PLoS ONE Alerts: New Articles

Expression of Concern: Immune Protection Induced on Day 10 Following Administration of the 2009 A/H1N1 Pandemic Influenza Vaccine

The PLoS ONE Editors — 2012-05-25T21:00:00Z

by The PLoS ONE Editors

Living on the Edge: Assessing the Extinction Risk of Critically Endangered Bonelli’s Eagle in Italy

2012-05-25T21:00:00Z

by Pascual López-López, Maurizio Sarà, Massimiliano Di Vittorio

Background

The population of Bonelli’s eagle (Aquila fasciata) has declined drastically throughout its European range due to habitat degradation and unnatural elevated mortality. There are less than 1500 breeding pairs accounted for in Europe, and the species is currently catalogued as Critically Endangered in Italy, where the 22 territories of Sicily, represent nearly 95% of the entire Italian population. However, despite national and European conservation concerns, the species currently lacks a specific conservation plan, and no previous attempts to estimate the risk of extinction have been made.

Methodology/Principal Findings

We incorporated the most updated demographic information available to assess the extinction risk of endangered Bonelli’s eagle in Italy through a Population Viability Analysis. Using perturbation analyses (sensitivity and elasticity), and a combination of demographic data obtained from an assortment of independent methods, we evaluated which demographic parameters have more influence on the population’s fate. We also simulated different scenarios to explore the effects of possible management actions. Our results showed that under the current conditions, Bonelli’s eagle is expected to become extinct in Italy in less than 50 years. Stand-alone juvenile mortality was the most critical demographic parameter with the strongest influence on population persistence with respect to other demographic parameters. Measures aimed at either decreasing juvenile mortality, adult mortality or decreasing both juvenile and adult mortality resulted in equivalent net positive effects on population persistence (population growth rate λ>1). In contrast, changes aimed at increasing breeding success had limited positive effects on demographic trends.

Conclusions/Significance

Our PVA provides essential information to direct the decision-making process and exposes gaps in our previous knowledge. To ensure the long-term persistence of the species in Italy, measures are urgently needed to decrease both adult mortality due to poaching and juvenile mortality due to nest plundering, the top ranking mortality causes.

Cardiac Explant-Derived Cells Are Regulated by Notch-Modulated Mesenchymal Transition

2012-05-25T21:00:00Z

by Liudmila Zakharova, Hikmet Nural-Guvener, Mohamed A. Gaballa

Background

Progenitor cell therapy is emerging as a novel treatment for heart failure. However the molecular mechanisms regulating the generation of cardiac progenitor cells is not fully understood. We hypothesized that cardiac progenitor cells are generated from cardiac explant via a process similar to epithelial to mesenchymal transition (EMT).

Methods/Findings

Explant-derived cells were generated from partially digested atrial tissue. After 21 days in culture, c-Kit+ cells were isolated from cell outgrowth. The majority of explant-originated c-Kit+ cells expressed the epicardial marker Wt1. Cardiac cell outgrowth exhibits a temporal up-regulation of EMT-markers. Notch stimulation augmented, while Notch inhibition suppressed, mesenchymal transition in both c-Kit+ and c-Kit- cells. In c-Kit+ cells, Notch stimulation reduced, while Notch inhibition up-regulated pluripotency marker expressions such as Nanog and Sox2. Notch induction was associated with degradation of β-catenin in c-Kit- cells. In contrast, Notch inhibition resulted in β-catenin accumulation, acquisition of epitheloid morphology, and up-regulation of Wnt target genes in c-Kit- cells.

Conclusion

Our study suggests that Notch-mediated reversible EMT process is a mechanism that regulates explant-derived c-Kit+ and c-Kit- cells.

Perivascular Expression and Potent Vasoconstrictor Effect of Dynorphin A in Cerebral Arteries

2012-05-25T21:00:00Z

by Éva Ruisanchez, Attila Cselenyák, Rege Sugárka Papp, Tamás Németh, Krisztina Káldi, Péter Sándor, Zoltán Benyó

Background

Numerous literary data indicate that dynorphin A (DYN-A) has a significant impact on cerebral circulation, especially under pathophysiological conditions, but its potential direct influence on the tone of cerebral vessels is obscure. The aim of the present study was threefold: 1) to clarify if DYN-A is present in cerebral vessels, 2) to determine if it exerts any direct effect on cerebrovascular tone, and if so, 3) to analyze the role of κ-opiate receptors in mediating the effect.

Methodology/Principal Findings

Immunohistochemical analysis revealed the expression of DYN-A in perivascular nerves of rat pial arteries as well as in both rat and human intraparenchymal vessels of the cerebral cortex. In isolated rat basilar and middle cerebral arteries (BAs and MCAs) DYN-A (1–13) and DYN-A (1–17) but not DYN-A (1–8) or dynorphin B (DYN-B) induced strong vasoconstriction in micromolar concentrations. The maximal effects, compared to a reference contraction induced by 124 mM K⁺, were 115±6% and 104±10% in BAs and 113±3% and 125±9% in MCAs for 10 µM of DYN-A (1–13) and DYN-A (1–17), respectively. The vasoconstrictor effects of DYN-A (1–13) could be inhibited but not abolished by both the κ-opiate receptor antagonist nor-Binaltorphimine dihydrochloride (NORBI) and blockade of G_i/o-protein mediated signaling by pertussis toxin. Finally, des-Tyr¹ DYN-A (2–13), which reportedly fails to activate κ-opiate receptors, induced vasoconstriction of 45±11% in BAs and 50±5% in MCAs at 10 µM, which effects were resistant to NORBI.

Conclusion/Significance

DYN-A is present in rat and human cerebral perivascular nerves and induces sustained contraction of rat cerebral arteries. This vasoconstrictor effect is only partly mediated by κ-opiate receptors and heterotrimeric G_i/o-proteins. To our knowledge our present findings are the first to indicate that DYN-A has a direct cerebral vasoconstrictor effect and that a dynorphin-induced vascular action may be, at least in part, independent of κ-opiate receptors.

