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1.  Comparative proteomic analysis implicates eEF2 as a novel target of PI3Kγ in the MDA-MB-231 metastatic breast cancer cell line 
Proteome Science  2013;11:4.
Cancer cell migration is fundamentally required for breast tumour invasion and metastasis. The insulin-like growth factor 1 tyrosine kinase receptor (IGF-1R) and the chemokine G-protein coupled receptor, CXCR4 have been shown to play an important role in breast cancer metastasis. Our previous study has shown that IGF-1R can transactivate CXCR4 via a physical association in the human MDA-MB-231 metastatic breast cancer cell line and that this plays a key role in IGF-I-induced migration of these cells. In the present study we used pharmacological inhibition and RNAi to identify PI3Kγ as an important migration signalling molecule downstream of receptor transactivation in MDA-MB-231 cells. To identify PI3Kγ-regulated proteins upon transactivation of CXCR4 by IGF-I, we undertook a comparative proteomics approach using 2-D- Fluorescence Difference Gel Electrophoresis (DIGE) and identified the proteins by mass spectrometry.
These experiments identified eukaryotic elongation factor 2 (eEF2) as a novel downstream target of PI3Kγ after activation of the IGF-1R-CXCR4 heterodimer by IGF-I. Further analysis demonstrated that eEF2 is phosphorylated in MDA-MB-231 cells in response to IGF-I and that this is dependent on PI3Kγ activity.
Our data imply a novel role for PI3Kγ in facilitating cell migration by regulating phosphorylation of eEF2.
PMCID: PMC3564858  PMID: 23320409
Receptor transactivation; Cell migration; IGF-I; CXCR4; PI3Kγ; eEF2; 2D-DIGE
2.  Research needs in allergy: an EAACI position paper, in collaboration with EFA 
Papadopoulos, Nikolaos G | Agache, Ioana | Bavbek, Sevim | Bilo, Beatrice M | Braido, Fulvio | Cardona, Victoria | Custovic, Adnan | deMonchy, Jan | Demoly, Pascal | Eigenmann, Philippe | Gayraud, Jacques | Grattan, Clive | Heffler, Enrico | Hellings, Peter W | Jutel, Marek | Knol, Edward | Lötvall, Jan | Muraro, Antonella | Poulsen, Lars K | Roberts, Graham | Schmid-Grendelmeier, Peter | Skevaki, Chrysanthi | Triggiani, Massimo | vanRee, Ronald | Werfel, Thomas | Flood, Breda | Palkonen, Susanna | Savli, Roberta | Allegri, Pia | Annesi-Maesano, Isabella | Annunziato, Francesco | Antolin-Amerigo, Dario | Apfelbacher, Christian | Blanca, Miguel | Bogacka, Ewa | Bonadonna, Patrizia | Bonini, Matteo | Boyman, Onur | Brockow, Knut | Burney, Peter | Buters, Jeroen | Butiene, Indre | Calderon, Moises | Cardell, Lars Olaf | Caubet, Jean-Christoph | Celenk, Sevcan | Cichocka-Jarosz, Ewa | Cingi, Cemal | Couto, Mariana | deJong, Nicolette | Del Giacco, Stefano | Douladiris, Nikolaos | Fassio, Filippo | Fauquert, Jean-Luc | Fernandez, Javier | Rivas, Montserrat Fernandez | Ferrer, Marta | Flohr, Carsten | Gardner, James | Genuneit, Jon | Gevaert, Philippe | Groblewska, Anna | Hamelmann, Eckard | Hoffmann, Hans Jürgen | Hoffmann-Sommergruber, Karin | Hovhannisyan, Lilit | Hox, Valérie | Jahnsen, Frode L | Kalayci, Ömer | Kalpaklioglu, Ayse Füsun | Kleine-Tebbe, Jörg | Konstantinou, George | Kurowski, Marcin | Lau, Susanne | Lauener, Roger | Lauerma, Antti | Logan, Kirsty | Magnan, Antoine | Makowska, Joanna | Makrinioti, Heidi | Mangina, Paraskevi | Manole, Felicia | Mari, Adriano | Mazon, Angel | Mills, Clare | Mingomataj, ErvinÇ | Niggemann, Bodo | Nilsson, Gunnar | Ollert, Markus | O'Mahony, Liam | O'Neil, Serena | Pala, Gianni | Papi, Alberto | Passalacqua, Gianni | Perkin, Michael | Pfaar, Oliver | Pitsios, Constantinos | Quirce, Santiago | Raap, Ulrike | Raulf-Heimsoth, Monika | Rhyner, Claudio | Robson-Ansley, Paula | Alves, Rodrigo Rodrigues | Roje, Zeljka | Rondon, Carmen | Rudzeviciene, Odilija | Ruëff, Franziska | Rukhadze, Maia | Rumi, Gabriele | Sackesen, Cansin | Santos, Alexandra F | Santucci, Annalisa | Scharf, Christian | Schmidt-Weber, Carsten | Schnyder, Benno | Schwarze, Jürgen | Senna, Gianenrico | Sergejeva, Svetlana | Seys, Sven | Siracusa, Andrea | Skypala, Isabel | Sokolowska, Milena | Spertini, Francois | Spiewak, Radoslaw | Sprikkelman, Aline | Sturm, Gunter | Swoboda, Ines | Terreehorst, Ingrid | Toskala, Elina | Traidl-Hoffmann, Claudia | Venter, Carina | Vlieg-Boerstra, Berber | Whitacker, Paul | Worm, Margitta | Xepapadaki, Paraskevi | Akdis, Cezmi A
In less than half a century, allergy, originally perceived as a rare disease, has become a major public health threat, today affecting the lives of more than 60 million people in Europe, and probably close to one billion worldwide, thereby heavily impacting the budgets of public health systems. More disturbingly, its prevalence and impact are on the rise, a development that has been associated with environmental and lifestyle changes accompanying the continuous process of urbanization and globalization. Therefore, there is an urgent need to prioritize and concert research efforts in the field of allergy, in order to achieve sustainable results on prevention, diagnosis and treatment of this most prevalent chronic disease of the 21st century.
