact: m.ragan@uq.edu.au

Hypertrophic osteoarthropathy is a paraneoplastic syndrome most often found in non-small cell lung cancer. Diagnosis is confirmed by the presence of clubbing on physical examination and periostitis on bone scintigram, and the syndrome generally resolves with treatment of the underlying malignancy. This article presents a case study and describes symptom management options, including nonsteroidal anti-inflammatory agents, octreotide, and bisphosphonates.

The purpose of this study was to evaluate the MRI findings of children with rheumatoid arthritis (JRA) affecting the temporomandibular joints (TMJ) and correlate these findings with symptoms. MRI studies of the TMJ in 26 children with a clinical diagnosis of JRA were retrospectively reviewed. All studies included oblique and sagittal T1, T2*, proton density/T2, and coronal T1-weighted images. T1 and proton density/T2-images were repeated with the mouth open. Post contrast sagittal and coronal T1-images were obtained in 19 patients. All studies were done on either 1.5 Tesla or 3.0 Tesla units with dedicated surface coils. By consensus, two radiologists evaluated the studies for abnormal condyles, bone erosions, presence or absence of discs, effusions, contrast enhancement and pannus. Open mouth views were assessed for incomplete or abnormal translation. Clinical records were reviewed to correlate symptoms with MRI findings. Abnormal condyles were seen in 49%. Discs were identifiable in 71%. Abnormal translation was seen in 71% and pannus in 49%. Erosions were seen in 37%, effusions in 24% and contrast enhancement in 50%. Correlation with clinical examination showed that of five asymptomatic patients, three had abnormal translation. Fifteen patients presented joint asymmetry on clinical examination and all showed abnormal translation on MRI. Our findings suggest that abnormal translation and joint enhancement may be the most common MRI findings in JRA patients with TMJ arthritis. Abnormalities may occur even in the absence of symptoms and the most common finding in symptomatic patients is abnormal translation.

Granulosa cell tumors of the ovary (GCT) represent ~5% of malignant ovarian tumors. The adult form is defined by a mutation in the FOXL2 gene. GCT exhibit many of the features of normal proliferating granulosa cells. We have profiled the expression of the 48 human nuclear receptors (NR) by quantitative RT-PCR in a panel of GCT and in two GCT-derived cell lines, COV434 and KGN. The highest level of expression is seen for COUP-TF2 with abundant expression of PPARγ, SF-1, and TR-α. Estrogen receptor (ER)-β is the most abundant of the steroid receptors with relatively high expression also of AR, ER-α, and PR. The concordance of expression for each NR across the tumors is remarkably high with same discordance between the cell lines and the tumors, particularly the COV434 line. No significant differences were observed with respect to tumor stage for NR expression. These findings provide a full profile of NR expression in GCT which will enable full characterization of their roles and potential as therapeutic targets.

Once-daily injections of teriparatide initially increase biochemical markers of bone formation and resorption, but markers peak after 6–12 months and then decline despite continued treatment. We sought to determine whether increasing teriparatide doses in a stepwise fashion could prolong skeletal responsiveness. We randomized 52 postmenopausal women with low spine and/or hip bone mineral density (BMD) to either a constant or an escalating subcutaneous teriparatide dose (30 mcg daily for 18 months or 20 mcg daily for 6 months, then 30 mcg daily for 6 months, then 40 mcg daily for 6 months). Serum procollagen I N-terminal propeptide, osteocalcin, and C-terminal telopeptide of type I collagen were assessed frequently. BMD of the spine, hip, radius, and total body was measured every 6 months. Acute changes in urinary cyclic AMP in response to teriparatide were examined in a subset of women in the constant dose group. All bone markers differed significantly between the two treatment groups. During the final six months, bone markers declined in the constant dose group but remained stable or increased in the escalating dose group (all markers, p

Among the ways corporations use their enormous wealth is to support private justice systems complete with investigative, adjudicatory, and sentencing powers. The present paper advances a sociological analysis of a private corporate justice system and the private police who run it. Our research setting is a full line store of one of the nation's largest corporations. Our focus is private justice for shoplifters in a civil recovery state. Analysis reveals that retail value of the item taken, neighborhood social class, and physical resistance are among the factors determining private corporate justice for shoplifters in a civil recovery state. The findings suggest the part played by private justice systems in shaping public images of crime and criminals.