Chronic Exposure of Corals to Fine Sediments: Lethal and Sub-Lethal Impacts

2012-05-25T21:00:00Z

by Florita Flores, Mia O. Hoogenboom, Luke D. Smith, Timothy F. Cooper, David Abrego, Andrew P. Negri

Understanding the sedimentation and turbidity thresholds for corals is critical in assessing the potential impacts of dredging projects in tropical marine systems. In this study, we exposed two species of coral sampled from offshore locations to six levels of total suspended solids (TSS) for 16 weeks in the laboratory, including a 4 week recovery period. Dose-response relationships were developed to quantify the lethal and sub-lethal thresholds of sedimentation and turbidity for the corals. The sediment treatments affected the horizontal foliaceous species (Montipora aequituberculata) more than the upright branching species (Acropora millepora). The lowest sediment treatments that caused full colony mortality were 30 mg l⁻¹ TSS (25 mg cm⁻² day⁻¹) for M. aequituberculata and 100 mg l⁻¹ TSS (83 mg cm⁻² day⁻¹) for A. millepora after 12 weeks. Coral mortality generally took longer than 4 weeks and was closely related to sediment accumulation on the surface of the corals. While measurements of damage to photosystem II in the symbionts and reductions in lipid content and growth indicated sub-lethal responses in surviving corals, the most reliable predictor of coral mortality in this experiment was long-term sediment accumulation on coral tissue.

Sexual Dimorphic Regulation of Body Weight Dynamics and Adipose Tissue Lipolysis

2012-05-25T21:00:00Z

by Verena Benz, Mandy Bloch, Sami Wardat, Christian Böhm, Lukas Maurer, Shokoufeh Mahmoodzadeh, Petra Wiedmer, Joachim Spranger, Anna Foryst-Ludwig, Ulrich Kintscher

Background

Successful reduction of body weight (BW) is often followed by recidivism to obesity. BW-changes including BW-loss and -regain is associated with marked alterations in energy expenditure (EE) and adipose tissue (AT) metabolism. Since these processes are sex-specifically controlled, we investigated sexual dimorphisms in metabolic processes during BW-dynamics (gain-loss-regain).

Research Design

Obesity was induced in C57BL/6J male (m) and female (f) mice by 15 weeks high-fat diet (HFD) feeding. Subsequently BW was reduced (-20%) by caloric restriction (CR) followed by adaptive feeding, and a regain-phase. Measurement of EE, body composition, blood/organ sampling were performed after each feeding period. Lipolysis was analyzed ex-vivo in gonadal AT.

Results

Male mice exhibited accelerated BW-gain compared to females (relative BW-gain m:140.5±3.2%; f:103.7±6.5%; p<0.001). In consonance, lean mass-specific EE was significantly higher in females compared to males during BW-gain. Under CR female mice reached their target-BW significantly faster than male mice (m:12.2 days; f:7.6 days; p<0.001) accompanied by a sustained sex-difference in EE. In addition, female mice predominantly downsized gonadal AT whereas the relation between gonadal and total body fat was not altered in males. Accordingly, only females exhibited an increased rate of forskolin-stimulated lipolysis in AT associated with significantly higher glycerol concentrations, lower RER-values, and increased AT expression of adipose triglyceride lipase (ATGL) and hormone sensitive lipase (HSL). Analysis of AT lipolysis in estrogen receptor alpha (ERα)–deficient mice revealed a reduced lipolytic rate in the absence of ERα exclusively in females. Finally, re-feeding caused BW-regain faster in males than in females.

Conclusion

The present study shows sex-specific dynamics during BW-gain-loss-regain. Female mice responded to CR with an increase in lipolytic activity, and augmented lipid-oxidation leading to more efficient weight loss. These processes likely involve ERα-dependent signaling in AT and sexual dimorphic regulation of genes involved in lipid metabolism.

Selection of Aptamers Specific for Adipose Tissue

2012-05-25T21:00:00Z

by Jun Liu, Huixia Liu, Kwame Sefah, Bo Liu, Ying Pu, Dimitri Van Simaeys, Weihong Tan

Background

Obesity has reached epidemic proportions, affecting more than one tenth of the world’s population. As such, adipose tissue is being increasingly recognized as an important therapeutic target for obesity and related metabolic disorders. While many potential targets of adipose tissue have been established and drugs developed, very few of those drugs specifically target adipose tissue without affecting other tissue. This results from a limited knowledge of both cell-surface markers and physicochemical traits specific to adipocytes that might otherwise be exploited by circulating drugs.

Methodology/Principal Findings

Here we report the use of cell-SELEX technology to select two aptamers that can specifically recognize mature adipocytes: adipo-1 and adipo-8. Adipo-8 shows high affinity for differentiated, mature 3T3-L1 adipocytes with a K_d value of 17.8±5.1 nM. The binding was sustained upon incubation at 37°C and insulin stimulation, but was lost upon trypsin treatment. The binding ability was also verified on frozen tissue slides with low background fluorescence and isolated adipocytes.

Conclusions/Significance

Aptamer adipo-8 selected from a random library appears to bind to mature differentiated adipocytes specifically. This aptamer holds great promise as a molecular recognition tool for adipocyte biomarker discovery or for targeted delivery of molecules to adipocytes.

Inhibition of Intestinal Bile Acid Transporter Slc10a2 Improves Triglyceride Metabolism and Normalizes Elevated Plasma Glucose Levels in Mice

2012-05-25T21:00:00Z

by Thomas Lundåsen, Eva-Marie Andersson, Michael Snaith, Helena Lindmark, Johanna Lundberg, Ann-Margret Östlund-Lindqvist, Bo Angelin, Mats Rudling

Interruption of the enterohepatic circulation of bile acids increases cholesterol catabolism, thereby stimulating hepatic cholesterol synthesis from acetate. We hypothesized that such treatment should lower the hepatic acetate pool which may alter triglyceride and glucose metabolism. We explored this using mice deficient of the ileal sodium-dependent BA transporter (Slc10a2) and ob/ob mice treated with a specific inhibitor of Slc10a2. Plasma TG levels were reduced in Slc10a2-deficient mice, and when challenged with a sucrose-rich diet, they displayed a reduced response in hepatic TG production as observed from the mRNA levels of several key enzymes in fatty acid synthesis. This effect was paralleled by a diminished induction of mature sterol regulatory element-binding protein 1c (Srebp1c). Unexpectedly, the SR-diet induced intestinal fibroblast growth factor (FGF) 15 mRNA and normalized bile acid synthesis in Slc10a2−/− mice. Pharmacologic inhibition of Slc10a2 in diabetic ob/ob mice reduced serum glucose, insulin and TGs, as well as hepatic mRNA levels of Srebp1c and its target genes. These responses are contrary to those reported following treatment of mice with a bile acid binding resin. Moreover, when key metabolic signal transduction pathways in the liver were investigated, those of Mek1/2 - Erk1/2 and Akt were blunted after treatment of ob/ob mice with the Slc10a2 inhibitor. It is concluded that abrogation of Slc10a2 reduces hepatic Srebp1c activity and serum TGs, and in the diabetic ob/ob model it also reduces glucose and insulin levels. Hence, targeting of Slc10a2 may be a promising strategy to treat hypertriglyceridemia and diabetes.