The European Academy of Allergy and Clinical Immunology (EAACI) is the leading professional organization in the field of allergy, promoting excellence in clinical care, education, training and basic and translational research, all with the ultimate goal of improving the health of allergic patients. The European Federation of Allergy and Airways Diseases Patients’ Associations (EFA) is a non-profit network of allergy, asthma and Chronic Obstructive Pulmonary Disorder (COPD) patients’ organizations. In support of their missions, the present EAACI Position Paper, in collaboration with EFA, highlights the most important research needs in the field of allergy to serve as key recommendations for future research funding at the national and European levels.
Although allergies may involve almost every organ of the body and an array of diverse external factors act as triggers, there are several common themes that need to be prioritized in research efforts. As in many other chronic diseases, effective prevention, curative treatment and accurate, rapid diagnosis represent major unmet needs. Detailed phenotyping/endotyping stands out as widely required in order to arrange or re-categorize clinical syndromes into more coherent, uniform and treatment-responsive groups. Research efforts to unveil the basic pathophysiologic pathways and mechanisms, thus leading to the comprehension and resolution of the pathophysiologic complexity of allergies will allow for the design of novel patient-oriented diagnostic and treatment protocols. Several allergic diseases require well-controlled epidemiological description and surveillance, using disease registries, pharmacoeconomic evaluation, as well as large biobanks. Additionally, there is a need for extensive studies to bring promising new biotechnological innovations, such as biological agents, vaccines of modified allergen molecules and engineered components for allergy diagnosis, closer to clinical practice. Finally, particular attention should be paid to the difficult-to-manage, precarious and costly severe disease forms and/or exacerbations. Nonetheless, currently arising treatments, mainly in the fields of immunotherapy and biologicals, hold great promise for targeted and causal management of allergic conditions. Active involvement of all stakeholders, including Patient Organizations and policy makers are necessary to achieve the aims emphasized herein.
PMCID: PMC3539924  PMID: 23121771
Allergy; Allergic diseases; Policy; Research needs; Research funding; Europe
3.  Specific antioxidant selenoproteins are induced in the heart during hypertrophy 
Selenium (Se) is thought to confer cardioprotective effects through the actions of antioxidant selenoprotein enzymes that directly limit levels of ROS such as hydrogen peroxide (H2O2) or that reverse oxidative damage to lipids and proteins. To determine how the selenoproteome responds to myocardial hypertrophy, two mouse models were employed: triidothyronine (T3)- or isoproterenol (ISO)-treatment. After 7 days of T3- and ISO-treatment, cardiac stress was demonstrated by increased H2O2 and caspase-3 activity. Neither treatment produced significant increases in phospholipid peroxidation or TUNEL-positive cells, suggesting that antioxidant systems were protecting the cardiomyocytes from damage. Many selenoprotein mRNAs were induced by T3- and ISO-treatment, with levels of methionine sulfoxide reductase 1 (MsrB1, also called SelR) mRNA showing the largest increases. MsrB enzymatic activity was also elevated in both models of cardiac stress, while glutathione peroxidase (GPx) activity and thioredoxin reductase (Trxrd) activity were moderately and nonsignificantly increased, respectively. Western blot assays revealed a marked increase in MsrB1 and moderate increases in GPx3, GPx4, and Trxrd1, particularly in T3-treated hearts. Thus, the main response of the selenoproteome during hypertrophy does not involve increased GPx1, but increased GPx3 for reducing extracellular H2O2 and increased GPx4, Trxrd1, and MsrB1 for minimizing intracellular oxidative damage.
PMCID: PMC3129375  PMID: 21621505
Selenoproteins; cardiac stress; hypertrophy; oxidative stress; antioxidant
4.  Selenoprotein K knockout mice exhibit deficient calcium flux in immune cells and impaired immune responses 
Selenoprotein K (Sel K) is a selenium-containing protein for which no function has been identified. We found that Sel K is an endoplasmic reticulum (ER) transmembrane protein expressed at relatively high levels in immune cells and is regulated by dietary selenium. Sel K−/− mice were generated and found to be similar to WT controls regarding growth and fertility. Immune system development was not affected by Sel K deletion, but specific immune cell defects were found in Sel K−/− mice. Receptor-mediated Ca2+ flux was decreased in T cells, neutrophils, and macrophages from Sel K−/− mice compare to controls. Ca2+-dependent functions including T cell proliferation, T cell and neutrophil migration, and Fcγ-receptor-mediated oxidative burst in macrophages were decreased in cells from Sel K−/− mice compared to controls. West Nile virus (WNV) infections were performed and Sel K−/− mice exhibited decreased viral clearance in the periphery and increased viral titers in brain. Furthermore, WNV-infected Sel K−/− mice demonstrated significantly lower survival (2/23; 8.7%) compared to WT controls (10/26; 38.5%). These results establish Sel K as an ER-membrane protein important for promoting effective Ca2+ flux during immune cell activation and provide insight into molecular mechanisms by which dietary selenium enhances immune responses.