Membrane organization describes the relationship of proteins to the membrane, that is, whether the protein crosses the membrane or is integral to the membrane and its orientation with respect to the membrane. Membrane organization is determined primarily by the presence of two features which target proteins to the secretory pathway: the endoplasmic reticulum signal peptide and the ?-helical transmembrane domain. In order to generate membrane organization annotation of high quality, confidence and throughput, the Membrane Organization (MemO) pipeline was developed, incorporating consensus feature prediction modules with integration and annotation rules derived from biological observations. The pipeline classifies proteins into six categories based on the presence or absence of predicted features: Soluble, intracellular proteins; Soluble, secreted proteins; Type I membrane proteins; Type II membrane proteins; Multi-span membrane proteins and Glycosylphosphatidylinositol anchored membrane proteins. The MemO pipeline represents an integrated strategy for the application of state-of-the-art bioinformatics tools to the annotation of protein membrane organization, a property which adds biological context to the large quantities of protein sequence information available.

e19059 Background: About 30-44% of patients with non-small cell lung cancer (NSCLC) will develop brain metastases, with a median survival if untreated of less than 3 months. Treatment options are l...

Application of a computational membrane organization prediction pipeline, MemO, identified putative type II membrane proteins as proteins predicted to encode a single alpha-helical transmembrane domain (TMD) and no signal peptides. MemO was applied to RIKEN's mouse isoform protein set to identify 1436 non-overlapping genomic regions or transcriptional units (TUs), which encode exclusively type II membrane proteins. Proteins with overlapping predicted InterPro and TMDs were reviewed to discard false positive predictions resulting in a dataset comprised of 1831 transcripts in 1408 TUs. This dataset was used to develop a systematic protocol to document subcellular localization of type II membrane proteins. This approach combines mining of published literature to identify subcellular localization data and a high-throughput, polymerase chain reaction (PCR)-based approach to experimentally characterize subcellular localization. These approaches have provided localization data for 244 and 169 proteins. Type II membrane proteins are localized to all major organelle compartments; however, some biases were observed towards the early secretory pathway and punctate structures. Collectively, this study reports the subcellular localization of 26% of the defined dataset. All reported localization data are presented in the LOCATE database (http://www.locate.imb.uq.edu.au).

Membrane organization describes the orientation of a protein with respect to the membrane and can be determined by the presence, or absence, and organization within the protein sequence of two features: endoplasmic reticulum signal peptides and alpha-helical transmembrane domains. These features allow protein sequences to be classified into one of five membrane organization categories: soluble intracellular proteins, soluble secreted proteins, type I membrane proteins, type II membrane proteins, and multi-spanning membrane proteins. Generation of protein isoforms with variable membrane organizations can change a protein's subcellular localization or association with the membrane. Application of MemO, a membrane organization annotation pipeline, to the FANTOM3 Isoform Protein Sequence mouse protein set revealed that within the 8,032 transcriptional units (TUs) with multiple protein isoforms, 573 had variation in their use of signal peptides, 1,527 had variation in their use of transmembrane domains, and 615 generated protein isoforms from distinct membrane organization classes. The mechanisms underlying these transcript variations were analyzed. While TUs were identified encoding all pairwise combinations of membrane organization categories, the most common was conversion of membrane proteins to soluble proteins. Observed within our high-confidence set were 156 TUs predicted to generate both extracellular soluble and membrane proteins, and 217 TUs generating both intracellular soluble and membrane proteins. The differential use of endoplasmic reticulum signal peptides and transmembrane domains is a common occurrence within the variable protein output of TUs. The generation of protein isoforms that are targeted to multiple subcellular locations represents a major functional consequence of transcript variation within the mouse transcriptome.