A Metabolomic Approach to the Study of Wine Micro-Oxygenation

2012-05-25T21:00:00Z

by Panagiotis Arapitsas, Matthias Scholz, Urska Vrhovsek, Stefano Di Blasi, Alessandra Biondi Bartolini, Domenico Masuero, Daniele Perenzoni, Adelio Rigo, Fulvio Mattivi

Wine micro-oxygenation is a globally used treatment and its effects were studied here by analysing by untargeted LC-MS the wine metabolomic fingerprint. Eight different procedural variations, marked by the addition of oxygen (four levels) and iron (two levels) were applied to Sangiovese wine, before and after malolactic fermentation. Data analysis using supervised and unsupervised multivariate methods highlighted some known candidate biomarkers, together with a number of metabolites which had never previously been considered as possible biomarkers for wine micro-oxygenation. Various pigments and tannins were identified among the known candidate biomarkers. Additional new information was obtained suggesting a correlation between oxygen doses and metal contents and changes in the concentration of primary metabolites such as arginine, proline, tryptophan and raffinose, and secondary metabolites such as succinic acid and xanthine. Based on these findings, new hypotheses regarding the formation and reactivity of wine pigment during micro-oxygenation have been proposed. This experiment highlights the feasibility of using unbiased, untargeted metabolomic fingerprinting to improve our understanding of wine chemistry.

Chlamydia trachomatis Incidence and Re-Infection among Young Women – Behavioural and Microbiological Characteristics

2012-05-25T21:00:00Z

by Jennifer Walker, Sepehr N. Tabrizi, Christopher K. Fairley, Marcus Y. Chen, Catriona S. Bradshaw, Jimmy Twin, Nicole Taylor, Basil Donovan, John M. Kaldor, Kathleen McNamee, Eve Urban, Sandra Walker, Marian Currie, Hudson Birden, Francis Bowden, Jane Gunn, Marie Pirotta, Lyle Gurrin, Veerakathy Harindra, Suzanne M. Garland, Jane S. Hocking

Background

This study aimed to estimate rates of chlamydia incidence and re-infection and to investigate the dynamics of chlamydia organism load in prevalent, incident and re-infections among young Australian women.

Methods

1,116 women aged 16 to 25 years were recruited from primary care clinics in Australia. Vaginal swabs were collected at 3 to 6 month intervals for chlamydia testing. Chlamydia organism load was measured by quantitative PCR.

Results

There were 47 incident cases of chlamydia diagnosed and 1,056.34 person years of follow up with a rate of 4.4 per 100 person years (95% CI: 3.3, 5.9). Incident infection was associated with being aged 16 to 20 years [RR = 3.7 (95%CI: 1.9, 7.1)], being employed [RR = 2.4 (95%CI: 1.1, 4.9)] and having two or more new sex partners [RR = 5.5 (95%CI: 2.6, 11.7)]. Recent antibiotic use was associated with a reduced incidence [RR:0.1 (95%CI: 0.0, 0.5)]. There were 14 re-infections with a rate of 22.3 per 100 person years (95%CI: 13.2, 37.6). The median time to re-infection was 4.6 months. Organism load was higher for prevalent than incident infections (p<0.01) and for prevalent than re-infections (p<0.01).

Conclusions

Chlamydia is common among young women and a high proportion of women are re-infected within a short period of time, highlighting the need for effective partner treatment and repeat testing. The difference in organism load between prevalent and incident infections suggests prevalent infection may be more important for ongoing transmission of chlamydia.

Involvement of SIK3 in Glucose and Lipid Homeostasis in Mice

2012-05-25T21:00:00Z

by Tatsuya Uebi, Yumi Itoh, Osamu Hatano, Ayako Kumagai, Masato Sanosaka, Tsutomu Sasaki, Satoru Sasagawa, Junko Doi, Keita Tatsumi, Kuniko Mitamura, Eiichi Morii, Katsuyuki Aozasa, Tomohiro Kawamura, Meinoshin Okumura, Jun Nakae, Hajime Takikawa, Toshio Fukusato, Minako Koura, Mayumi Nish, Anders Hamsten, Angela Silveira, Alejandro M. Bertorello, Kazuo Kitagawa, Yasuo Nagaoka, Hidehisa Kawahara, Takeshi Tomonaga, Tetsuji Naka, Shigeo Ikegawa, Noriyuki Tsumaki, Junichiro Matsuda, Hiroshi Takemori

Salt-inducible kinase 3 (SIK3), an AMP-activated protein kinase-related kinase, is induced in the murine liver after the consumption of a diet rich in fat, sucrose, and cholesterol. To examine whether SIK3 can modulate glucose and lipid metabolism in the liver, we analyzed phenotypes of SIK3-deficent mice. Sik3^−/− mice have a malnourished the phenotype (i.e., lipodystrophy, hypolipidemia, hypoglycemia, and hyper-insulin sensitivity) accompanied by cholestasis and cholelithiasis. The hypoglycemic and hyper-insulin-sensitive phenotypes may be due to reduced energy storage, which is represented by the low expression levels of mRNA for components of the fatty acid synthesis pathways in the liver. The biliary disorders in Sik3^−/− mice are associated with the dysregulation of gene expression programs that respond to nutritional stresses and are probably regulated by nuclear receptors. Retinoic acid plays a role in cholesterol and bile acid homeostasis, wheras ALDH1a which produces retinoic acid, is expressed at low levels in Sik3^−/− mice. Lipid metabolism disorders in Sik3^−/− mice are ameliorated by the treatment with 9-cis-retinoic acid. In conclusion, SIK3 is a novel energy regulator that modulates cholesterol and bile acid metabolism by coupling with retinoid metabolism, and may alter the size of energy storage in mice.

Evidence for an African Cluster of Human Head and Body Lice with Variable Colors and Interbreeding of Lice between Continents

2012-05-25T21:00:00Z

by Aurélie Veracx, Amina Boutellis, Vicky Merhej, Georges Diatta, Didier Raoult

Background

Human head lice and body lice have been classified based on phenotypic characteristics, including geographical source, ecotype (preferred egg laying site hair or clothes), shape and color. More recently, genotypic studies have been based on mitochondrial genes, nuclear genes and intergenic spacers. Mitochondrial genetic analysis reclassified lice into three genotypes (A, B and C). However, no previous study has attempted to correlate both genotypic and phenotypic data.

Materials and Methods

Lice were collected in four African countries: Senegal, Burundi, Rwanda and Ethiopia and were photographed to compare their colors. The Multi-Spacer-Typing (MST) method was used to genotype lice belonging to the worldwide Clade A, allowing a comparison of phenotypic and genotypic data.

