COMMUNICATION
TO THE
EDITOR
Variable Electric Fields for High Throughput Electroporation Protocol Design in Curvilinear Coordinates Francois Fernand, Liel Rubinsky, Alex Golberg, Boris Rubinsky Department of Mechanical Engineering, Etcheverry Hall, 6124, University of California at Berkeley, 2521 Hearst Ave, Berkeley, California 94720; telephoneQ2: XXX; fax: XXX; e-mail:
[email protected] Received 14 October 2011; revision received 5 January 2012; accepted 9 February 2012 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bit.24479
ABSTRACT: The mathematical solution to the electric field equation in cylindrical coordinates, has suggested to us a new experimental methodology and device for reducing experimental effort in designing electroporation protocols. Using a new cylindrical electroporation system, we show, with an Escherichia coli cell model, how key electroporation parameters emerge precisely from single experiments rather than through interpolation from numerous experiments in the conventional Cartesian electroporation system. Biotechnol. Bioeng. 2012;9999: 1–6. ß 2012 Wiley Periodicals, Inc. KEYWORDS: high throughput; electroporation; protocol design
The permeabilization of the cell membrane using electric fields applied across the membrane is known as electroporation (Neumann et al., 1982) or electropermeabilization (Stopper et al., 1985). Electroporation is reversible when cells survive the electropermeabilization and irreversible when they do not. Reversible electroporation is commonly used in biotechnology and medicine for such applications as gene or drug delivery into cells (Dev et al., 2000). Irreversible electroporation is important for non-thermal sterilization in the food industry, biotechnology and medicine, and for tissue ablation in medicine (Pakhomov et al., 2010; Rubinsky, 2010). The outcome of an electroporation protocol, whether reversible or irreversible, depends on the parameters of the electric field such as strength, pulse length, number of pulses, time interval between pulses, frequency; on solution composition, pH, temperature and on cell type, shape and size. Because electroporation depends on so many parameters, designing optimal electroporation protocols requires tedious and lengthy efforts. To illustrate the complexity of Correspondence to: A. Golberg
ß 2012 Wiley Periodicals, Inc.
protocol design, Figure 1 shows a theoretical curve adapted from (Dev et al., 2000), which correlates electric field strength, single pulse length and the biophysical phenomenon that occurs when the particular parameters are applied across a cell. One of the most important features of the figure is the line that separates between the reversible and irreversible electroporation domains, which is critical in designing optimal electroporation protocols. In optimal reversible electroporation it is desirable to be close to and below that line while in optimal irreversible electroporation it is desirable to be close to and above that line. Conventional methods for the systematic development of optimal electroporation protocols employ experimental systems made of two parallel electrodes, bounding the media of interest, in a one-dimensional Cartesian configuration (e.g., Sale and Hamilton, 1967; Hamilton and Sale, 1967). The solution to the simple Laplace equation (r2 ’ ¼ 0; where w is the potential) for a homogeneous Cartesian system, subject to constant voltage boundary conditions on the electrodes, V2 and V1, gives an expression for the electric field between the planar electrodes. It is, ðV2 V1 Þ=L where L is the distance between the electrodes. It is evident that the Cartesian configuration produces a constant electric field in the treated medium between the electrodes. Identifying the electric field parameters that separate between reversible and irreversible electroporation requires numerous constant electric field experiments, in which the electric field strength is continuously changed in separate experiments until the interface is detected approximately, through interpolation between experiments (Rubinsky et al., 2008). Several approaches were introduced for multiparameter optimization of in vitro and in vivo electroporation. Heiser (1999) published an extensive review on electroporation parameters for various cell lines and general guidelines for electroporation protocol optimizations in vitro (Heiser, 1999). A review and guidelines for optimization of in vivo electroporation applications was reported by Gehl (2003).
Biotechnology and Bioengineering, Vol. 9999, No. 9999, 2012
BIT-11-943.R1(24479)
1
Figure 1.
Theoretical mapping of biophysical effects experienced by cells as a function of electroporation parameters.