PMCID: PMC3088479  PMID: 21220695
5.  Fluorescence Dequenching Makes Haem-Free Soluble Guanylate Cyclase Detectable in Living Cells 
PLoS ONE  2011;6(8):e23596.
In cardiovascular disease, the protective NO/sGC/cGMP signalling-pathway is impaired due to a decreased pool of NO-sensitive haem-containing sGC accompanied by a reciprocal increase in NO-insensitive haem-free sGC. However, no direct method to detect cellular haem-free sGC other than its activation by the new therapeutic class of haem mimetics, such as BAY 58-2667, is available. Here we show that fluorescence dequenching, based on the interaction of the optical active prosthetic haem group and the attached biarsenical fluorophor FlAsH can be used to detect changes in cellular sGC haem status. The partly overlap of the emission spectrum of haem and FlAsH allows energy transfer from the fluorophore to the haem which reduces the intensity of FlAsH fluorescence. Loss of the prosthetic group, e.g. by oxidative stress or by replacement with the haem mimetic BAY 58-2667, prevented the energy transfer resulting in increased fluorescence. Haem loss was corroborated by an observed decrease in NO-induced sGC activity, reduced sGC protein levels, and an increased effect of BAY 58-2667. The use of a haem-free sGC mutant and a biarsenical dye that was not quenched by haem as controls further validated that the increase in fluorescence was due to the loss of the prosthetic haem group. The present approach is based on the cellular expression of an engineered sGC variant limiting is applicability to recombinant expression systems. Nevertheless, it allows to monitor sGC's redox regulation in living cells and future enhancements might be able to extend this approach to in vivo conditions.
PMCID: PMC3157391  PMID: 21858179
6.  Efficacy and outcome of expanded newborn screening for metabolic diseases - Report of 10 years from South-West Germany * 
National newborn screening programmes based on tandem-mass spectrometry (MS/MS) and other newborn screening (NBS) technologies show a substantial variation in number and types of disorders included in the screening panel. Once established, these methods offer the opportunity to extend newborn screening panels without significant investment and cost. However, systematic evaluations of newborn screening programmes are rare, most often only describing parts of the whole process from taking blood samples to long-term evaluation of outcome.
In a prospective single screening centre observational study 373 cases with confirmed diagnosis of a metabolic disorder from a total cohort of 1,084,195 neonates screened in one newborn screening laboratory between January 1, 1999, and June 30, 2009 and subsequently treated and monitored in five specialised centres for inborn errors of metabolism were examined. Process times for taking screening samples, obtaining results, initiating diagnostic confirmation and starting treatment as well as the outcome variables metabolic decompensations, clinical status, and intellectual development at a mean age of 3.3 years were evaluated.
Optimal outcome is achieved especially for the large subgroup of patients with medium-chain acyl-CoA dehydrogenase deficiency. Kaplan-Meier-analysis revealed disorder related patterns of decompensation. Urea cycle disorders, organic acid disorders, and amino acid disorders show an early high and continuous risk, medium-chain acyl-CoA dehydrogenase deficiency a continuous but much lower risk for decompensation, other fatty acid oxidation disorders an intermediate risk increasing towards the end of the first year. Clinical symptoms seem inevitable in a small subgroup of patients with very early disease onset. Later decompensation can not be completely prevented despite pre-symptomatic start of treatment. Metabolic decompensation does not necessarily result in impairment of intellectual development, but there is a definite association between the two.
Physical and cognitive outcome in patients with presymptomatic diagnosis of metabolic disorders included in the current German screening panel is equally good as in phenylketonuria, used as a gold standard for NBS. Extended NBS entails many different interrelated variables which need to be carefully evaluated and optimized. More reports from different parts of the world are needed to allow a comprehensive assessment of the likely benefits, harms and costs in different populations.
PMCID: PMC3141366  PMID: 21689452
newborn screening; tandem-mass spectrometry
7.  The impact of stroke on emotional intelligence 
BMC Neurology  2010;10:103.
Emotional intelligence (EI) is important for personal, social and career success and has been linked to the frontal anterior cingulate, insula and amygdala regions.
To ascertain which stroke lesion sites impair emotional intelligence and relation to current frontal assessment measurements.
One hundred consecutive, non aphasic, independently functioning patients post stroke were evaluated with the Bar-On emotional intelligence test, "known as the Emotional Quotient Inventory (EQ-i)" and frontal tests that included the Wisconsin Card Sorting Test (WCST) and Frontal Systems Behavioral Inventory (FRSBE) for correlational validity. The results of a screening, bedside frontal network syndrome test (FNS) and NIHSS to document neurological deficit were also recorded. Lesion location was determined by the Cerefy digital, coxial brain atlas.