Results

No congruence between louse color and genotype has been identified. Phylogenetic analysis of the spacer PM2, performed including lice from other sources, showed the existence of an African cluster of human lice. However, the analysis of other spacers suggested that lice from different areas are interbreeding.

Conclusions

We identified two geotypes of Clade A head and body lice including one that is specifically African, that can be either black or grey and can live on the head or in clothing. We also hypothesized that lice from different areas are interbreeding.

Regulation of RKIP Function by Helicobacter pylori in Gastric Cancer

2012-05-25T21:00:00Z

by Erika L. Moen, Sicheng Wen, Talha Anwar, Sam Cross-Knorr, Kate Brilliant, Faith Birnbaum, Sherida Rahaman, John M. Sedivy, Steven F. Moss, Devasis Chatterjee

Helicobacter pylori (H. pylori) is a gram-negative, spiral-shaped bacterium that infects more than half of the world’s population and is a major cause of gastric adenocarcinoma. The mechanisms that link H. pylori infection to gastric carcinogenesis are not well understood. In the present study, we report that the Raf-kinase inhibitor protein (RKIP) has a role in the induction of apoptosis by H. pylori in gastric epithelial cells. Western blot and luciferase transcription reporter assays demonstrate that the pathogenicity island of H. pylori rapidly phosphorylates RKIP, which then localizes to the nucleus where it activates its own transcription and induces apoptosis. Forced overexpression of RKIP enhances apoptosis in H. pylori-infected cells, whereas RKIP RNA inhibition suppresses the induction of apoptosis by H. pylori infection. While inducing the phosphorylation of RKIP, H. pylori simultaneously targets non-phosphorylated RKIP for proteasome-mediated degradation. The increase in RKIP transcription and phosphorylation is abrogated by mutating RKIP serine 153 to valine, demonstrating that regulation of RKIP activity by H. pylori is dependent upon RKIP’s S153 residue. In addition, H. pylori infection increases the expression of Snail, a transcriptional repressor of RKIP. Our results suggest that H. pylori utilizes a tumor suppressor protein, RKIP, to promote apoptosis in gastric cancer cells.

Equity and Geography: The Case of Child Mortality in Papua New Guinea

2012-05-25T21:00:00Z

by Anna E. Bauze, Linda N. Tran, Kim-Huong Nguyen, Sonja Firth, Eliana Jimenez-Soto, Laura Dwyer-Lindgren, Andrew Hodge, Alan D. Lopez

Background

Recent assessments show continued decline in child mortality in Papua New Guinea (PNG), yet complete subnational analyses remain rare. This study aims to estimate under-five mortality in PNG at national and subnational levels to examine the importance of geographical inequities in health outcomes and track progress towards Millennium Development Goal (MDG) 4.

Methodology

We performed retrospective data validation of the Demographic and Health Survey (DHS) 2006 using 2000 Census data, then applied advanced indirect methods to estimate under-five mortality rates between 1976 and 2000.

Findings

The DHS 2006 was found to be unreliable. Hence we used the 2000 Census to estimate under-five mortality rates at national and subnational levels. During the period under study, PNG experienced a slow reduction in national under-five mortality from approximately 103 to 78 deaths per 1,000 live births. Subnational analyses revealed significant disparities between rural and urban populations as well as inter- and intra-regional variations. Some of the provinces that performed the best (worst) in terms of under-five mortality included the districts that performed worst (best), with district-level under-five mortality rates correlating strongly with poverty levels and access to services.

Conclusions

The evidence from PNG demonstrates substantial within-province heterogeneity, suggesting that under-five mortality needs to be addressed at subnational levels. This is especially relevant in countries, like PNG, where responsibility for health services is devolved to provinces and districts. This study presents the first comprehensive estimates of under-five mortality at the district level for PNG. The results demonstrate that for countries that rely on few data sources even greater importance must be given to the quality of future population surveys and to the exploration of alternative options of birth and death surveillance.

Down-Regulation of GEP100 Causes Increase in E-Cadherin Levels and Inhibits Pancreatic Cancer Cell Invasion

2012-05-25T21:00:00Z

by Chuan-gao Xie, Shu-mei Wei, Jia-min Chen, Xuan-fu Xu, Jian-ting Cai, Qin-yu Chen, Li-tao Jia

Aims

Invasion and metastasis are major reasons for pancreatic cancer death and identifying signaling molecules that are specifically used in tumor invasion is of great significance. The purpose of this study was to elucidate the role of GEP100 in pancreatic cancer cell invasion and metastasis and the corresponding molecular mechanism.

Methods

Stable cell lines with GEP100 knocked-down were established by transfecting GEP100 shRNA vector into PaTu8988 cells and selected by puromycin. qRT-PCR and Western blot were performed to detect gene expression. Matrigel-invasion assay was used to detect cancer cell invasion in vitro. Liver metastasis in vivo was determined by splenic injection of indicated cell lines followed by spleen resection. Immunofluorescence study was used to detect the intracellular localization of E-cadherin.

Results

We found that the expression level of GEP100 protein was closely related to the invasive ability of a panel of 6 different human pancreatic cancer cell lines. Down-regulation of GEP100 in PaTu8988 cells significantly decreased invasive activity by Matrigel invasion assay, without affecting migration, invasion and viability. The inhibited invasive activity was rescued by over-expression of GEP100 cDNA. In vivo study showed that liver metastasis was significantly decreased in the PaTu8988 cells with GEP100 stably knocked-down. In addition, an epithelial-like morphological change, mimicking a mesenchymal to epithelial transition (MET) was induced by GEP100 down-regulation. The expression of E-cadherin protein was increased 2–3 folds accompanied by its redistribution to the cell-cell contacts, while no obvious changes were observed for E-cadherin mRNA. Unexpectedly, the mRNA of Slug was increased by GEP100 knock-down.

Conclusion

These findings provided important evidence that GEP100 plays a significant role in pancreatic cancer invasion through regulating the expression of E-cadherin and the process of MET, indicating the possibility of it becoming a potential therapeutic target against pancreatic cancer.