The ability of breast cancer cells to switch between epithelial and mesenchymal phenotypes may be key to their survival in new environments, resistance to therapies and ability to form metastases. Epithelial mesenchymal plasticity (EMP) is instrumental in embryological development and has been implicated in stemness, therapy resistance and metastasis of breast cancer. EMP markers are enriched in basal-like, triple negative breast cancer, which is a type of breast cancer associated with early recurrence and poor prognosis, and established as a common phenotype in women with BRCA1 mutations. The EMPathy Breast Cancer Network (BCN) is a national collaborative effort including scientists, surgeons, medical oncologists and a consumer advocate investigating the role of EMP in breast cancer recurrence. The 7 thematic research projects of EMPathy BCN, including the 9 program-funded ‘Satellite’ projects, are aligned with the Cooperative Research Centre for Cancer Therapeutics (CTx) (www.cancercrc.com/index), so that any potential drug targets identified may progress into the CTx drug development program. Multiple parallel approaches in the Target Discovery theme were used to identify candidate regulators and effectors of EMP. A total of 10 functional or gene expression experiments provided 7,950 significant events in any one system, which were cross referenced against 10 public breast cancer datasets relevant to EMP and/or breast cancer stem cells. A series of criteria were used to select a panel of 127 candidates that were combined with 123 ad hoc candidates (mainly hits close to the cut-off and breast cancer context genes) to give a total of 250 candidates to be analysed in breast cancer tissues using Nanostring technology. The 2,301 ‘significant events’ in any functional screen were further cross-referenced to 10 public functional datasets relevant to EMP in any system and a series of criteria were used to select a panel of 320 candidates that are to be analysed in an siRNA ‘functional screen' of multiple breast cancer cell lines, to support the choice of Candidate Targets for drug development. Ongoing studies will address the biology behind selected Candidates. This work and the EMPathy BCN is supported by a NBCF National Collaborative Research Program Grant. Citation Format: Tony Blick, Gayle Phillip, Eva Tomaskovic-Crook, Annet Hammacher, Nicholas Wong, Izhak Haviv, The EMPathy Breast Cancer Network, Greg Goodall, Melissa Davis, Erik W. Thompson. Integrated target discovery in the EMPathy Breast Cancer Network - Multidimensional analysis of epithelial mesenchymal plasticity (EMP) in experimental systems. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1060. doi:10.1158/1538-7445.AM2014-1060

We present here LOCATE, a curated, web-accessible database that houses data describing the membrane organization and subcellular localization of proteins from the FANTOM3 Isoform Protein Sequence set. Membrane organization is predicted by the high-throughput, computational pipeline MemO. The subcellular locations of selected proteins from this set were determined by a high-throughput, immunofluorescence-based assay and by manually reviewing >1700 peer-reviewed publications. LOCATE represents the first effort to catalogue the experimentally verified subcellular location and membrane organization of mammalian proteins using a high-throughput approach and provides localization data for approximately 40% of the mouse proteome. It is available at http://locate.imb.uq.edu.au.