Furthermore, several statistical methodologies were proposed to reduce the number of experiments required for protocol optimizations. Multifactorial experimental design for optimizing transformation protocols was introduced by (Marciset and Mollet, 1994). Keng-Shiang et al. (2007) used the Taguchi Method for the optimization of gene electrotransfer (Keng-Shiang et al., 2007). Recently, a central composite design was used to optimize electroporation protocols (Madeira et al., 2010). In this study we developed a different approach to multiparameter optimization, based on the use in a single experiment of a well-defined variable electric field topology in the curvilinear coordinate system. The concept will be illustrated with a simple to implement cylindrical coordinate system. The electric field calculated from the solution to the one-dimensional Laplace equation in cylindrical coordinates, in a medium between two cylinders of radiuses R1 and R2 on which electric potentials of V1 and V2 are imposed, respectively, is given by,
experiment is performed with cylindrical (or spherical) electrodes, the results of a single experiment produce continuous information on the effect of a wide range of electric fields, which are quantified by the radius at which they are produced. In contrast, to produce similar information, the conventional Cartesian electrode system requires a very large number of experiments and the interpolation of results between the studied discrete data points. Figure 2 illustrates results obtained from a study performed with a one-dimensional cylindrical system, using Escherichia coli BL21 (D13) PSJS1244, an ampicillin stable strain. The Figure 2d shows the electric field at the reversible/ irreversible interface as a function of the number of pulses. The microorganisms were spread on a Petri dish and a constant pulsed electric potential was imposed on two concentric metal cylinders, in contact with the surface on which the microorganism was plated. In the one-dimensional cylindrical electrode system used, the outer diameter of the inner cylinder was 1.18 mm and the inner diameter of the outer cylinder was 22.15 mm. The electric pulse was delivered by a BTX (BTX ECM 830, Harvard Apparatus, MAQ3). Four sites were treated in each Petri dish, after which the samples were incubated for 18 h at 378C and examined. Figure 2a and d reports on results with a pH buffered plate, at which pH did not change after the application of electric field, and in which 2,200 V pulses were applied between the concentric electrodes in 40 ms pulse duration at 1 Hz frequency. Five repeats were performed for each condition.
V1 V 2 r ln RR21 where r is the variable radius inside the domain of interest. Obviously, the electric field varies continuously as an inverse function of the radius. (In one-dimensional spherical coordinates the electric field varies as one over the radius squared.) Therefore, in a single experiment in onedimensional cylindrical or spherical electrode systems, the cells between the electrodes will experience a continuously variable electric field, that is, nevertheless, well defined as a function of the radius. The response of the cells to any electroporation protocol can be evaluated as a function of their relative location (defined by radius) and thereby correlated to the electric field. Therefore, when an
2
Biotechnology and Bioengineering, Vol. 9999, No. 9999, 2012
Figure 2. Electroporation parameters analysis: (a) image of a plate electroporated in one-dimensional cylindrical coordinates, after incubation. The central part has no cell colonies, (b) a schematic view of the analyzed cylindrical system. Two cylindrical electrodes with radiuses R1 and R2 were used. The radius (r) of irreversible electroporated zone was measured. c: Calculated electric field-radius (r) correlation for the experiment, (d) electric parameters that identify the conditions in which irreversible electroporation occurs during electroporation of E. coli in pH buffered medium (error bar 1 standard deviation).