After exclusions (n = 8), patients tested (n = 92, mean age 50.1, CI: 52.9, 47.3 years) revealed that EQ-i scores were correlated (negatively) with all FRSBE T sub-scores (apathy, disinhibition, executive, total), with self-reported scores correlating better than family reported scores. Regression analysis revealed age and FRSBE total scores as the most influential variables. The WCST error percentage T score did not correlate with the EQ-i scores. Based on ANOVA, there were significant differences among the lesion sites with the lowest mean EQ-i scores associated with temporal (71.5) and frontal (87.3) lesions followed by subtentorial (91.7), subcortical gray (92.6) and white (95.2) matter, and the highest scores associated with parieto-occipital lesions (113.1).
1) Stroke impairs EI and is associated with apathy, disinhibition and executive functioning. 2) EI is associated with frontal, temporal, subcortical and subtentorial stroke syndromes.
PMCID: PMC2987854  PMID: 21029468
8.  A New Approach for Analyzing Cellular Infiltration during Allergic Airway Inflammation 
Journal of immunological methods  2007;328(1-2):21-33.
A mouse model for allergic airway inflammation involving ovalbumin (OVA) sensitization and challenge has been developed that reproduces hallmark features of human asthma and has provided valuable insight into the mechanisms by which this disease occurs. Cellular infiltrate in lungs of mice used in this model have conventionally been evaluated using histological examination of tissue sections and light microscopic analysis of lung lavage samples. As an alternative or complementary approach for characterizing cellular infiltrate, we developed a multicolor fluorescence-activated cell sorter (FACS) method involving the simultaneous detection of seven different markers on lung cell suspensions: CD4, CD8, B220, CD11b, Gr-1, CD49b, and FcεRI. Only some of these cell types increased in OVA-challenged mice compared to PBS controls, including the CD4+, B220+, CD11b+, and FcεRI+ groups. We also examined subpopulations of cells for coexpression of these markers and dissected heterogeneous populations as further evaluation procedures to characterize the cellular infiltrate resulting from OVA challenge. Finally, we combined FACS with real-time PCR to analyze certain cell types in terms of mRNA levels for factors involved in asthma, including GATA-3 and IL-1β. Overall, these FACS-based techniques provide a powerful approach for analyzing cellular profiles in lung tissue from mice used in the mouse model of asthma and may also prove valuable in evaluating cellular infiltrates for other models of inflammation and immune responses.
PMCID: PMC2864229  PMID: 17825315
Asthma; allergy; airway inflammation; FACS; cellular infiltration
9.  The selenoproteome exhibits widely varying, tissue-specific dependence on selenoprotein P for selenium supply 
Nucleic Acids Research  2007;35(12):3963-3973.
Selenoprotein P (Sel P) is a selenium-rich glycoprotein believed to play a key role in selenium (Se) transport throughout the body. Development of a Sel P knockout mouse model has supported this notion and initial studies have indicated that selenium supply to various tissues is differentially affected by genetic deletion of Sel P. Se in the form of the amino acid, selenocysteine, is incorporated into selenoproteins at UGA codons. Thus, Se availability affects not only selenoprotein levels, but also the turnover of selenoprotein mRNAs via the nonsense-mediated decay pathway. We investigated how genetic deletion of Sel P in mice affected levels of the mRNAs encoding all known members of the murine selenoprotein family, as well as three non-selenoprotein factors involved in their synthesis, selenophosphate synthetase 1 (SPS1), SECIS-binding protein 2 (SBP2) and SECp43. Our findings present a comprehensive description of selenoprotein mRNA expression in the following murine tissues: brain, heart, intestine, kidney, liver, lung, spleen and testes. We also describe how abundance of selenoproteins and selenoprotein-synthesis factors are affected by genetic deletion of Sel P in some of these tissues, providing insight into how the presence of this selenoprotein influences selenoprotein mRNA levels, and thus, the selenoproteome.
PMCID: PMC1919489  PMID: 17553827
10.  Induction of TFF1 gene expression in pancreas overexpressing transforming growth factor α 
Gut  1999;45(1):105-111.
BACKGROUND/AIMS—Chronic pancreatitis is an inflammatory disease of the exocrine pancreas associated with extensive fibrosis, enlarged pancreatic ducts, acinar cell degeneration, and the formation of tubular complexes. The molecular and biochemical alterations associated with these histological changes are not kown. Generally, the new family of TFF peptides (formerly known as P-domain peptides or trefoil factors) is aberrantly expressed during chronic inflammatory diseases of the gastrointestinal tract.
METHODS—Using human pancreatic tissues obtained from patients with chronic pancreatitis and murine pancreatic tissues obtained from transgenic mice overexpressing transforming growth factor α (TGF-α), the expression and cellular distribution of TFF1 was analysed using northern blot analysis, polymerase chain reaction (PCR), and immunohistochemistry.