Isoaspartyl dipeptidase (IAD) is a member of the amidohydrolase superfamily and catalyzes the hydrolytic cleavage of beta-aspartyl dipeptides. Structural studies of the wild-type enzyme have demonstrated that the active site consists of a binuclear metal center positioned at the C-terminal end of a (beta/alpha)(8)-barrel domain. Steady-state kinetic parameters for the hydrolysis of beta-aspartyl dipeptides were obtained at pH 8.1. The pH-rate profiles for the hydrolysis of beta-Asp-Leu were obtained for the Zn/Zn-, Co/Co-, Ni/Ni-, and Cd/Cd-substituted forms of IAD. Bell-shaped profiles were observed for k(cat) and k(cat)/K(m) as a function of pH for all four metal-substituted forms. The pK(a) of the group that must be unprotonated for catalytic activity varied according to the specific metal ion bound in the active site, whereas the pK(a) of the group that must be protonated for catalytic activity was relatively independent of the specific metal ion present. The identity of the group that must be unprotonated for catalytic activity was consistent with the hydroxide that bridges the two divalent cations of the binuclear metal center. The identity of the group that must be protonated for activity was consistent with the free alpha-amino group of the dipeptide substrate. Kinetic constants were obtained for the mutant enzymes at conserved residues Glu77, Tyr137, Arg169, Arg233, Asp285, and Ser289. The catalytic properties of the wild-type and mutant enzymes, coupled with the X-ray crystal structure of the D285N mutant complexed with beta-Asp-His, are consistent with a chemical reaction mechanism for the hydrolysis of dipeptides that is initiated by the polarization of the amide bond via complexation to the beta-metal ion of the binuclear metal center. Nucleophilic attack by the bridging hydroxide is facilitated by abstraction of its proton by the side chain carboxylate of Asp285. Collapse of the tetrahedral intermediate and cleavage of the carbon-nitrogen bond occur with donation of a proton from the protonated form of Asp285.

We describe a phased screening system for discovery of compounds with antiviral activity against hepatitis C virus (HCV). The primary assay utilizes dicistronic subgenomic HCV replicons in which the upstream cistron was modified to express the human immunodeficiency virus (HIV) tat protein. When these replicons are stably transfected into Huh-7-derived cells that express secreted alkaline phosphatase (SEAP) under transcriptional control of the HIV long terminal repeat promoter, there is a strong correlation between intracellular HCV RNA abundance and the activity of SEAP secreted into the culture medium. Thus, active compounds are easily identified by direct enzymatic quantification of SEAP in the medium without post-assay processing. Compounds that reduce SEAP activity without causing cellular toxicity are next evaluated in a second Huh-7-derived cell line constitutively expressing SEAP under control of the tat-HIV promoter axis, independent of HCV RNA replication. This specificity control identifies compounds that cause reductions in SEAP that are unrelated to suppression of HCV RNA replication. Compounds showing HCV-specific activity in primary assays are next evaluated by real-time RT-PCR to directly quantify reductions in HCV RNA. We have found excellent agreement between the SEAP and RT-PCR assays. This phased system provides an efficient and cost-effective screen for compounds with antiviral activity against HCV.

Although many of the molecular interactions in kidney development are now well understood, the molecules involved in the specification of the metanephric mesenchyme from surrounding intermediate mesoderm and, hence, the formation of the renal progenitor population are poorly characterized. In this study, cDNA microarrays were used to identify genes enriched in the murine embryonic day 10.5 (E10.5) uninduced metanephric mesenchyme, the renal progenitor population, in comparison with more rostral derivatives of the intermediate mesoderm. Microarray data were analyzed using R statistical software to determine accurately genes differentially expressed between these populations. Microarray outliers were biologically verified, and the spatial expression pattern of these genes at E10.5 and subsequent stages of early kidney development was determined by RNA in situ hybridization. This approach identified 21 genes preferentially expressed by the E10.5 metanephric mesenchyme, including Ewing sarcoma homolog, 14-3-3 theta, retinoic acid receptor-alpha, stearoyl-CoA desaturase 2, CD24, and cadherin-11, that may be important in formation of renal progenitor cells. Cell surface proteins such as CD24 and cadherin-11 that were strongly and specifically expressed in the uninduced metanephric mesenchyme and mark the renal progenitor population may prove useful in the purification of renal progenitor cells by FACS. These findings may assist in the isolation and characterization of potential renal stem cells for use in cellular therapies for kidney disease.