It should be emphasized that each data point on the curves was obtained from a single experiment (with five repeats). In contrast, obtaining such a single data point with Cartesian electrodes would require numerous single electric field experiments and interpolation. Figure 2b and c shows how the plot in Figure 2d was obtained. Figure 2a shows the appearance of a treated cylinder after incubation. It is evident that the cells in the central area did not survive the electric fields to which they were exposed and did not form colonies. To determine the radius of cell death we measured the innermost radius of the colonies that survived electroporation, as described in the Materials and Methods Section. Figure 2b shows the model of the analyzed system. Two cylindrical electrodes, with radiuses of R1 and R2, and a measured radius (r) of a zone where irreversible electroporation takes place, are shown on Figure 2b. Then, the mathematical expression for the electric field as a function of radius in cylindrical coordinates was used to produce Figure 2c. Figure 2c is used to correlate the radius of cell death in Figure 2a with the electric field at that radius. The electric field at the radius of cell death is than plotted as a function of number of pulses in Figure 2d. This plots the electric parameters at which irreversible electroporation begins. In summary, we propose that the use of cylindrical onedimensional electrodes will substantially reduce the number of experiments needed to design optimal electroporation protocols, over those obtained with the use of traditional Cartesian electrodes. In this report we have shown the use of the concept for obtaining the reversible/irreversible interface. Obviously a similar experiment with fluorescence dies or genes can be used to determine the parameters at the interface between reversible and no effect electric fields. Furthermore, this method provides a means to examine in a single experiment, various colonies that have undergone electroporation with a wide range of well-defined electroporation parameters. The relative location of each colony of interest identifies the electroporation conditions it has experienced. It should be noted that the idea of a welldefined topological space of variable electric fields could be extended through further research in topology to the design of systems of more complex surfaces than the cylinder or sphere, which may produce in a single experiment complex ranges of parameters of interest.
Dickinson & Co, NJQ4) was cut 1 cm from the top, to form the inner, 0.6 mm radius cylinder. The needle was then inserted through the center of the plastic square in the middle of the brass ring forming two concentric cylinders.
Materials and Methods
Plate Preparation
Experimental Device The cylindrical one-dimensional electroporation electrodes were manufactured using a Perspex ‘‘square’’ (3 cm by 3 cm) basis. A half cm notch carved in the side of the square was attached to the top of a brass ring using a heated glue gun. The brass ring had an inside diameter of 22.15 mm, an outside diameter of 25.40 mm, and a height of 4 mm. The tip of an 18 gauge steel needle (Precision Glide needle, Becton
Electroporation Procedure The study was performed using E. coli BL21 (D13) PSJS1244 an ampicillin stable strain. A single E. coli colony was used to inoculate 50 mL of sterile LB Broth (Ditco, NYQ5) containing 100 mg/mL of ampicillin (American SystemQ6). The sample was placed in a Thermo Scientific MaxQ 4450 shaker-incubator. The temperature was maintained at 378C. The shaker speed was 200 rpm to allow aeration for optimal growth. The sample was kept in the shaker-incubator for 14 h to reach stationary phase. The final concentration of approximately 106 CFU/mL was determined by viable count method. After 14 h in the shaker-incubator a 100 mL sample was removed and diluted in 10 mL of sterile water (100 dilution) 100 mL of the diluted sample was plated on to each pre-prepared agar plate and spread using glass beads (Novagen, CAQ7). The glass beads were removed and the electroporation device was inserted into the agar in one quadrant of the Petri dish. The device was pushed into the agar plate until the ring and needle touched the Petri dish bottom in order to ensure they were at the same depth. Alligator clips were attached to the brass ring and the 18G needle. The alligator clips were never in direct contact with the agar to ensure no direct discharge into the gel. This allowed the field to be equally distributed around the needle. The clips were hooked up to the BTX (BTX-model 610, BTX ECM 830 square-wave electroporator, Harvard Apparatus). The electroporation parameters used were 2,200 V, 40 ms pulse duration, 1 Hz frequency. The numbers of pulses were changed between experiments. Statistical analysis was done with the final parameters recorded from the BTX device. Following the electroporation the needle and the ring were removed from the agar gel. (A similar experiment was than performed in another quadrant. A total of four experiments were performed per dish.) A total of five experiments per parameter were performed. After the experiment the Petri dish was incubated at 378C for 18 h. Following the incubation period the dishes were removed and IRE curve radius was measured.