Donor Age of Human Platelet Lysate Affects Proliferation and Differentiation of Mesenchymal Stem Cells

2012-05-25T21:00:00Z

by Michael Lohmann, Gudrun Walenda, Hatim Hemeda, Sylvia Joussen, Wolf Drescher, Stefan Jockenhoevel, Gabriele Hutschenreuter, Martin Zenke, Wolfgang Wagner

The regenerative potential declines upon aging. This might be due to cell-intrinsic changes in stem and progenitor cells or to influences by the microenvironment. Mesenchymal stem cells (MSC) raise high hopes in regenerative medicine. They are usually culture expanded in media with fetal calf serum (FCS) or other serum supplements such as human platelet lysate (HPL). In this study, we have analyzed the impact of HPL-donor age on culture expansion. 31 single donor derived HPLs (25 to 57 years old) were simultaneously compared for culture of MSC. Proliferation of MSC did not reveal a clear association with platelet counts of HPL donors or growth factors concentrations (PDGF-AB, TGF-β1, bFGF, or IGF-1), but it was significantly higher with HPLs from younger donors (<35 years) as compared to older donors (>45 years). Furthermore, HPLs from older donors increased activity of senescence-associated beta-galactosidase (SA-βgal). HPL-donor age did not affect the fibroblastoid colony-forming unit (CFU-f) frequency, immunophenotype or induction of adipogenic differentiation, whereas osteogenic differentiation was significantly lower with HPLs from older donors. Concentrations of various growth factors (PDGF-AB, TGF-β1, bFGF, IGF-1) or hormones (estradiol, parathormone, leptin, 1,25 vitamin D3) were not associated with HPL-donor age or MSC growth. Taken together, our data support the notion that aging is associated with systemic feedback mechanisms acting on stem and progenitor cells, and this is also relevant for serum supplements in cell culture: HPLs derived from younger donors facilitate enhanced expansion and more pronounced osteogenic differentiation.

Integrative Gene Regulatory Network Analysis Reveals Light-Induced Regional Gene Expression Phase Shift Programs in the Mouse Suprachiasmatic Nucleus

2012-05-25T21:00:00Z

by Haisun Zhu, Rajanikanth Vadigepalli, Rachel Rafferty, Gregory E. Gonye, David R. Weaver, James S. Schwaber

We use the multigenic pattern of gene expression across suprachiasmatic nuclei (SCN) regions and time to understand the dynamics within the SCN in response to a circadian phase-resetting light pulse. Global gene expression studies of the SCN indicate that circadian functions like phase resetting are complex multigenic processes. While the molecular dynamics of phase resetting are not well understood, it is clear they involve a “functional gene expression program”, e.g., the coordinated behavior of functionally related genes in space and time. In the present study we selected a set of 89 of these functionally related genes in order to further understand this multigenic program. By use of high-throughput qPCR we studied 52 small samples taken by anatomically precise laser capture from within the core and shell SCN regions, and taken at time points with and without phase resetting light exposure. The results show striking regional differences in light response to be present in the mouse SCN. By using network-based analyses, we are able to establish a highly specific multigenic correlation between genes expressed in response to light at night and genes normally activated during the day. The light pulse triggers a complex and highly coordinated network of gene regulation. The largest differences marking neuroanatomical location are in transmitter receptors, and the largest time-dependent differences occur in clock-related genes. Nighttime phase resetting appears to recruit transcriptional regulatory processes normally active in the day. This program, or mechanism, causes the pattern of core region gene expression to transiently shift to become more like that of the shell region.

Rhodopsin Expression Level Affects Rod Outer Segment Morphology and Photoresponse Kinetics

2012-05-25T21:00:00Z

by Clint L. Makino, Xiao-Hong Wen, Norman A. Michaud, Henry I. Covington, Emmanuele DiBenedetto, Heidi E. Hamm, Janis Lem, Giovanni Caruso

Background

The retinal rod outer segment is a sensory cilium that is specialized for the conversion of light into an electrical signal. Within the cilium, up to several thousand membranous disks contain as many as a billion copies of rhodopsin for efficient photon capture. Disks are continually turned over, requiring the daily synthesis of a prodigious amount of rhodopsin. To promote axial diffusion in the aqueous cytoplasm, the disks have one or more incisures. Across vertebrates, the range of disk diameters spans an order of magnitude, and the number and length of the incisures vary considerably, but the mechanisms controlling disk architecture are not well understood. The finding that transgenic mice overexpressing rhodopsin have enlarged disks lacking an incisure prompted us to test whether lowered rhodopsin levels constrain disk assembly.

Methodology/Principal Findings

The structure and function of rods from hemizygous rhodopsin knockout (R+/−) mice with decreased rhodopsin expression were analyzed by transmission electron microscopy and single cell recording. R+/− rods were structurally altered in three ways: disk shape changed from circular to elliptical, disk surface area decreased, and the single incisure lengthened to divide the disk into two sections. Photocurrent responses to flashes recovered more rapidly than normal. A spatially resolved model of phototransduction indicated that changes in the packing densities of rhodopsin and other transduction proteins were responsible. The decrease in aqueous outer segment volume and the lengthened incisure had only minor effects on photon response amplitude and kinetics.

Conclusions/Significance

Rhodopsin availability limits disk assembly and outer segment girth in normal rods. The incisure may buffer the supply of structural proteins needed to form larger disks. Decreased rhodopsin level accelerated photoresponse kinetics by increasing the rates of molecular collisions on the membrane. Faster responses, together with fewer rhodopsins, combine to lower overall sensitivity of R+/− rods to light.

Bmp2, Bmp4 and Bmp7 Are Co-Required in the Mouse AER for Normal Digit Patterning but Not Limb Outgrowth

2012-05-25T21:00:00Z

by Kyung-Suk Choi, Chanmi Lee, Danielle M. Maatouk, Brian D. Harfe

Outgrowth and patterning of the vertebrate limb requires a functional apical ectodermal ridge (AER). The AER is a thickening of ectodermal tissue located at the distal end of the limb bud. Loss of this structure, either through genetic or physical manipulations results in truncation of the limb. A number of genes, including Bmps, are expressed in the AER. Previously, it was shown that removal of the BMP receptor Bmpr1a specifically from the AER resulted in complete loss of hindlimbs suggesting that Bmp signaling in the AER is required for limb outgrowth. In this report, we genetically removed the three known AER-expressed Bmp ligands, Bmp2, Bmp4 and Bmp7 from the AER of the limb bud using floxed conditional alleles and the Msx2-cre allele. Surprisingly, only defects in digit patterning and not limb outgrowth were observed. In triple mutants, the anterior and posterior AER was present but loss of the central region of the AER was observed. These data suggest that Bmp ligands expressed in the AER are not required for limb outgrowth but instead play an essential role in maintaining the AER and patterning vertebrate digits.