RESULTS—In the normal human pancreas, TFF1 was scarce, with only a few ducts exhibiting cytoplasmic TFF1 immunoreactivity. In contrast, human chronic pancreatitis tissue specimens exhibited strong TFF1 immunoreactivity in ductal cells, areas of ductal hyperplasia, and tubular complexes. Semiquantitative PCR analysis of TFF1 mRNA levels showed enhanced expression of TFF1 in the pancreas of patients with chronic pancreatitis. Furthermore, TFF1 mRNA levels were detectable in the pancreas in four of five transgenic mice overexpressing TGF-α. In contrast, four of five wild type mice did not exhibit a TFF1 mRNA transcript. In addition, while no specific TFF1 immunoreactivity was present in the pancreas of the wild type mice, ductal epithelial cells and duct-like tubular complexes in the pancreas of the transgenic mice overexpressing TGF-α exhibited pronounced TFF1 immunoreactivity.
CONCLUSIONS—Ductal cells and tubular complexes in pancreatic fibrosis express TFF1. As the 5'-flanking region of TFF1 contains an epidermal growth factor responsive enhancer region and the expression of epidermal growth factor and TGF-α is enhanced in pancreatic fibrosis, the enhanced expression of TFF1 in pancreatic fibrosis may be mediated by TGF-α.

Keywords: fibrosis; pS2; pancreatitis; expression; transforming growth factor α; TFF peptides
PMCID: PMC1727567  PMID: 10369712
12.  Neurodevelopmental Outcomes for Children With Hypoplastic Left Heart Syndrome at the Age of 5 Years 
Pediatric cardiology  2013;34(7):1597-1604.
This study aimed to determine the neurodevelopmental (ND) outcome for children with hypoplastic left heart syndrome (HLHS) at early school age. English-speaking patients who underwent the Norwood procedure between 2000 and 2005 were eligible at 4–6 years of age for ND testing. Of the 72 eligible patients, 44 (61 %) agreed to participate, and 37 completed ND testing before the close of the study. Three subjects were excluded from analyses due to late stroke. The ND testing included intelligence, visual motor integration, memory and motor and language skills. Parents and teachers completed measures of behavior and attention problems. Subjects’ scores and parent/teacher ratings were converted to z-scores and compared with test norms. Higher scores on child measures represent better outcomes, whereas higher scores on parent and teacher rating scales indicate more problems. The average ND performance of the tested cohort fell within one standard deviation of the test norms for all measures. However, the subjects performed significantly lower than the test norms on measures of visual-motor integration, fine motor skills, memory, and word structure (z = −0.42 to −0.54; p < 0.005). On the parent and teacher completed measures, the subjects scored higher than the test norms on attention problems (z = 0.40–0.62; p < 0.005). Although the overall ND performance of the cohort was normal, the subjects showed relative weakness in visual motor and attention skills. Ongoing developmental monitoring of these children is recommended to guide interventions that may improve individual outcomes and to assess the impact of changes in clinical management strategies on functional outcomes.
PMCID: PMC3982227  PMID: 23503929
Congenital heart disease/defects; Developmental outcomes; Hypoplastic left heart syndrome
13.  Automated Quantification Reveals Hyperglycemia Inhibits Endothelial Angiogenic Function 
PLoS ONE  2014;9(4):e94599.
Diabetes Mellitus (DM) has reached epidemic levels globally. A contributing factor to the development of DM is high blood glucose (hyperglycemia). One complication associated with DM is a decreased angiogenesis. The Matrigel tube formation assay (TFA) is the most widely utilized in vitro assay designed to assess angiogeneic factors and conditions. In spite of the widespread use of Matrigel TFAs, quantification is labor-intensive and subjective, often limiting experiential design and interpretation of results. This study describes the development and validation of an open source software tool for high throughput, morphometric analysis of TFA images and the validation of an in vitro hyperglycemic model of DM.
Approach and Results
Endothelial cells mimic angiogenesis when placed onto a Matrigel coated surface by forming tube-like structures. The goal of this study was to develop an open-source software algorithm requiring minimal user input (Pipeline v1.3) to automatically quantify tubular metrics from TFA images. Using Pipeline, the ability of endothelial cells to form tubes was assessed after culture in normal or high glucose for 1 or 2 weeks. A significant decrease in the total tube length and number of branch points was found when comparing groups treated with high glucose for 2 weeks versus normal glucose or 1 week of high glucose.
Using Pipeline, it was determined that hyperglycemia inhibits formation of endothelial tubes in vitro. Analysis using Pipeline was more accurate and significantly faster than manual analysis. The Pipeline algorithm was shown to have additional applications, such as detection of retinal vasculature.
PMCID: PMC3981811  PMID: 24718615
14.  Selectins Mediate Small Cell Lung Cancer Systemic Metastasis 
PLoS ONE  2014;9(4):e92327.
Metastasis formation is the major reason for the extremely poor prognosis in small cell lung cancer (SCLC) patients. The molecular interaction partners regulating metastasis formation in SCLC are largely unidentified, however, from other tumor entities it is known that tumor cells use the adhesion molecules of the leukocyte adhesion cascade to attach to the endothelium at the site of the future metastasis. Using the human OH-1 SCLC line as a model, we found that these cells expressed E- and P-selectin binding sites, which could be in part attributed to the selectin binding carbohydrate motif sialyl Lewis A. In addition, protein backbones known to carry these glycotopes in other cell lines including PSGL-1, CD44 and CEA could be detected in in vitro and in vivo grown OH1 SCLC cells. By intravital microscopy of murine mesenterial vasculature we could capture SCLC cells while rolling along vessel walls demonstrating that SCLC cells mimic leukocyte rolling behavior in terms of selectin and selectin ligand interaction in vivo indicating that this mechanism might indeed be important for SCLC cells to seed distant metastases. Accordingly, formation of spontaneous distant metastases was reduced by 50% when OH-1 cells were xenografted into E-/P-selectin-deficient mice compared with wild type mice (p = 0.0181). However, as metastasis formation was not completely abrogated in selectin deficient mice, we concluded that this adhesion cascade is redundant and that other molecules of this cascade mediate metastasis formation as well. Using several of these adhesion molecules as interaction partners presumably make SCLC cells so highly metastatic.
