NonViral Gene Delivery of Growth and Differentiation Factor 6 (GDF6) to whole bovine Intervertebral Disc

Question: Low back pain is an increasing global health problem, which is associated with intervertebral disc (IVD) damage and de- generation. Major changes occur in the nucleus pulposus (NP), with the degradation of the extracellular matrix (ECM) [1]. Further studies showed that growth factors from the transforming growth factor (TGF) and bone morphogenic proteins (BMP) family may induce chondrogenic differentiation of mesenchymal stem cells (MSC) [2]. Focusing on non-viral gene therapies and their possible translation into the clinics, we investigated if GDF6 (syn. BMP13 or CDMP2) can induce regeneration of degraded NP. We hypothesized that IVD transfected with plasmid over-expressing GDF6 also up-regulates other NP- and chondrogenic cell markers and enhances ECM deposition. Methods: Bovine IVD cells were isolated by pronase/collagenase II overnight digestion. After monolayer expansion up to passage 3, cells were transfected with the plasmid pGDF6 (RG211366, Origene, SF) or with green fluorescence protein (GFP) control using the NeonÒ transfection system (Invitrogen, Basel), both equipped with a Cy- tomegalovirus (CMV) promotor to induce over-expression. We tested a range of yet unpublished parameters for each of the primary disc cells to optimize efficiency. To test a non-viral gene therapy applied directly to 3D whole organ culture, bovine IVDs were harvested from fresh tails obtained from the abattoir within 5 h post-mortem [3]. Discs were then pre-incubated for 24 h in high glucose Dulbecco’s Modified Eagle Medium and 5 % fetal calf serum. Each disc was transfected by injection of 5 lg of plasmid GDF6 (Origene, RG211366) into the center by 25G needle and using Hamilton sy- ringe. Electroporation was performed using 2-needle array electrode or tweezertrodes; 8 pulses at 200mv/cm with an interval of 10 ms were applied using ECM830 Square Wave Electroporation System (Harvard Apparatus, MA) (Fig. 1). After transfection discs were cultured for 72 h to allow expression of GFP or GDF6. Discs were then fixed, cryosectioned and analysed by immunofluorescence against GDF6. Results: We successfully transfected bovine NP and AF cells in monolayer culture with the two plasmids using a 1,400 V, 20 ms and 2 pulses with a *25 % efficiency using 0.15 M cells and 3 lg DNA (Fig. 1). Organ IVD culture transfection revealed GFP6 positive staining in the centre of the disc using 2-needle array electrode. Results from tweezertrodes did not show any GFP posi- tive cells. Conclusions: We identified novel parameters to successfully transfect primary bovine IVD cells. For transfection of whole IVD explants electroporation parameters need to be further optimized. Acknowledgments: This study was supported by the Lindenhof Foundation ‘‘Forschung und Lehre’’ (Project no. 13-02-F). References 1. Roughly PJ (2004) Spine (Phila) 29:2691–2699 2. 3. Clarke LE, McConell JC, Sherratt MJ, Derby B, Richardson SM, Hoyland JA (2014) Arthritis Res Ther 16:R67 Chan SC, Gantenbein-Ritter B (2012) J Vis Exp 60(60):e3490

Development and implementation of a dynamic heterogeneous proton equivalent anthropomorphic thorax phantom for the assessment of scanned proton beam therapy