A general overview of the protein sequence set for the mouse transcriptome produced during the FANTOM2 sequencing project is presented here. We applied different algorithms to characterize protein sequences derived from a nonredundant representative protein set (RPS) and a variant protein set (VPS) of the mouse transcriptome. The functional characterization and assignment of Gene Ontology terms was done by analysis of the proteome using InterPro. The Superfamily database analyses gave a detailed structural classification according to SCOP and provide additional evidence for the functional characterization of the proteome data. The MDS database analysis revealed new domains which are not presented in existing protein domain databases. Thus the transcriptome gives us a unique source of data for the detection of new functional groups. The data obtained for the RPS and VPS sets facilitated the comparison of different patterns of protein expression. A comparison of other existing mouse and human protein sequence sets (e.g., the International Protein Index) demonstrates the common patterns in mammalian proteomes. The analysis of the membrane organization within the transcriptome of multiple eukaryotes provides valuable statistics about the distribution of secretory and transmembrane proteins

We have developed a computational strategy to identify the set of soluble proteins secreted into the extracellular environment of a cell. Within the protein sequences predominantly derived from the RIKEN representative transcript and protein set, we identified 2033 unique soluble proteins that are potentially secreted from the cell. These proteins contain a signal peptide required for entry into the secretory pathway and lack any transmembrane domains or intracellular localization signals. This class of proteins, which we have termed the mouse secretome, included >500 novel proteins and 92 proteins

Background Cell survival and development are orchestrated by complex interlocking programs of gene activation and repression. Understanding how this gene regulatory network (GRN) functions in normal states, and is altered in cancers subtypes, offers fundamental insight into oncogenesis and disease progression, and holds great promise for guiding clinical decisions. Inferring a GRN from empirical microarray gene expression data is a challenging task in cancer systems biology. In recent years, module-based approaches for GRN inference have been proposed to address this challenge. Despite the demonstrated success of module-based approaches in uncovering biologically meaningful regulatory interactions, their application remains limited a single condition, without supporting the comparison of multiple disease subtypes/conditions. Also, their use remains unnecessarily restricted to computational biologists, as accurate inference of modules and their regulators requires integration of diverse tools and heterogeneous data sources, which in turn requires scripting skills, data infrastructure and powerful computational facilities. New analytical frameworks are required to make module-based GRN inference approach more generally useful to the research community. Results We present the RMaNI (Regulatory Module Network Inference) framework, which supports cancer subtype-specific or condition specific GRN inference and differential network analysis. It combines both transcriptomic as well as genomic data sources, and integrates heterogeneous knowledge resources and a set of complementary bioinformatic methods for automated inference of modules, their condition specific regulators and facilitates downstream network analyses and data visualization. To demonstrate its utility, we applied RMaNI to a hepatocellular microarray data containing normal and three disease conditions. We demonstrate that how RMaNI can be employed to understand the genetic architecture underlying three disease conditions. RMaNI is freely available at http://inspect.braembl.org.au/bi/inspect/rmani Conclusion RMaNI makes available a workflow with comprehensive set of tools that would otherwise be challenging for non-expert users to install and apply. The framework presented in this paper is flexible and can be easily extended to analyse any dataset with multiple disease conditions.