Agar Petri plates were prepared to maintain pH 7 after the application of electric pulses. The mixture was composed of 0.1 g/L NaCl (Spectrum Chemical, Mfg Corp, CAQ8), 10 g/L Bactotryptone, 5 g/L Yeast Extract, 15 g/L Bacto Agar (Becton Dickinson & Co, NJQ9), 0.5 g/L glucose was dissolved in distilled water and heated at 1218C in an autoclave for 15 min. After cooling down and reaching 508C, 23.83 g/L HEPES (Sigma-Aldrich, CQ10A) and ampicillin (American BioanalyticalQ11) at 10 mg/mL was added to
Fernand et al.: VariableQ1 Electric Fields Biotechnology and Bioengineering
3
100 mg/mL final concentration. The buffered agar was then poured into a 100 mm Petri dish and the drying time between the pouring and the closing of the plates was 6.5 min. In fact, the evaporation of water during storage must be taken into account because it changes the NaCl concentration and of course the conductivity of the medium.
Radius Measurement and Statistical Analysis Electroporated plates were removed from the incubator after 18 h. Digital images of the plates and scale reference were taken and then used to determine the death zone diameter. The error on the electric field estimate includes the diameter measurement errors (precision of 0.05 mm) and the BTX device output error (20 V). The reported radius is an average of five repeats with a standard deviation calculated from the five measurements.
References Dev S, Rabussay D, Widera D, Hofmann G. 2000. Medical applications of electroporation. IEEE Trans Plasma Sci 28(1):206–223. Gehl J. 2003. Electroporation: Theory and methods, perspectives for drug delivery, gene therapy and research. Acta Physiol Scand 177(4): 437–447.
4
Biotechnology and Bioengineering, Vol. 9999, No. 9999, 2012
Hamilton WA, Sale AJH. 1967. Effect of high electric field on microorganisms. II. Mechanism of action of the lethal effect. Biochim Biophys Acta 148:788–800. Heiser WC. 1999. Optimizing electroporation conditions for the transformation of mammalian cells. In: Tymms MJ, editor. Transcription factor protocols. Totowa, New Jersey: Springer. p 117–134. Keng-Shiang H, Sheng-Chung Y, Hung-Yi C, Yu-Cheng L. 2007. Optimization of Gene Transfection Condition using Taguchi Method for an Electroporation Microchip; June 10–14, 2007. p 847–850. Madeira C, Ribeiro S, Turk M, Cabral J. 2010. Optimization of gene delivery to HEK293T cells by microporation using a central composite design methodology. Biotechnol Lett 32(10):1393–1399. Marciset O, Mollet B. 1994. Multifactorial experimental design for optimizing transformation: Electroporation of Streptococcus thermophilus. Biotechnol Bioeng 43(6):490–496. Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH. 1982. Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J 1(7):841–845. Pakhomov AG, Miklavcic D, Markov MM. 2010. Advanced electroporation techniques in biology and medicine. Boca Raton: CRC Press. 521 p. Rubinsky B, editor. ‘‘Irreversible Electroporation’’ Springer. Series in Biomedical Engineering 2010, XIV, 314 p. Hardcover Chennai ISBN: 978-3-642-05419-8. Rubinsky J, Onik G, Mikus P, Rubinsky B. 2008. Optimal parameters for the destruction of prostate cancer using irreversible electroporation. J Urol 180(6):2668–2674. Sale AJH, Hamilton WA. 1967. Effect of high electric field on microorganisms. I. Killing of bacteria and yeast. Biochim Biophys Acta 148:781–788. Stopper H, Zimmermann U, Wecker E. 1985. High yields of DNA-transfer into mouse L-cells by electropermeabilization. Z Naturforsch 40(11–12):929–932.