DEVELOPMENT AND IMPLEMENTATION OF A DYNAMIC HETEROGENEOUS PROTON EQUIVALENT ANTHROPOMORPHIC THORAX PHANTOM FOR THE ASSESSMENT OF SCANNED PROTON BEAM THERAPY by James Leroy Neihart, B.S. APPROVED: ______________________________David Followill, Ph.D. ______________________________Peter Balter, Ph.D. ______________________________Narayan Sahoo, Ph.D. ______________________________Kenneth Hess, Ph.D. ______________________________Paige Summers, M.S. APPROVED: ____________________________ Dean, The University of Texas Graduate School of Biomedical Sciences at Houston DEVELOPMENT AND IMPLEMENTATION OF A DYNAMIC HETEROGENEOUS PROTON EQUIVALENT ANTHROPOMORPHIC THORAX PHANTOM FOR THE ASSESSMENT OF SCANNED PROTON BEAM THERAPY A THESIS Presented to the Faculty of The University of Texas Health Science Center at Houston andThe University of TexasMD Anderson Cancer CenterGraduate School of Biomedical Sciences in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE by James Leroy Neihart, B.S. Houston, Texas Date of Graduation August, 2013 Acknowledgments I would like to acknowledge my advisory committee members, chair David Followill, Ph.D., Peter Balter, Ph.D, Narayan Sahoo, Ph.D., Kenneth Hess, Ph.D., Paige Summers M.S. and, for their time and effort contributed to this project. I would additionally like to thank the faculty and staff at the PTC-H and the RPC who assisted in many aspects of this project. Falk Pӧnisch, Ph.D. for his breath hold proton therapy treatment expertise, Matt Palmer and Jaques Bluett for proton dosimetry assistance, Matt Kerr for verification plan assistance, Carrie Amador, Nadia Hernandez, Trang Nguyen, Andrea Molineu, Lynda McDonald for TLD and film dosimetry assistance. Finally, I would like to thank my wife and family for their support and encouragement during my research and studies. Development and implementation of a dynamic heterogeneous proton equivalent anthropomorphic thorax phantom for the assessment of scanned proton beam therapy By: James Leroy Neihart, B.S. Chair of Advisory Committee: David Followill, Ph.D Proton therapy has been gaining ground recently in radiation oncology. To date, the most successful utilization of proton therapy is in head and neck cases as well as prostate cases. These tumor locations do not suffer from the resulting difficulties of treatment delivery as a result of respiratory motion. Lung tumors require either breath hold or motion tracking, neither of which have been assessed with an end-to-end phantom for proton treatments. Currently, the RPC does not have a dynamic thoracic phantom for proton therapy procedure assessment. Additionally, such a phantom could be an excellent means of assessing quality assurance of the procedures of proton therapy centers wishing to participate in clinical trials. An eventual goal of this phantom is to have a means of evaluating and auditing institutions for the ability to start clinical trials utilizing proton therapy procedures for lung cancers. Therefore, the hypothesis of this study is that a dynamic anthropomorphic thoracic phantom can be created to evaluate end-to-end proton therapy treatment procedures for lung cancer to assure agreement between the measured and calculated dose within 5% / 5 mm with a reproducibility of 2%. Multiple materials were assessed for thoracic heterogeneity equivalency. The phantom was designed from the materials found to be in greatest agreement. The phantom was treated in an end-to-end treatment four times, which included simulation, treatment planning and treatment delivery. Each treatment plan was delivered three times to assess reproducibility. The dose measured within the phantom was compared to that of the treatment plan. The hypothesis was fully supported for three of the treatment plans, but failed the reproducibility requirement for the most aggressive treatment plan.

Do pingers cause stress in fish? An experimental tank study with European sardine, Sardina pilchardus (Walbaum, 1792) (Actinopterygii, Clupeidae), exposed to a 70 kHz dolphin pinger

Date of acceptance: 06/12/2014 Acknowledgments The study was funded by the Portuguese Ministry of Science (Fundac¸a˜o para a Cieˆncia e Tecnologia– FCT) through a PhD Grant of SG (SFRH/BD/47931/2008). We would like to thank the captain of the purse-seiner (Jose´ Manuel Saveedra) and his crew for facilitating the capture and transport of live fish. Moreover, we want to thank Ana Marc¸alo for suggestions on the experimental design, Manuel Garci for technical advice on underwater video recordings and James Turner from the company Future Oceans for providing technical details on the 70 kHz dolphin pingers. We would also like to acknowledge the scientific advice of Dr. Jose´ Iglesias and the technical and logistic support for the preparation of the laboratory and the materials for tank experiments by Enrique Martı´nez Gonza´lez, Ricardo Pazo´and other staff at the aquaculture facilities of the Spanish Institute for Oceanography (IEO) and the Marine Sciences Station of Toralla (ECIMAT) in Vigo. Furthermore, we are grateful to Francisco de la Granda Grandoso for his practical assistance during the fish tank experiments and to Juan Santos Blanco for helping with statistical analysis. Finally, we would like to thank Pilar Riobo´ Agula, Amelia Fernandez Villamarin, Jose´ Franco Soler, Jose´ Luis Mun˜oz, Angela Benedetti, Marcos Antonio Lopez Patin˜o and Marta Conde Sieira for scientific advice and practical support with cortisol analysis and Rosana Rodrı´guez for preparing histological samples for us.