Drug-nutrient interactions affecting chromium were investigated in this study. Rats were injected with indomethacin to reduce endogenous prostaglandin synthesis and dosed with prostaglandin analogues or prostacyclin. Effects on absorption, tissue distribution and urinary excretion of 51 Cr from 51 CrCl 3 were evaluated using a 2 x 4 factorial experimental design. Forty-eight adult male rats were food deprived for 12 h and then injected intraperitoneally with indomethacin (5 mg/kg body wt) or placebo. Thirty minutes later, rats were intubated and dosed with one of four treatments: a prostaglandin E 1 analogue (misoprostol) at 50 μg/kg body wt; a prostaglandin E 2 analogue (16,16-dimethylprostaglandin E 2 ) at 7.5 μg/kg body wt; prostacyclin at 20 μg/kg body wt; or control (7.64 mmol/L Tween-80 suspended in 0.15 mol/L NaCI containing 0.48 mol/L ethanol). Immediately after intubation, rats were dosed with 3.7 mBq of 51 CrCl 3 by micropipette. Blood was collected from the tail at intervals after 51 Cr dosing. Six hours after dosing, 51 Cr rats were exsanguinated by cardiac puncture. Indomethacin, an inhibitor of prostaglandin synthesis, significantly increased (P < 0.05) 51 Cr in blood at all time periods tested except at 15 min. In tissues, indomethacin significantly increased 51 Cr retention. Urinary 51 Cr excretion at 6 h was higher (P < 0.05) in indomethacin-pretreated rats than in control rats. Administration of indomethacin, which blocks prostaglandin synthesis, enhanced 51 Cr absorption, whereas dosing with 16,16-dimethylprostaglandin E 2 decreased 51 Cr absorption.

Objective Sustained hemodynamic stress mediated by high blood flow promotes arteriogenesis, the outward remodeling of existing arteries. Here, we examined whether Ca2+/calmodulin-dependent kinase II (CaMKII) regulates arteriogenesis. Methods and Results Ligation of the left common carotid led to an increase in vessel diameter and perimeter of internal and external elastic lamina in the contralateral, right common carotid. Deletion of CaMKIIδ (CaMKIIδ−/−) abolished this outward remodeling. Carotid ligation increased CaMKII expression and was associated with oxidative activation of CaMKII in the adventitia and endothelium. Remodeling was abrogated in a knock-in model in which oxidative activation of CaMKII is abolished. Early after ligation, matrix metalloproteinase 9 (MMP9) was robustly expressed in the adventitia of right carotid arteries of WT but not CaMKIIδ−/− mice. MMP9 mainly colocalized with adventitial macrophages. In contrast, we did not observe an effect of CaMKIIδ deficiency on other proposed mediators of arteriogenesis such as expression of adhesion molecules or smooth muscle proliferation. Transplantation of WT bone marrow into CaMKIIδ−/− mice normalized flow-mediated remodeling. Conclusion CaMKIIδ is activated by oxidation under high blood flow conditions and is required for flow-mediated remodeling through a mechanism that includes increased MMP9 expression in bone marrow-derived cells invading the arterial wall.

Background There are no rigorously confirmed effective medical therapies for calcific aortic stenosis. Hypercholesterolemic Ldlr (-/-) Apob (100/100) mice develop calcific aortic stenosis and valvular cardiomyopathy in old age. Osteoprotegerin (OPG) modulates calcification in bone and blood vessels, but its effect on valve calcification and valve function is not known. Objectives To determine the impact of pharmacologic treatment with OPG upon aortic valve calcification and valve function in aortic stenosis-prone hypercholesterolemic Ldlr (-/-) Apob (100/100) mice. Methods Young Ldlr (-/-) Apob (100/100) mice (age 2 months) were fed a Western diet and received exogenous OPG or vehicle (N = 12 each) 3 times per week, until age 8 months. After echocardiographic evaluation of valve function, the aortic valve was evaluated histologically. Older Ldlr (-/-) Apob (100/100) mice were fed a Western diet beginning at age 2 months. OPG or vehicle (N = 12 each) was administered from 6 to 12 months of age, followed by echocardiographic evaluation of valve function, followed by histologic evaluation. Results In Young Ldlr (-/-) Apob (100/100) mice, OPG significantly attenuated osteogenic transformation in the aortic valve, but did not affect lipid accumulation. In Older Ldlr (-/-) Apob (100/100) mice, OPG attenuated accumulation of the osteoblast-specific matrix protein osteocalcin by ∼80%, and attenuated aortic valve calcification by ∼ 70%. OPG also attenuated impairment of aortic valve function. Conclusions OPG attenuates pro-calcific processes in the aortic valve, and protects against impairment of aortic valve function in hypercholesterolemic aortic stenosis-prone Ldlr (-/-) Apob (100/100) mice.