Aetiologies of Central Nervous System Infection in Viet Nam: A Prospective Provincial Hospital-Based Descriptive Surveillance Study

2012-05-25T21:00:00Z

by Nghia Ho Dang Trung, Tu Le Thi Phuong, Marcel Wolbers, Hoang Nguyen Van Minh, Vinh Nguyen Thanh, Minh Pham Van, Nga Tran Vu Thieu, Tan Le Van, Diep To Song, Phuong Le Thi, Thao Nguyen Thi Phuong, Cong Bui Van, Vu Tang, Tuan Hoang Ngoc Anh, Dong Nguyen, Tien Phan Trung, Lien Nguyen Thi Nam, Hao Tran Kiem, Tam Nguyen Thi Thanh, James Campbell, Maxine Caws, Jeremy Day, Menno D. de Jong, Chau Nguyen Van Vinh, H. Rogier Van Doorn, Hien Tran Tinh, Jeremy Farrar, Constance Schultsz, the VIZIONS CNS Infection Network

Background

Infectious diseases of the central nervous system (CNS) remain common and life-threatening, especially in developing countries. Knowledge of the aetiological agents responsible for these infections is essential to guide empiric therapy and develop a rational public health policy. To date most data has come from patients admitted to tertiary referral hospitals in Asia and there is limited aetiological data at the provincial hospital level where most patients are seen.

Methods

We conducted a prospective Provincial Hospital-based descriptive surveillance study in adults and children at thirteen hospitals in central and southern Viet Nam between August 2007– April 2010. The pathogens of CNS infection were confirmed in CSF and blood samples by using classical microbiology, molecular diagnostics and serology.

Results

We recruited 1241 patients with clinically suspected infection of the CNS. An aetiological agent was identified in 640/1241 (52%) of the patients. The most common pathogens were Streptococcus suis serotype 2 in patients older than 14 years of age (147/617, 24%) and Japanese encephalitis virus in patients less than 14 years old (142/624, 23%). Mycobacterium tuberculosis was confirmed in 34/617 (6%) adult patients and 11/624 (2%) paediatric patients. The acute case fatality rate (CFR) during hospital admission was 73/617 (12%) in adults and to 42/624 (7%) in children.

Conclusions

Zoonotic bacterial and viral pathogens are the most common causes of CNS infection in adults and children in Viet Nam.

Is the Self Always Better than a Friend? Self-Face Recognition in Christians and Atheists

2012-05-25T21:00:00Z

by Yina Ma, Shihui Han

Early behavioral studies found that human adults responded faster to their own faces than faces of familiar others or strangers, a finding referred to as self-face advantage. Recent research suggests that the self-face advantage is mediated by implicit positive association with the self and is influenced by sociocultural experience. The current study investigated whether and how Christian belief and practice affect the processing of self-face in a Chinese population. Christian and Atheist participants were recruited for an implicit association test (IAT) in Experiment 1 and a face-owner identification task in Experiment 2. Experiment 1 found that atheists responded faster to self-face when it shared the same response key with positive compared to negative trait adjectives. This IAT effect, however, was significantly reduced in Christians. Experiment 2 found that atheists responded faster to self-face compared to a friend’s face, but this self-face advantage was significantly reduced in Christians. Hierarchical regression analyses further showed that the IAT effect positively predicted self-face advantage in atheists but not in Christians. Our findings suggest that Christian belief and practice may weaken implicit positive association with the self and thus decrease the advantage of the self over a friend during face recognition in the believers.

p53 Modulation as a Therapeutic Strategy in Gastrointestinal Stromal Tumors

2012-05-25T21:00:00Z

by Joern Henze, Thomas Mühlenberg, Susanne Simon, Florian Grabellus, Brian Rubin, Georg Taeger, Martin Schuler, Juergen Treckmann, Maria Debiec-Rychter, Takahiro Taguchi, Jonathan A. Fletcher, Sebastian Bauer

The KIT-inhibitor imatinib mesylate (IM) has greatly improved the treatment of metastatic gastrointestinal stromal tumors (GIST). IM exhibits strong antiproliferative effects but fails to induce sufficient levels of apoptosis resulting in low pathologic complete remission rates and a high rate of secondary progression in the metastatic setting. Upregulation of p53 by MDM2 inhibitors has been shown to induce apoptosis in p53 wildtype tumors. Analyzing a series of 62 mostly untreated, localized and metastatic GIST we detected a low rate (3%) of inactivating p53 mutations, thus providing a rationale for further exploration of p53-directed therapeutic strategies. To this end, we studied nutlin-3, an inhibitor of the p53 antagonist MDM2, and RITA, a putative p53 activator, in GIST cell lines. Nutlin-3 effectively induced p53 at therapeutically relevant levels, which resulted in moderate antiproliferative effects and cell cycle arrest in p53 wildtype GIST cell lines GIST430, GIST48 and GIST48B. P53 reactivation substantially improved the apoptotic response after effective KIT inhibition with sunitinib and 17-AAG in IM-resistant cell lines. The commonly used imatinib-sensitive cell lines GIST882 and GIST-T1 were shown to harbor defective p53 and therefore failed to respond to nutlin-3 treatment. RITA induced p53 in GIST48B, followed by antiproliferative effects and a strong induction of apoptosis. Surprisingly, GIST-T1 was also highly sensitive to RITA despite lacking functional p53. This suggested a more complex, p53-independent mechanism of action for the latter compound. No antagonistic effects from p53-activating drugs were seen with any drug combination. Our data provide first evidence that modulation of the MDM2/p53 pathway may be therapeutically useful to improve the apoptotic response of KIT-inhibitory drugs in the treatment of naïve GIST, with p53 mutation status being a predictive factor of response.

Hyaluronan Binding Motifs of USP17 and SDS3 Exhibit Anti-Tumor Activity

2012-05-25T21:00:00Z

by Suresh Ramakrishna, Bharathi Suresh, Su-Mi Bae, Woong-Shick Ahn, Key-Hwan Lim, Kwang-Hyun Baek

Background

We previously reported that the USP17 deubiquitinating enzyme having hyaluronan binding motifs (HABMs) interacts with human SDS3 (suppressor of defective silencing 3) and specifically deubiquitinates Lys-63 branched polyubiquitination of SDS3 resulting in negative regulation of histone deacetylase (HDAC) activity in cancer cells. Furthermore, USP17 and SDS3 mutually interact with each other to block cell proliferation in HeLa cells but the mechanism for this inhibition in cell proliferation is not known. We wished to investigate whether the HABMs of USP17 were responsible for tumor suppression activity.

Methodology/Principal Findings

Similarly to USP17, we have identified that SDS3 also has three consecutive HABMs and shows direct binding with hyaluronan (HA) using cetylpyridinium chloride (CPC) assay. Additionally, HA oligosaccharides (6-18 sugar units) competitively block binding of endogenous HA polymer to HA binding proteins. Thus, administration of HA oligosaccharides antagonizes the interaction between HA and USP17 or SDS3. Interestingly, HABMs deleted USP17 showed lesser interaction with SDS3 but retain its deubiquitinating activity towards SDS3. The deletion of HABMs of USP17 could not alter its functional regulation on SDS3-associated HDAC activity. Furthermore, to explore whether HABMs in USP17 and SDS3 are responsible for the inhibition of cell proliferation, we investigated the effect of USP17 and SDS3-lacking HABMs on cell proliferation by soft agar, apoptosis, cell migration and cell proliferation assays.