Revival of oscillation from mean-field-induced death : Theory and experiment

Submitted ACKNOWLEDGMENTS T. B. acknowledges the financial support from SERB, Department of Science and Technology (DST), India [Project Grant No.: SB/FTP/PS-005/2013]. D. G. acknowledges DST, India, for providing support through the INSPIRE fellowship. J. K. acknowledges Government of the Russian Federation (Agreement No. 14.Z50.31.0033 with Institute of Applied Physics RAS).

Something fishy about homecooked infant feeding recipes

Acknowledgments The authors would like to thank the statistical team within the Division of Applied Health Sciences at the University of Aberdeen for their support in analysing the data. This work was supported by The Scottish Government’s Rural and Environment Science and Analytical Services (RESAS) division (LC grant). Substantial contributions to the conception or design of the work; analysis, and interpretation of data for the work were conducted by Sharon Carstairs (SC) under the supervision of Dr K Kiezebrink (KK), Dr D Marais (DM) and Dr L Craig (LC). Data collection was conducted by SC and a 10% duplicate data extraction by DM. Financial Support This work was funded by The Seafish Authority and Interface Food and Drink as part of a Doctorate Scholarship undertaken at the University of Aberdeen (grant number HS053 RBZ0214).

Spatial network surrogates for disentangling complex system structure from spatial embedding of nodes

ACKNOWLEDGMENTS MW and RVD have been supported by the German Federal Ministry for Education and Research (BMBF) via the Young Investigators Group CoSy-CC2 (grant no. 01LN1306A). JFD thanks the Stordalen Foundation and BMBF (project GLUES) for financial support. JK acknowledges the IRTG 1740 funded by DFG and FAPESP. MT Gastner is acknowledged for providing his data on the airline, interstate, and Internet network. P Menck thankfully provided his data on the Scandinavian power grid. We thank S Willner on behalf of the entire zeean team for providing the data on the world trade network. All computations have been performed using the Python package pyunicorn [41] that is available at https://github.com/pik-copan/pyunicorn.

Do the recent severe droughts in the Amazonia have the same period of length?

5 figures Acknowledgments This work was partially supported by the NNSFC (Grant Nos. 11305062, 11135001), the DFG/FAPESP (Grant No. IRTG 1740/TRP 2011/50151-0), and Government of the Russian Federation (Agreement No. 14.Z50.31.0033 with Institute of Applied Physics RAS). All data for this paper is properly cited and referred to in the reference list.

Hunger and Satiety Mechanisms and Their Potential Exploitation in the Regulation of Food Intake

Acknowledgments Tehmina Amin is the Project Manager and Julian Mercer is Project Coordinator for Full4Health. Both are funded by the Full4Health project (grant agreement no. 266408) under the EU Seventh Framework Programme (FP7/2007–2013). Julian Mercer is funded by the Scottish Government, Rural and Environment Science and Analytical Services Division, Food, Land and People programme. He is also a partner in FP7 projects: NeuroFAST (grant agreement no. 245099) and SATIN (grant agreement no. 289800).

NEAT - A New, Evolutive API and Transport-Layer Architecture for the Internet

ACKNOWLEDGMENTS This work has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 644334 (NEAT). The views expressed are solely those of the authors.