Prostate cancer (PCa) is one of the leading causes of cancer death in men. CD151 is a member of the tetraspanin family and is associated with regulation of migration of normal and tumour cells via cell surface microdomain formation. Previous studies from our laboratory revealed that expression of CD151 differs across histological grades of PCa and high levels of expression are linked to shorter survival, independent of Gleason score1. In vitro motility assays of human PCa cell lines suggest CD151 is a motility promoter2. We have sought to develop a number of CD151 inhibitors and examine their ability to modulate prostate cancer motility and metastasis. The human PCa cell line, LNCaP (low endogenous level of CD151), transfected with CD151 shows increased motility and invasion compared to control LNCaP, whilst CD151 siRNA knock-down (KD) of PC-3 cells (high endogenous level of CD151), reduces motility compared to control PC-3. We have conducted in silico screening with compounds predicted to bind a model of the large extracellular domain (EC2) of CD151 and found that a number of these small molecules possess bioactivity in vitro and in vivo in inhibiting prostate cancer motility and progression. LNCaP growth and migration was confirmed by stimulating with 1 and 10 nM DHT at 24 hrs in charcoal stripped FBS. It was found that cell proliferation and motility of LNCaP cells was significantly increased after stimulation by 10 nM DHT at 24 hrs (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 469. doi:1538-7445.AM2012-469

Expression of caveolin-1 is up-regulated in prostate cancer metastasis and is associated with aggressive recurrence of the disease. Intriguingly, caveolin-1 is also secreted from prostate cancer cell lines and has been identified in secreted prostasomes. Caveolin-1 is the major structural component of the plasma membrane invaginations called caveolae. Co-expression of the coat protein Polymerase I and transcript release factor (PTRF) is required for caveolae formation. We recently found that expression of caveolin-1 in the aggressive prostate cancer cell line PC-3 is not accompanied by PTRF, leading to noncaveolar caveolin-1 lipid rafts. Moreover, ectopic expression of PTRF in PC-3 cells sequesters caveolin-1 into caveolae. Here we quantitatively analyzed the effect of PTRF expression on the PC-3 proteome using stable isotope labeling by amino acids in culture and subcellular proteomics. We show that PTRF reduced the secretion of a subset of proteins including secreted proteases, cytokines, and growth regulatory proteins, partly via a reduction in prostasome secretion. To determine the cellular mechanism accounting for the observed reduction in secreted proteins we analyzed total membrane and the detergent-resistant membrane fractions. Our data show that PTRF expression selectively impaired the recruitment of actin cytoskeletal proteins to the detergent-resistant membrane, which correlated with altered cholesterol distribution in PC-3 cells expressing PTRF. Consistent with this, modulating cellular cholesterol altered the actin cytoskeleton and protein secretion in PC-3 cells. Intriguingly, several proteins that function in ER to Golgi trafficking were reduced by PTRF expression. Taken together, these results suggest that the noncaveolar caveolin-1 found in prostate cancer cells generates a lipid raft microenvironment that accentuates secretion pathways, possibly at the step of ER sorting/exit. Importantly, these effects could be modulated by PTRF expression.

Transcriptomics continues to provide ever-more evidence that in morphologically complex eukaryotes, each protein-coding genetic locus can give rise to multiple transcripts that differ in length, exon content and/or other sequence features. In humans, more than 60% of loci give rise to multiple transcripts in this way. Motifs that mediate protein-protein interactions can be present or absent in these transcripts. Analysis of protein interaction networks has been a valuable development in systems biology. Interactions are typically recorded for representative proteins or even genes, although exploratory transcriptomics has revealed great spatiotemporal diversity in the output of genes at both the transcript and protein-isoform levels. The increasing availability of high-resolution protein structures has made it possible to identify the domain-domain interactions that underpin many protein interactions. To explore the impact of transcript and isoform diversity we use full-length human cDNAs to interrogate the protein-coding transcriptional output of genes, identifying variation in the inclusion of protein interaction domains. We map these data to a set of high-quality protein interactions, and characterise the variation in network connectivity likely to result. We find strong evidence for altered interaction potential in nearly 20% of genes, suggesting that transcriptional variation can significantly rewire the human interactome.