PMCID: PMC3974710  PMID: 24699516
16.  Genome-wide linkage analysis is a powerful prenatal diagnostic tool in families with unknown genetic defects 
Genome-wide linkage analysis is an established tool to map inherited diseases. To our knowledge it has not been used in prenatal diagnostics of any genetic disorder. We present a family with a severe recessive mental retardation syndrome, where the mother wished pregnancy termination to avoid delivering another affected child. By genome-wide scanning using the Affymetrix (Santa Clara, CA, USA) 10k chip we were able to establish the disease haplotype. Without knowing the exact genetic defect, we excluded the condition in the fetus. The woman finally gave birth to a healthy baby. We suggest that genome-wide linkage analysis – based on either SNP mapping or full-genome sequencing – is a very useful tool in prenatal diagnostics of diseases.
PMCID: PMC3598323  PMID: 23032112
linkage analysis; prenatal diagnostics; mental retardation
17.  Minimally invasive and open surgical treatment of proximal tibia fractures using a polyaxial locking plate system: a prospective multi-centre study 
International Orthopaedics  2013;37(4):701-708.
The purpose of this study was to determine whether a polyaxial locking plate of the latest generation (NCB PT®, Zimmer Inc.) which can be applied both open and minimally invasively, can be used as a routine method of treatment for proximal tibia fractures.
Eighty-six patients (35 women, 51 men; mean age 51 years) were enrolled in this prospective multicentre trial. Ninety-six percent of the fractures were intra-articular (AO type B and C); 36 % were treated open and 64 % minimally invasively. Follow-up was obtained three, six and 12 months after surgery.
No implant failure occurred. At 12 months, the functional result using a knee-specific score was good to excellent in 95 %, and 99 % of the fractures were radiologically healed.
The system is a versatile implant for proximal tibia fracture treatment. Polyaxiality and a specific locking mechanism are compatible with different fracture patterns. The minimally invasive technique effectively protects soft tissues but should not be performed at the expense of fracture reduction. Early functional results and complication rate are comparable to those in the literature.
PMCID: PMC3609995  PMID: 23417521
18.  Nutrient status: a missing factor in phenological and pollen research? 
Journal of Experimental Botany  2013;64(7):2081-2092.
Phenology ranks among the best ecosystem processes for fingerprinting climate change since temperature explains a high percentage of the interannual or spatial variation in phenological onset dates. However, roles of other environmental variables, such as foliar nutrient concentrations, are far from adequately understood. This observational study examined the effects of air temperature and 11 nutrients on spring phenology of Betula pendula Roth (birch) along an urban–rural gradient in Munich, Germany, during the years 2010/2011. Moreover, the influence of temperature, nutrients, and air pollutants (NO2 and O3) on the amounts of pollen and catkin biomass in 2010 was evaluated. In addition to the influence of higher temperatures advancing phenological onset dates, higher foliar concentrations of potassium, boron, zinc, and calcium were statistically significantly linked to earlier onset dates. Since flushing of leaves is a turgor-driven process and all the influential nutrients are involved in cell extension, membrane function, and stability, there might be a reasonable physiological interpretation of the observed association. The amounts of pollen were negatively correlated with temperature, atmospheric NO2, and foliar iron concentration, suggesting that these variables restrict pollen production. The results of this study suggested an influence of nutritional status on both phenology and pollen production. The interaction of urbanization and climate change should be considered in the assessment of the impact of global warming on ecosystems and human health.
PMCID: PMC3638828  PMID: 23630329
Betula pendula Roth; birch; catkin biomass; Munich; nutrients; phenology; pollen; temperature; urban heat island.
19.  Classification of Human Coronary Atherosclerotic Plaques Ex-Vivo with T1, T2 and Ultra-short TE MRI 
JACC. Cardiovascular imaging  2013;6(4):466-474.
To determine whether the classification of human coronary atherosclerotic plaques with T1, T2 and ultrashort TE (UTE) MRI would correlate well with atherosclerotic plaque classification by histology.
MRI has been extensively used to classify carotid plaque but its ability to characterize coronary plaque remains unknown. In addition, the detection of plaque calcification by MRI remains challenging. Here we used T1, T2 and UTE MRI to evaluate atherosclerotic plaques in fixed post-mortem human coronary arteries. We hypothesized that the combination of T1, T2 and UTE MRI would allow both calcified and lipid-rich coronary plaques to be accurately detected.