Conclusions

Our results have demonstrated that these HABMs in USP17 and its substrate SDS3 are mainly involved in the inhibition of anchorage-independent tumor growth.

Lipoxin A4 Stimulates Calcium-Activated Chloride Currents and Increases Airway Surface Liquid Height in Normal and Cystic Fibrosis Airway Epithelia

2012-05-25T21:00:00Z

by Valia Verrière, Gerard Higgins, Mazen Al-Alawi, Richard W. Costello, Paul McNally, Raphaël Chiron, Brian J. Harvey, Valérie Urbach

Cystic Fibrosis (CF) is a genetic disease characterised by a deficit in epithelial Cl⁻ secretion which in the lung leads to airway dehydration and a reduced Airway Surface Liquid (ASL) height. The endogenous lipoxin LXA₄ is a member of the newly identified eicosanoids playing a key role in ending the inflammatory process. Levels of LXA₄ are reported to be decreased in the airways of patients with CF. We have previously shown that in normal human bronchial epithelial cells, LXA₄ produced a rapid and transient increase in intracellular Ca²⁺. We have investigated, the effect of LXA₄ on Cl⁻ secretion and the functional consequences on ASL generation in bronchial epithelial cells obtained from CF and non-CF patient biopsies and in bronchial epithelial cell lines. We found that LXA₄ stimulated a rapid intracellular Ca²⁺ increase in all of the different CF bronchial epithelial cells tested. In non-CF and CF bronchial epithelia, LXA₄ stimulated whole-cell Cl⁻ currents which were inhibited by NPPB (calcium-activated Cl⁻ channel inhibitor), BAPTA-AM (chelator of intracellular Ca²⁺) but not by CFTRinh-172 (CFTR inhibitor). We found, using confocal imaging, that LXA₄ increased the ASL height in non-CF and in CF airway bronchial epithelia. The LXA₄ effect on ASL height was sensitive to bumetanide, an inhibitor of transepithelial Cl⁻ secretion. The LXA₄ stimulation of intracellular Ca²⁺, whole-cell Cl⁻ currents, conductances and ASL height were inhibited by Boc-2, a specific antagonist of the ALX/FPR2 receptor. Our results provide, for the first time, evidence for a novel role of LXA₄ in the stimulation of intracellular Ca²⁺ signalling leading to Ca²⁺-activated Cl⁻ secretion and enhanced ASL height in non-CF and CF bronchial epithelia.

Lobelia siphilitica Plants That Escape Herbivory in Time Also Have Reduced Latex Production

2012-05-25T21:00:00Z

by Amy L. Parachnowitsch, Christina M. Caruso, Stuart A. Campbell, André Kessler

Flowering phenology is an important determinant of a plant’s reproductive success. Both assortative mating and niche construction can result in the evolution of correlations between phenology and other reproductive, functional, and life history traits. Correlations between phenology and herbivore defence traits are particularly likely because the timing of flowering can allow a plant to escape herbivory. To test whether herbivore escape and defence are correlated, we estimated phenotypic and genetic correlations between flowering phenology and latex production in greenhouse-grown Lobelia siphilitica L. (Lobeliaceae). Lobelia siphilitica plants that flower later escape herbivory by a specialist pre-dispersal seed predator, and thus should invest fewer resources in defence. Consistent with this prediction, we found that later flowering was phenotypically and genetically correlated with reduced latex production. To test whether herbivore escape and latex production were costly, we also measured four fitness correlates. Flowering phenology was negatively genetically correlated with three out of four fitness estimates, suggesting that herbivore escape can be costly. In contrast, we did not find evidence for costs of latex production. Generally, our results suggest that herbivore escape and defence traits will not evolve independently in L. siphilitica.

Metabolic Profiling of the Protozoan Parasite Entamoeba invadens Revealed Activation of Unpredicted Pathway during Encystation

2012-05-25T21:00:00Z

by Ghulam Jeelani, Dan Sato, Afzal Husain, Aleyla Escueta-de Cadiz, Masahiro Sugimoto, Tomoyoshi Soga, Makoto Suematsu, Tomoyoshi Nozaki

Encystation, which is cellular differentiation from the motile, proliferative, labile trophozoite form to the dormant, resistant cyst form, is a crucial process found in parasitic and free-living protozoa such as Entamoeba, Giardia, Acanthamoeba, and Balamuthia. Since encystation is an essential process to deal with the adverse external environmental changes during the life cycle, and often integral to the transmission of the diseases, biochemical understanding of the process potentially provides useful measures against the infections caused by this group of protozoa. In this study, we investigated metabolic and transcriptomic changes that occur during encystation in Entamoeba invadens, the reptilian sibling of mammal-infecting E. histolytica, using capillary electrophoresis-tandem mass spectrometry-based metabolite profiling and DNA microarray-based expression profiling. As the encystation progressed, the levels of majority of metabolites involved in glycolysis and nucleotides drastically decreased, indicating energy generation is ceased. Furthermore, the flux of glycolysis was redirected toward chitin wall biosynthesis. We found remarkable temporal increases in biogenic amines such as isoamylamine, isobutylamine, and cadaverine, during the early period of encystation, when the trophozoites form large multicellular aggregates (precyst). We also found remarkable induction of γ-aminobutyric acid (GABA) during encystation. This study has unveiled for the first time the dynamics of the transcriptional and metabolic regulatory networks during encystation, and should help in better understanding of the process in pathogenic eukaryotes, and further development of measures controlling infections they cause.

