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 Milan Češka
David Šafránek (Eds.)
LNBI 11095

Computational Methods
in Systems Biology
16th International Conference, CMSB 2018
Brno, Czech Republic, September 12–14, 2018
Proceedings

123
Lecture Notes in Bioinformatics 11095

Subseries of Lecture Notes in Computer Science

LNBI Series Editors

Sorin Istrail
Brown University, Providence, RI, USA
Pavel Pevzner
University of California, San Diego, CA, USA
Michael Waterman
University of Southern California, Los Angeles, CA, USA

LNBI Editorial Board

Søren Brunak
Technical University of Denmark, Kongens Lyngby, Denmark
Mikhail S. Gelfand
IITP, Research and Training Center on Bioinformatics, Moscow, Russia
Thomas Lengauer
Max Planck Institute for Informatics, Saarbrücken, Germany
Satoru Miyano
University of Tokyo, Tokyo, Japan
Eugene Myers
Max Planck Institute of Molecular Cell Biology and Genetics, Dresden,
Germany
Marie-France Sagot
Université Lyon 1, Villeurbanne, France
David Sankoff
University of Ottawa, Ottawa, Canada
Ron Shamir
Tel Aviv University, Ramat Aviv, Tel Aviv, Israel
Terry Speed
Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia
Martin Vingron
Max Planck Institute for Molecular Genetics, Berlin, Germany
W. Eric Wong
University of Texas at Dallas, Richardson, TX, USA
More information about this series at http://www.springer.com/series/5381
Milan Češka David Šafránek (Eds.)
•

Computational Methods
in Systems Biology
16th International Conference, CMSB 2018
Brno, Czech Republic, September 12–14, 2018
Proceedings

123
Editors
Milan Češka David Šafránek
Brno University of Technology Masaryk University
Brno Brno
Czech Republic Czech Republic

ISSN 0302-9743 ISSN 1611-3349 (electronic)

Lecture Notes in Bioinformatics
ISBN 978-3-319-99428-4 ISBN 978-3-319-99429-1 (eBook)
https://doi.org/10.1007/978-3-319-99429-1

Library of Congress Control Number: 2018951890

LNCS Sublibrary: SL8 – Bioinformatics

© Springer Nature Switzerland AG 2018

Chapters 3, 9 and 10 are licensed under the terms of the Creative Commons Attribution 4.0 International
License (http://creativecommons.org/licenses/by/4.0/). For further details see license information in the
chapters.
This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the
material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,
broadcasting, reproduction on microfilms or in any other physical way, and transmission or information
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The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication
does not imply, even in the absence of a specific statement, that such names are exempt from the relevant
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The publisher, the authors and the editors are safe to assume that the advice and information in this book are
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The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland
 Preface

This volume contains the papers presented at CMSB 2018, the 16th Conference on
Computational Methods in Systems Biology, held during September 12–14, 2018, at
the Faculty of Informatics, Masaryk University, Brno, Czech Republic.
The CMSB annual conference series, initiated in 2003, provides a unique discussion
forum for computer scientists, biologists, mathematicians, engineers, and physicists
interested in a system-level understanding of biological processes. Topics covered by
the CMSB proceedings include: formalisms for modeling biological processes; models
and their biological applications; frameworks for model verification, validation, anal-
ysis, and simulation of biological systems; high-performance computational systems
biology and parallel implementations; model inference from experimental data; model
integration from biological databases; multi-scale modeling and analysis methods;
computational approaches for synthetic biology; case studies in systems and synthetic
biology.
There were 73 submissions in total for the five conference tracks. In particular, 46
submissions were submitted to the proceedings tracks and 27 submissions to the
presentation tracks. The submissions were as follows: 37 regular paper submissions,
five tool paper submissions, four original work poster submissions, 14 poster sub-
missions, and 13 presentation-only submissions describing recently published work.
Each regular submission and tool paper submission was reviewed by at least three
Program Committee members. Each original work poster submission was reviewed by
at least two Program Committee members. For the proceedings, the committee decided
to accept 15 regular papers, four tool papers, and three original posters. Moreover, the
committee selected three presentation-only submissions. In addition, 16 poster
presentations were selected from poster submissions and rejected presentation-only
submissions. Finally, out of the selected posters, the committee accepted nine posters to
be presented in the form of flash talks together with the three original work poster
submissions accepted for the proceedings.
In view of the broad scope of the CMSB conference series, we selected the fol-
lowing five high-profile invited speakers: Ilka M. Axmann (Heinrich Heine University
Düsseldorf, Germany), Mustafa Khammash (ETH Zurich, Switzerland), Chris J. Myers
(University of Utah, USA), Andrew Phillips (Microsoft Research, UK), and Andrew
Turberfield (University of Oxford, UK). Their invited talks stimulated fruitful dis-
cussions among the conference attendees and were the highlights of the CMSB 2018
program.
Further details on CMSB 2018 are available on the following website:
https://cmsb2018.fi.muni.cz/
Finally, as the program co-chairs, we are extremely grateful to the members of the
Program Committee and the external reviewers for their peer reviews and the valuable
feedback they provided to the authors. Our special thanks go to François Fages, Jérȏme
Feret, Ezio Bartocci, and all the members of the CMSB Steering Committee, for their
VI Preface