XXXQ12
F. Fernand, L. Rubinsky, A. Golberg*, B. Rubinsky. . . . . . . . . . . . xx–xx Variable Electric Fields for High Throughput Electroporation Protocol Design in Curvilinear Coordinates
Q1: Author: A running head short title was not supplied; please check if this one is suitable and, if not, please supply a short title of up to 45 characters that can be used instead. Q2: Author: Please provide the corresponding author’s telephone and fax number. Q3: Author: Please provide the manufacturer’s state name. Q4: Author: Please provide the manufacturer’s city name. Q5: Author: Please provide the manufacturer’s city name. Q6: Author: Please provide the manufacturer’s city and state names. Q7: Author: Please provide the manufacturer’s city name. Q8: Author: Please provide the manufacturer’s city name. Q9: Author: Please provide the manufacturer’s city name. Q10: Author: Please provide the manufacturer’s city name. Q11: Author: Please provide the manufacturer’s city name. Q12: Author: New for 2012: Biotechnology and Bioengineering is initiating publication of a graphical Table of Contents. Please supply an image and text for this purpose that adheres to the following guidelines: Image: A colorful (if possible), mostly square image from your article. Please ensure images are of high enough quality for online publication. Text: Descriptive text (2-3 sentences; 80 word maximum) which should capture the most compelling aspects of your work, aimed at a broad readership. Submit the requested image and text with your page proofs. The Editorial Office will select the image and text if they are not supplied.
USING E-ANNOTATION TOOLS FOR ELECTRONIC PROOF CORRECTION Required Software Adobe Acrobat Professional or Acrobat Reader (version 7.0 or above) is required to e-annotate PDFs. Acrobat 8 Reader is a free download: http://www.adobe.com/products/acrobat/readstep2.html. For help with system requirements, go to: http://www.adobe.com/support/. Once you have Acrobat Reader on your PC and open the proof, you will see the Commenting Toolbar (if it does not appear automatically go to Tools>Commenting>Commenting Toolbar). If these options are not available in your Adobe Reader menus then it is possible that your Adobe version is lower than 7 or the PDF has not been prepared properly.
PDF Annotations (Adobe Reader version 7 or 8) – Commenting Toolbars look like this:
(PC, Adobe version 7)
(PC, Adobe version 8, rightͲclick on title bar (Comment & Markup) to show additional icons)
(Mac)
PDF Annotations (Adobe Reader version 9) If you experience problems annotating files in Adobe Acrobat Reader 9 then you may need to change a preference setting in order to edit.
The default for the Commenting toolbar is set to ‘off’ in version 9. To change this setting select ‘Edit | Preferences’, then ‘Documents’ (at left under ‘Categories’), then select the option ‘Never’ for ‘PDF/A View Mode’. (the Commenting toolbar is the same as in version 8).
PLEASE DO NOT ATTEMPT TO EDIT THE ARTICLE TEXT ITSELF TO INDICATE INSERT, REPLACE, OR REMOVE TEXT x
Insert text
button on the Commenting toolbar. Click to set the cursor location in the Click the ‘Text Edits’ text and simply start typing. The text will appear in a commenting box. You may also cut-and-paste text from another file into the commenting box. Close the box by clicking on ‘x’ in the top right-hand corner. It can be deleted by right clicking (for the PC, ctrl-click on the Mac) on it and selecting ‘Delete’. x
Replace text
Click the ‘Text Edits’ button on the Commenting toolbar. To highlight the text to be replaced, click and drag the cursor over the text. Then simply type in the replacement text. The replacement text will appear in a commenting box. You may also cut-and-paste text from another file into this box. To replace formatted text (an equation for example) please Attach a file (see below). x
Remove text
Click the ‘Text Edits’ button on the Commenting toolbar. Click and drag over the text to be deleted. Then press the delete button on your keyboard. The text to be deleted will then be struck through.
HIGHLIGHT TEXT/MAKE A COMMENT Click on the ‘Highlight’ button on the commenting toolbar. Click and drag over the text. To make a comment, double click on the highlighted text and simply start typing.
ATTACH A FILE Click on the ‘Attach a file’ button on the commenting toolbar. Click on the figure, table or formatted text to be replaced. A window will automatically open allowing you to attach a file. To make a comment, go to ‘General’ and then ‘Description’ in the ‘Properties’ window. A graphic will appear indicating the insertion of a file.
LEAVE A NOTE/COMMENT Click on the ‘Note Tool’ button on the commenting toolbar. Click to set the location of the note on the document and simply start typing. Do not use this feature to make text edits.