Nilotinib Counteracts P-Glycoprotein-Mediated Multidrug Resistance and Synergizes the Antitumoral Effect of Doxorubicin in Soft Tissue Sarcomas

2012-05-25T21:00:00Z

by Victor Hugo Villar, Oliver Vögler, Jordi Martínez-Serra, Rafael Ramos, Silvia Calabuig-Fariñas, Antonio Gutiérrez, Francisca Barceló, Javier Martín-Broto, Regina Alemany

The therapeutic effect of doxorubicin (DXR) in the treatment of soft tissue sarcomas (STS) is limited by its toxicity and the development of multidrug resistance (MDR), the latter mainly induced by high expression of efflux pumps (e.g., P-glycoprotein [P-gp]). Therefore, the search for alternative therapies, which sensitize these tumors to chemotherapy while maintaining a low toxicity profile, is a rational approach. We assessed efficacy and molecular mechanisms involved in the antiproliferative effects of the tyrosine kinase inhibitors, nilotinib and imatinib, as single agents or in combination with DXR, in human synovial sarcoma SW982 and leiomyosarcoma SK-UT-1 cells. As single compound nilotinib (1–10 µM) was more potent than imatinib inhibiting the growth of SK-UT-1 and SW982 cells by 33.5–59.6%, respectively. Importantly, only nilotinib synergized the antitumoral effect of DXR (0.05–0.5 µM) by at least 2-fold, which clearly surpassed the mere sum of effects according to isobolographic analysis. Moreover, nilotinib in combination with DXR had a sustained effect on cell number (−70.3±5.8%) even 12 days after withdrawal of drugs compared to DXR alone. On the molecular level, only nilotinib fully blocked FBS-induced ERK1 and p38 MAPK activation, hence, reducing basal and DXR-induced up-regulation of P-gp levels. Moreover, efflux activity of the MDR-related proteins P-gp and MRP-1 was inhibited, altogether resulting in intracellular DXR retention. In high-risk STS tumors 53.8% and 15.4% were positive for P-gp and MRP-1 expression, respectively, with high incidence of P-gp in synovial sarcoma (72.7%). In summary, nilotinib exhibits antiproliferative effects on cellular models of STS and sensitizes them to DXR by reverting DXR-induced P-gp-mediated MDR and inhibiting MRP-1 activity, leading to a synergistic effect with potential for clinical treatment.

Identification, Expression and Target Gene Analyses of MicroRNAs in Spodoptera litura

2012-05-25T21:00:00Z

by Zhongchen Rao, Wenyin He, Lin Liu, Sichun Zheng, Lihua Huang, Qili Feng

MicroRNAs (miRNAs) are small RNAs widely present in animals and plants and involved in post-transcriptional regulation of gene transcripts. In this study we identified and validated 58 miRNAs from an EST dataset of Spodoptera litura based on the computational and experimental analysis of sequence conservation and secondary structure of miRNA by comparing the miRNA sequences in the miRbase. RT-PCR was conducted to examine the expression of these miRNAs and stem-loop RT-PCR assay was performed to examine expression of 11 mature miRNAs (out of the 58 putative miRNA) that showed significant changes in different tissues and stages of the insect development. One hundred twenty eight possible target genes against the 11 miRNAs were predicted by using computational methods. Binding of one miRNA (sli-miR-928b) with the three possible target mRNAs was confirmed by Southern blotting, implying its possible function in regulation of the target genes.

Ulcerative Colitis Impairs the Acylethanolamide-Based Anti-Inflammatory System Reversal by 5-Aminosalicylic Acid and Glucocorticoids

2012-05-25T21:00:00Z

by Juan Suárez, Yanina Romero-Zerbo, Lucia Márquez, Patricia Rivera, Mar Iglesias, Francisco J. Bermúdez-Silva, Montserrat Andreu, Fernando Rodríguez de Fonseca

Studies in animal models and humans suggest anti-inflammatory roles on the N-acylethanolamide (NAE)-peroxisome proliferators activated receptor alpha (PPARα) system in inflammatory bowel diseases. However, the presence and function of NAE-PPARα signaling system in the ulcerative colitis (UC) of humans remain unknown as well as its response to active anti-inflammatory therapies such as 5-aminosalicylic acid (5-ASA) and glucocorticoids. Expression of PPARα receptor and PPARα ligands-biosynthetic (NAPE-PLD) and -degrading (FAAH and NAAA) enzymes were analyzed in untreated active and 5-ASA/glucocorticoids/immunomodulators-treated quiescent UC patients compared to healthy human colonic tissue by RT-PCR and immunohistochemical analyses. PPARα, NAAA, NAPE-PLD and FAAH showed differential distributions in the colonic epithelium, lamina propria, smooth muscle and enteric plexus. Gene expression analysis indicated a decrease of PPARα, PPARγ and NAAA, and an increase of FAAH and iNOS in the active colitis mucosa. Immunohistochemical expression in active colitis epithelium confirmed a PPARα decrease, but showed a sharp NAAA increase and a NAPE-PLD decrease, which were partially restored to control levels after treatment. We also characterized the immune cells of the UC mucosa infiltrate. We detected a decreased number of NAAA-positive and an increased number of FAAH-positive immune cells in active UC, which were partially restored to control levels after treatment. NAE-PPARα signaling system is impaired during active UC and 5-ASA/glucocorticoids treatment restored its normal expression. Since 5-ASA actions may work through PPARα and glucocorticoids through NAE-producing/degrading enzymes, the use of PPARα agonists or FAAH/NAAA blockers that increases endogenous PPARα ligands may yield similar therapeutics advantages.

Pan-Pathway Based Interaction Profiling of FDA-Approved Nucleoside and Nucleobase Analogs with Enzymes of the Human Nucleotide Metabolism

2012-05-25T21:00:00Z

by Louise Egeblad, Martin Welin, Susanne Flodin, Susanne Gräslund, Liya Wang, Jan Balzarini, Staffan Eriksson, Pär Nordlund

To identify interactions a nucleoside analog library (NAL) consisting of 45 FDA-approved nucleoside analogs was screened against 23 enzymes of the human nucleotide metabolism using a thermal shift assay. The method was validated with deoxycytidine kinase; eight interactions known from the literature were detected and five additional interactions were revealed after the addition of ATP, the second substrate. The NAL screening gave relatively few significant hits, supporting a low rate of “off target effects.” However, unexpected ligands were identified for two catabolic enzymes guanine deaminase (GDA) and uridine phosphorylase 1 (UPP1). An acyclic guanosine prodrug analog, valaciclovir, was shown to stabilize GDA to the same degree as the natural substrate, guanine, with a ΔT_agg around 7°C. Aciclovir, penciclovir, ganciclovir, thioguanine and mercaptopurine were also identified as ligands for GDA. The crystal structure of GDA with valaciclovir bound in the active site was determined, revealing the binding of the long unbranched chain of valaciclovir in the active site of the enzyme. Several ligands were identified for UPP1: vidarabine, an antiviral nucleoside analog, as well as trifluridine, idoxuridine, floxuridine, zidovudine, telbivudine, fluorouracil and thioguanine caused concentration-dependent stabilization of UPP1. A kinetic study of UPP1 with vidarabine revealed that vidarabine was a mixed-type competitive inhibitor with the natural substrate uridine. The unexpected ligands identified for UPP1 and GDA imply further metabolic consequences for these nucleoside analogs, which could also serve as a starting point for future drug design.