A Toolbox for Quantitative Gene Expression in Varroa destructor : RNA Degradation in Field Samples and Systematic Analysis of Reference Gene Stability

Funding: This project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no. 613960 (SMARTBEES) (http://www.smartbees-fp7.eu/) and Veterinary Medicines Directorate, Department for Environment Food & Rural Affairs (Project # VM0517) (https://www.gov.uk/government/organisations/veterinary-medicines-directorate). CHM was supported by a Biosciences Knowledge Transfer Network Biotechnology and Biological Sciences Research Council (KTN-BBSRC CASE) Studentship (BB/L502467/1) (http://www.bbsrc.ac.uk/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Acknowledgments We gratefully acknowledge Mr Sebastian Bacz’s expert help and advice with beekeeping.

Stability threshold approach for complex dynamical systems

Acknowledgments This paper was developed within the scope of the IRTG 1740/TRP 2011/50151-0, funded by the DFG/FAPESP, and supported by the Government of the Russian Federation (Agreement No. 14.Z50.31.0033 with the Institute of Applied Physics RAS). The first author thanks Dr Roman Ovsyannikov for valuable discussions regarding estimation of the mistake probability.

Maternal smoking dysregulates protein expression in second trimester human fetal livers in a sex-specific manner

Acknowledgments The staff at Grampian National Health Service Pregnancy Counseling Service were essential for collecting fetuses. We thank the Aberdeen Proteomics Core Facility (University of Aberdeen) for their expert assistance. Support for the study was provided by the Chief Scientist Office (Scottish Executive, CZG/1/109, & CZG/4/742), National Health Service Grampian Endowments (08/02), the European Community's Seventh Framework Programme (FP7/2007–2013) under grant agreement no 212885, and the Medical Research Council, UK (MR/L010011/1).

Unified underpinning of human mobility in the real world and cyberspace

Acknowledgments This work is supported by National Science Foundation of China (Grant No. 61573064, 61074116 and 11547188), the Youth Scholars Program of Beijing Normal University (grant No. 2014NT38), and the Fundamental Research Funds for the Central Universities Beijing Nova Programme, China. XYY acknowledges the support from the National Natural Science Foundation of China (Grant No. 61304177) and the Fundamental Research Funds of BJTU (Grant No. 2015RC042).

Biomechanical factors may explain why grasping violates Weber's law

Copyright © 2015. Published by Elsevier Ltd. Acknowledgments The experiment was part of N. Aschenneller’s MD thesis. The study was funded by the Staedtler Stiftung (Nuremberg, Germany).

Anisakis infection in allis shad, Alosa alosa (Linnaeus, 1758), and twaite shad, Alosa fallax (Lacépède, 1803), from Western Iberian Peninsula Rivers : zoonotic and ecological implications

Acknowledgments The authors would like to thank M. N. Cueto and J.M. Antonio (ECOBIOMAR) for their excellent technical support and also Rodrigo López for making the map of the study area. We also thank the personal of the Vigo IEO, for providing information about shad captures at sea collected on the basis of national program (AMDES) included in the European Data Collection Framework (DCF) project. We are also grateful to Comandancia Naval de Tui for providing fishing data. M. Bao is supported by a PhD grant from the University of Aberdeen and also by financial support of the contract from the EU Project PARASITE (grant number 312068). This study was partially supported by a PhD grant from the Portuguese Foundation for Science and Technology (FCT) SFRH/BD/44892/2008) and partially supported by the European Regional Development Fund (ERDF) through the COMPETE—Operational Competitiveness Programme and national funds through Foundation for Science and Technology (FCT), under the project BPEst-C/MAR/ LA0015/2013. The authors thank the staff of the Station of Hydrobiology of the USC BEncoro do Con^ due their participation in the surveys. This work has been partially supported by the project 10PXIB2111059PR of the Xunta de Galicia and the project MIGRANET of the Interreg IV BSUDOE (South-West Europe) Territorial Cooperation Programme (SOE2/P2/E288). D.J. Nachón is supported by a PhD grant from the Xunta de Galicia (PRE/2011/198)