REVIEW To review your changes, click on the ‘Show’ button on the commenting toolbar. Choose ‘Show Comments List’. Navigate by clicking on a correction in the list. Alternatively, double click on any mark-up to open the commenting box.
UNDO/DELETE CHANGE To undo any changes made, use the right click button on your mouse (for PCs, Ctrl-Click for Mac). Alternatively click on the ‘Edit’ in the main Adobe menu and then ‘Undo’. You can also delete edits using the right click (Ctrl-Click on the Mac) and selecting ‘Delete’.
SEND YOUR ANNOTATED PDF FILE BACK TO WILEY VIA
[email protected] Save the annotations to your file and return as an e-mail. Before returning, please ensure you have answered any questions raised on the Query form that you have inserted all the corrections: later inclusion of any subsequent corrections cannot be guaranteed. Note: Comprehensive instructions are provided within your PDF file: to access these instructions please click on the Comments and Markup menu in the main tool bar, or click on Help.
Color figures were included with the final manuscript files that we received for your article. Because of the high cost of color printing, we can only print figures in color if authors cover the expense. Please indicate if you would like your figures to be printed in color or black and white. Color images will be reproduced online in Wiley Online Library at no charge, whether or not you opt for color printing.
Failure to return this form will result in the publication of your figures in black and white. JOURNAL
VOLUME
BIOTECHNOLOGY AND BIOENGINEERING
ISSUE
TITLE OF MANUSCRIPT
MS. NO.
No. Color Pages 1 2 3 4
NO. OF COLOR PAGES
Color Charges 500 1000 1500 2000
AUTHOR(S)
No. Color Pages 5 6 7 8
Color Charges 2500 3000 3500 4000
No. Color Pages 9 10 11 12
***Please contact
[email protected] for a quote if you have more than 12 pages of color***
Please print my figures in black and white Please print my figures in color BILL TO: Name
Purchase Order No.
Institution
Phone
Address Fax E-mail
Color Charges 4500 5000 5500 6000
COPYRIGHT TRANSFER AGREEMENT Date:
Contributor name:
Contributor address: Manuscript number (Editorial office only): Re: Manuscript entitled (the “Contribution”) for publication in published by
(the “Journal”) (“Wiley-Blackwell”).
Dear Contributor(s): Thank you for submitting your Contribution for publication. In order to expedite the editing and publishing process and enable Wiley-Blackwell to disseminate your Contribution to the fullest extent, we need to have this Copyright Transfer Agreement signed and returned as directed in the Journal’s instructions for authors as soon as possible. If the Contribution is not accepted for publication, or if the Contribution is subsequently rejected, this Agreement shall be null and void. Publication cannot proceed without a signed copy of this Agreement.
A. COPYRIGHT 1. The Contributor assigns to Wiley-Blackwell, during the full term of copyright and any extensions or renewals, all copyright in and to the Contribution, and all rights therein, including but not limited to the right to publish, republish, transmit, sell, distribute and otherwise use the Contribution in whole or in part in electronic and print editions of the Journal and in derivative works throughout the world, in all languages and in all media of expression now known or later developed, and to license or permit others to do so. 2. Reproduction, posting, transmission or other distribution or use of the final Contribution in whole or in part in any medium by the Contributor as permitted by this Agreement requires a citation to the Journal and an appropriate credit to Wiley-Blackwell as Publisher, and/or the Society if applicable, suitable in form and content as follows: (Title of Article, Author, Journal Title and Volume/Issue, Copyright © [year], copyright owner as specified in the Journal). Links to the final article on Wiley-Blackwell’s website are encouraged where appropriate.
B . R E TA I N E D R I G H T S Notwithstanding the above, the Contributor or, if applicable, the Contributor’s Employer, retains all proprietary rights other than copyright, such as patent rights, in any process, procedure or article of manufacture described in the Contribution.