28 plaques from human donor hearts with proven coronary artery disease were imaged at 9.4 Tesla with a T1 weighted 3D FLASH sequence (250 um resolution), a T2 weighted Rare sequence (in plane resolution 0.156mm), and an UTE sequence (300um resolution). Plaques showing selective hypointensity on T2 weighted MRI were classified as lipid-rich. Areas of hypointensity on the T1 weighted images but not the UTE images were classified as calcified. Hyperintensity on the T1 weighted and UTE images was classified as hemorrhage. Following MRI, histological characterization of the plaques was performed with a pentachrome stain and established AHA criteria.
MRI showed high sensitivity and specificity for the detection of calcification (100% and 90%) and lipid-rich necrotic cores (90% and 75%). Only two lipid-rich foci were missed by MRI, both of which were extremely small. Overall, MRI based classification of plaque was in complete agreement with the histological classification in 22/28 cases (weighted κ =0.6945, p<0.0001).
The utilization of UTE MRI allows plaque calcification in the coronary arteries to be robustly detected. High-resolution MRI with T1, T2 and UTE contrast enables accurate classification of human coronary atherosclerotic plaque.
PMCID: PMC3661771  PMID: 23498670
atherosclerosis; coronary artery; MRI; ultra-short TE; plaque classification
20.  Intramuscular Fat and Associations with Metabolic Risk Factors in the Framingham Heart Study 
Intramuscular fat accumulates between muscle fibers or within muscle cells. We investigated the association of intramuscular fat with other ectopic fat deposits and metabolic risk factors.
Approach and Results
Participants (n = 2945; 50.2% women; mean age 50.8 years) from the Framingham Heart Study underwent multidetector computed tomography scanning of the abdomen. Regions of interest were placed on the left and right paraspinous muscle and the muscle attenuation (MA) in Hounsfield units were averaged. We examined the association between MA and metabolic risk factors in multivariable models and additionally adjusted for BMI and visceral fat (VAT) in separate models. MA was associated with dysglycemia, dyslipidemia, and hypertension in both sexes. In women, per standard deviation decrease in MA, there was a 1.34 (95% CI 1.10–1.64) increase in the odds of diabetes, a 1.40 (95% CI 1.22 – 1.61) increase in the odds of high triglycerides, and a 1.29 (95% CI 1.12 – 1.48) increase in the odds of hypertension. However, none of these associations persisted after adjustment for BMI or VAT. In men, we observed similar patterns for most risk factors. The exception was metabolic syndrome, which retained association in women even after adjustment for BMI and VAT, and low HDL and high triglycerides in men, whose associations also persisted after adjustment for BMI and VAT.
MA was associated with metabolic risk factors, but most of these associations were lost after adjustment for BMI or VAT. However, a unique association remained for metabolic syndrome in women and lipids in men.
PMCID: PMC3696991  PMID: 23349188
Metabolism; obesity; intramuscular fat; epidemiology
21.  The RhoD to centrosomal duplication 
Small GTPases  2013;4(2):116-122.
The main functional roles attributed to the centrosome, the major microtubule organizing center (MTOC) of metazoans, are related to cell locomotion, sensory perception and division. The role of vesicular trafficking in the regulation of the centrosome cycle has been largely unexplored. Recently, however, several studies have indicated the involvement of molecules and/or complexes of the trafficking routes in centrosome positioning, duplication and regulation. Functional screens have revealed communication between the outer nuclear envelope, the Golgi apparatus, the endosomal recycling compartment and centrosomes, while other studies underline the involvement of the ESCRT complex proteins in centrosome function. In this commentary, we discuss our recent study, which shows the involvement of an endosomal Rho protein, namely RhoD, in centrosome duplication and possible links between the centrosome’s structural and functional integrity to vesicular trafficking.
PMCID: PMC3747252  PMID: 23422264
Rho GTPase; RhoD; centrosome; recycling endosome; trafficking
22.  Collagen Binding Provides a Sensitive Screen for Variant Von Willebrand Disease 
Clinical chemistry  2013;59(4):10.1373/clinchem.2012.199000.
Von Willebrand factor (VWF) is a multimeric protein that binds platelets and collagen, facilitating hemostasis at sites of vessel injury. Measurement of VWF multimer distribution is critical for diagnosis of variant von Willebrand disease (VWD), particularly types 2A and 2B, but the typical measurement by gel electrophoresis is technically difficult and time consuming. A comparison of VWF collagen binding (VWF:CB) and VWF multimer distribution was performed to evaluate the utility of VWF:CB as a diagnostic test.
Participants were enrolled in the Zimmerman Program for the Molecular and Clinical Biology of VWD. VWF:CB was analyzed with type III collagen and multimer distribution by agarose gel electrophoresis. The study population included 146 healthy controls, 351 individuals with type 1 VWD, and 77 with type 2 VWD. Differences between normal and abnormal multimer groups were assessed with Mann-Whitney tests.
The mean VWF:CB/VWF antigen ratio was 1.10 for individuals with normal multimer distribution and 0.51 for those with abnormal multimer distribution (P<0.001). Sensitivity of VWF:CB for multimer abnormalities was 100% for healthy controls, 99% for type 1, and 100% for type 2A and type 2B VWD using a VWF:CB/VWF antigen cutoff ratio of 0.6, and decreased to 99% for all with a ratio of 0.7. With the exception of individuals with novel or unclassified mutations, the VWF:CB was able to correctly categorize participants with variant VWD.