A cDNA clone encoding the mature polypeptide for the chicken liver alpha-N-acetylgalactosaminidase enzyme was isolated by screening a cDNA library with a synthetic sequence derived from the human alpha-N-acetylgalactosaminidase gene. The deduced amino acid sequence of the chicken liver clone was identical to the amino-terminal and cyanogen bromide derived peptide sequences obtained from the purified enzyme. The chicken enzyme has 75% identity with the human alpha-N-acetylgalactosaminidase and 56-33% identity with alpha-galactosidases from several sources.

We studied American Oystercatchers (Haematopus palliatus) by examining reproductive success and the factors affecting it at one location, and by examining the status and trends of breeding population numbers from Florida to Nova Scotia on the east coast of North America. We conducted our field research on Cape Lookout National Seashore, Carteret County, North Carolina from 1997 to 1999, and we documented larger scale breeding population trends by contacting state biologists and reviewing the literature and historical accounts. Presence of eggs and young were checked two to five times per week (by observing from a distance), and efforts were made to determine the reasons for loss of the nest contents. We monitored a total of 245 nests and found low productivity. At least one egg in 32 (13%) nests hatched and one or more chicks fledged from 12 broods. Overall, 14 chicks successfully fledged (nine from North Core Banks and five from South Core Banks) during the three years of the study. Of the 213 clutches that did not hatch, 163 (76%) failed because of predation, and 46 (22%) because of overwash or severe weather. Our minimum estimate for the number of oystercatchers breeding along the entire Atlantic coast and the Gulf coast of Florida is 1,624 pairs. North of Virginia, numbers appear to be stable or slowly increasing and the species has expanded as far north as Cape Sable Island in Nova Scotia. From Virginia south, breeding numbers show a decline in recent years. The number of oystercatchers breeding on barrier islands in Virginia has decreased by more than 50% in the last 20 years. Given their relatively small numbers and inherently low productivity, American Oystercatchers are at risk in rapidly changing coastal ecosystems. Received 1 June 2000, accepted 11 October 2000.

In many instances, kidney dysgenesis results as a secondary consequence to defects in the development of the ureter. Through the use of mouse genetics a number of genes associated with such malformations have been identified, however, the cause of many other abnormalities remain unknown. In order to identify novel genes involved in ureter development we compared gene expression in embryonic day (E) 12.5, E15.5 and postnatal day (P) 75 ureters using the Compugen mouse long oligo microarrays. A total of 248 genes were dynamically upregulated and 208 downregulated between E12.5 and P75. At E12.5, when the mouse ureter is comprised of a simple cuboidal epithelium surrounded by ureteric mesenchyme, genes previously reported to be expressed in the ureteric mesenchyme, foxC1 and foxC2 were upregulated. By E15.5 the epithelial layer develops into urothelium, impermeable to urine, and smooth muscle develops for the peristaltic movement of urine towards the bladder. The development of these two cell types coincided with the upregulation of UPIIIa, RAB27b and PPARgamma reported to be expressed in the urothelium, and several muscle genes, Acta1, Tnnt2, Myocd, and Tpm2. In situ hybridization identified several novel genes with spatial expression within the smooth muscle, Acta1; ureteric mesenchyme and smooth muscle, Thbs2 and Col5a2; and urothelium, Kcnj8 and Adh1. This study marks the first known report defining global gene expression of the developing mouse ureter and will provide insight into the molecular mechanisms underlying kidney and lower urinary tract malformations.