C. PERMITTED USES BY CONTRIBUTOR 1. Submitted Version. Wiley-Blackwell licenses back the following rights to the Contributor in the version of the Contribution as originally submitted for publication: a. After publication of the final article, the right to self-archive on the Contributor’s personal website or in the Contributor’s institution’s/employer’s institutional repository or archive. This right extends to both intranets and the Internet. The Contributor may not update the submission version or replace it with the published Contribution. The version posted must contain a legend as follows: This is the pre-peer reviewed version of the following article: FULL CITE, which has been published in final form at [Link to final article]. b. The right to transmit, print and share copies with colleagues. 2. Accepted Version. Re-use of the accepted and peer-reviewed (but not final) version of the Contribution shall be by separate agreement with WileyBlackwell. Wiley-Blackwell has agreements with certain funding agencies governing reuse of this version. The details of those relationships, and other offerings allowing open web use, are set forth at the following website: http://www.wiley.com/go/funderstatement. NIH grantees should check the box at the bottom of this document.
3. Final Published Version. Wiley-Blackwell hereby licenses back to the Contributor the following rights with respect to the final published version of the Contribution: a. Copies for colleagues. The personal right of the Contributor only to send or transmit individual copies of the final published version in any format to colleagues upon their specific request provided no fee is charged, and further-provided that there is no systematic distribution of the Contribution, e.g. posting on a listserve, website or automated delivery. b. Re-use in other publications. The right to re-use the final Contribution or parts thereof for any publication authored or edited by the Contributor (excluding journal articles) where such re-used material constitutes less than half of the total material in such publication. In such case, any modifications should be accurately noted. c. Teaching duties. The right to include the Contribution in teaching or training duties at the Contributor’s institution/place of employment including in course packs, e-reserves, presentation at professional conferences, in-house training, or distance learning. The Contribution may not be used in seminars outside of normal teaching obligations (e.g. commercial seminars). Electronic posting of the final published version in connection with teaching/training at the Contributor’s institution/place of employment is permitted subject to the implementation of reasonable access control mechanisms, such as user name and password. Posting the final published version on the open Internet is not permitted. d. Oral presentations. The right to make oral presentations based on the Contribution. 4. Article Abstracts, Figures, Tables, Data Sets, Artwork and Selected Text (up to 250 words). a. Contributors may re-use unmodified abstracts for any non-commercial purpose. For on-line uses of the abstracts, Wiley-Blackwell encourages but does not require linking back to the final published versions. b. Contributors may re-use figures, tables, data sets, artwork, and selected text up to 250 words from their Contributions, provided the following conditions are met: (i) Full and accurate credit must be given to the Contribution. (ii) Modifications to the figures, tables and data must be noted. Otherwise, no changes may be made. (iii) The reuse may not be made for direct commercial purposes, or for financial consideration to the Contributor. (iv) Nothing herein shall permit dual publication in violation of journal ethical practices.
CTA-A
D. CONTRIBUTIONS OWNED BY EMPLOYER 1. If the Contribution was written by the Contributor in the course of the Contributor’s employment (as a “work-made-for-hire” in the course of employment), the Contribution is owned by the company/employer which must sign this Agreement (in addition to the Contributor’s signature) in the space provided below. In such case, the company/employer hereby assigns to Wiley-Blackwell, during the full term of copyright, all copyright in and to the Contribution for the full term of copyright throughout the world as specified in paragraph A above. 2. In addition to the rights specified as retained in paragraph B above and the rights granted back to the Contributor pursuant to paragraph C above, WileyBlackwell hereby grants back, without charge, to such company/employer, its subsidiaries and divisions, the right to make copies of and distribute the final published Contribution internally in print format or electronically on the Company’s internal network. Copies so used may not be resold or distributed externally. However the company/employer may include information and text from the Contribution as part of an information package included with software or other products offered for sale or license or included in patent applications. Posting of the final published Contribution by the institution on a public access website may only be done with Wiley-Blackwell’s written permission, and payment of any applicable fee(s). Also, upon payment of Wiley-Blackwell’s reprint fee, the institution may distribute print copies of the published Contribution externally.