These findings suggest VWF:CB may substitute for multimer distribution in initial VWD testing, although further studies are needed to validate its clinical utility.
PMCID: PMC3852672  PMID: 23340442
von Willebrand disease; von Willebrand factor; collagen
23.  Confirmation of TNIP1 but not RHOB and PSORS1C1 as systemic sclerosis risk factors in a large independent replication study 
Annals of the rheumatic diseases  2012;72(4):10.1136/annrheumdis-2012-201888.
A recent genome-wide association study in European systemic sclerosis (SSc) patients identified three loci (PSORS1C1, TNIP1 and RHOB) as novel genetic risk factors for the disease. The aim of this study was to replicate the previously mentioned findings in a large multicentre independent SSc cohort of Caucasian ancestry.
4389 SSc patients and 7611 healthy controls from different European countries and the USA were included in the study. Six single nucleotide polymorphisms (SNP): rs342070, rs13021401 (RHOB), rs2233287, rs4958881, rs3792783 (TNIP1) and rs3130573 (PSORS1C1) were analysed. Overall significance was calculated by pooled analysis of all the cohorts. Haplotype analyses and conditional logistic regression analyses were carried out to explore further the genetic structure of the tested loci.
Pooled analyses of all the analysed SNPs in TNIP1 revealed significant association with the whole disease (rs2233287 pMH=1.94×10−4, OR 1.19; rs4958881 pMH=3.26×10−5, OR 1.19; rs3792783 pMH=2.16×10−4, OR 1.19). These associations were maintained in all the subgroups considered. PSORS1C1 comparison showed association with the complete set of patients and all the subsets except for the anti-centromere-positive patients. However, the association was dependent on different HLA class II alleles. The variants in the RHOB gene were not associated with SSc or any of its subsets.
These data confirmed the influence of TNIP1 on an increased susceptibility to SSc and reinforced this locus as a common autoimmunity risk factor.
PMCID: PMC3887516  PMID: 22896740
24.  Tick-borne encephalitis virus (TBEV) – findings on cross reactivity and longevity of TBEV antibodies in animal sera 
By using animal sera as sentinels, natural TBEV foci could be identified and further analyses including investigations of ticks could be initiated. However, antibody response against TBEV-related flaviviruses might adversely affect the readout of such a monitoring. Therefore, the cross-reactivity of the applied TBEV serology test systems – enzyme linked immunosorbent assay (ELISA) and virus neutralization test (VNT) – as well as the longevity of TBEV antibody titres in sheep and goats were investigated in this study.
Cross-reactivity of the TBEV antibody test systems with defined antibody-positive samples against selected members of the Flaviviridae family (e.g. Louping ill virus, West Nile virus) was observed for Louping-ill-positive sera only. In contrast, the commercial West Nile virus (WNV) competitive ELISA showed a high level of cross-reactivity with TBEV-specific positive sera.
To assess the longevity of TBEV antibody titres, sera from two sheep and two goats, which had been immunized four times with a commercially available TBEV vaccine, were tested routinely over 28 months. In three of the four animals, TBEV-specific antibody titres could be detected over the whole test period.
In addition, sera from the years 2010 and 2011 were collected in flocks in different villages of Baden-Württemberg and Thuringia to allow re-examination two to four years after the initial analysis. Interestingly, in most cases the results of the former investigations were confirmed, which may be caused by steadily existing natural TBEV foci.
Cross-reactivity must be taken into consideration, particularly for TBEV serology in regions with a prevalence of Louping ill virus and for serological testing of WNV by cross-reactive ELISAs. Furthermore, over-interpretation of single TBEV-positive serological results should be avoided, especially in areas without a TBEV history.
PMCID: PMC3978054  PMID: 24690234
Tick borne encephalitis; Animal sera; Virus neutralization test; ELISA; Cross-reactivity; Flaviviridae
25.  Evaluation of a Novel Dog Adoption Program in Two US Communities 
PLoS ONE  2014;9(3):e91959.
Millions of dogs enter animal welfare organizations every year and only a fraction of them are adopted. Despite the most recent American Pet Products Association (APPA) data that nearly half the US population owns a dog, only 20% acquired their dog from an animal welfare organization. Studies show that people consider adopting from an animal shelter more often than they actually do, which indicates a potential market increase if programs can make shelter dogs more visible to adopters. This research focused on a novel adoption program where shelter dogs were transferred into foster homes who were tasked with finding an adopter. Shelter dogs were placed in the path of potential adopters and bypassed the need for the adopter to go to the shelter. The results show that this novel program was effective in a variety of ways including getting dogs adopted. Although length of stay was significantly longer for dogs in the program, the dogs were in a home environment, not taking up kennel space in the shelter. The program also had a lower rate of returns than dogs adopted at the shelter. The foster program tapped adopters in different geographical segments of the community than the dogs adopted from the shelter. By bringing shelter dogs to where adopters spend their time (ex: restaurants, parks, hair salons), the program potentially captured a segment of the population who might have obtained their dog from other sources besides the shelter (such as breeders or pet stores). This novel approach can be an effective method for adoption, has many benefits for shelters, and can tap into a new adopter market by engaging their community in a new way.
PMCID: PMC3963870  PMID: 24663804

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