E. GOVERNMENT CONTRACTS In the case of a Contribution prepared under U.S. Government contract or grant, the U.S. Government may reproduce, without charge, all or portions of the Contribution and may authorize others to do so, for official U.S. Govern-
ment purposes only, if the U.S. Government contract or grant so requires. (U.S. Government, U.K. Government, and other government employees: see notes at end)
F. C O P Y R I G H T N O T I C E The Contributor and the company/employer agree that any and all copies of the final published version of the Contribution or any part thereof distributed or posted by them in print or electronic format as permitted herein will include the notice of copyright as stipulated in the Journal and a full citation to the Journal as published by Wiley-Blackwell.
G . C O N T R I B U T O R ’ S R E P R E S E N TAT I O N S The Contributor represents that the Contribution is the Contributor’s original work, all individuals identified as Contributors actually contributed to the Contribution, and all individuals who contributed are included. If the Contribution was prepared jointly, the Contributor agrees to inform the co-Contributors of the terms of this Agreement and to obtain their signature to this Agreement or their written permission to sign on their behalf. The Contribution is submitted only to this Journal and has not been published before. (If excerpts from copyrighted works owned by third parties are included, the Contributor will obtain written permission from the copyright owners for all uses as set forth in WileyBlackwell’s permissions form or in the Journal’s Instructions for Contributors, and show credit to the sources in the Contribution.) The Contributor also warrants that the Contribution contains no libelous or unlawful statements, does not infringe upon the rights (including without limitation the copyright, patent or trademark rights) or the privacy of others, or contain material or instructions that might cause harm or injury.
CHECK ONE BOX: Contributor-owned work AT TA C H A D D I T I O N A L S I G N AT U R E PA G E S A S N E C E S S A RY
Contributor’s signature
Date
Type or print name and title
Co-contributor’s signature
Date
Type or print name and title
Company/Institution-owned work (made-for-hire in the course of employment)
Company or Institution (Employer-for-Hire)
Date
Authorized signature of Employer
Date
U.S. Government work
Note to U.S. Government Employees A contribution prepared by a U.S. federal government employee as part of the employee’s official duties, or which is an official U.S. Government publication, is called a “U.S. Government work,” and is in the public domain in the United States. In such case, the employee may cross out Paragraph A.1 but must sign (in the Contributor’s signature line) and return this Agreement. If the Contribution was not prepared as part of the employee’s duties or is not an official U.S. Government publication, it is not a U.S. Government work.
U.K. Government work (Crown Copyright)
Note to U.K. Government Employees The rights in a Contribution prepared by an employee of a U.K. government department, agency or other Crown body as part of his/her official duties, or which is an official government publication, belong to the Crown. U.K. government authors should submit a signed declaration form together with this Agreement. The form can be obtained via http://www.opsi.gov.uk/advice/crown-copyright/copyright-guidance/ publication-of-articles-written-by-ministers-and-civil-servants.htm
Other Government work
Note to Non-U.S., Non-U.K. Government Employees If your status as a government employee legally prevents you from signing this Agreement, please contact the editorial office.
NIH Grantees
Note to NIH Grantees Pursuant to NIH mandate, Wiley-Blackwell will post the accepted version of Contributions authored by NIH grant-holders to PubMed Central upon acceptance. This accepted version will be made publicly available 12 months after publication. For further information, see www.wiley.com/go/nihmandate. CTA-A
Additional reprint and journal issue purchases
Should you wish to purchase additional copies of your article, please click on the link and follow the instructions provided: https://caesar.sheridan.com/reprints/redir.php?pub=10089&acro=BIT Corresponding authors are invited to inform their co‐authors of the reprint options available. Please note that regardless of the form in which they are acquired, reprints should not be resold, nor further disseminated in electronic form, nor deployed in part or in whole in any marketing, promotional or educational contexts without authorization from Wiley. Permissions requests should be directed to mailto:
[email protected] For information about ‘Pay‐Per‐View and Article Select’ click on the following link: http://wileyonlinelibrary.com/ppv