advice on organizing and running the conference. We acknowledge the support of the
EasyChair conference system during the reviewing process and the production of these
proceedings. We also thank Springer for publishing the CMSB proceedings in its
Lecture Notes in Computer Science series. Our gratitude also goes to the tool track
chair, Samuel Pastva, and all the members of the Tool Evaluation Committee, for the
careful checking of the submitted tools. Additionally, we would like to thank the
administrative staff of the Faculty of Informatics, Masaryk University, for helping us
with the financial management of the conference. Moreover, we are pleased to
acknowledge the financial support kindly obtained from the National Center for Sys-
tems Biology of Czech Republic (C4SYS), Microsoft Research, and the Faculty of
Informatics, Masaryk University, where this year’s event was hosted. Finally, we
would like to thank all the participants of the conference. It was the quality of their
presentations and their contribution to the discussions that made the meeting a scientific
success.

September 2018 Milan Češka

David Šafránek
 Organization

Steering Committee
Ezio Bartocci (Guest) Vienna University of Technology, Austria
Finn Drabløs NTNU, Norway
François Fages Inria/Université Paris-Saclay, France
Jérôme Feret (Guest) Inria, France
David Harel Weizmann Institute of Science, Israel
Monika Heiner Brandenburg Technical University, Germany
Heinz Koeppl (Guest) TU Darmstadt, Germany
Pietro Liò (Guest) University of Cambridge, UK
Tommaso Mazza IRCCS Casa Sollievo della Sofferenza, Mendel, Italy
Satoru Miyano The University of Tokyo, Japan
Nicola Paoletti (Guest) Stony Brook University, USA
Gordon Plotkin The University of Edinburgh, UK
Corrado Priami CoSBi/Microsoft Research, University of Trento, Italy
Carolyn Talcott SRI International, USA
Adelinde Uhrmacher University of Rostock, Germany

Program Committee Co-chairs

Milan Češka Brno University of Technology, Czech Republic
David Šafránek Masaryk University, Czech Republic

Tools Track Chair

Samuel Pastva Masaryk University, Czech Republic

Local Organization Chair

David Šafránek Masaryk University, Czech Republic

Program Committee
Alessandro Abate University of Oxford, UK
Ezio Bartocci Vienna University of Technology, Austria
Nikola Beneš Masaryk University, Czech Republic
Luca Bortolussi University of Trieste, Italy
Luca Cardelli Microsoft Research, UK
Claudine Chaouiya Insituto Gulbenkian de Ciência, Portugal
Eugenio Cinquemani Inria, France
Milan Češka Brno University of Technology, Czech Republic
VIII Organization

Thao Dang CNRS-VERIMAG, France

Hidde De Jong Inria, France
François Fages Inria/Université Paris-Saclay, France
Jérôme Feret Inria, France
Christoph Flamm University of Vienna, Austria
Tomáš Gedeon Montana State University, USA
Radu Grosu Stony Brook University, USA
Monika Heiner Brandenburg Technical University, Germany
Jane Hillston The University of Edinburgh, UK
Heinz Koeppl TU Darmstadt, Germany
Jean Krivine IRIF, France
Oded Maler CNRS-VERIMAG, France
Tommaso Mazza IRCCS Casa Sollievo della Sofferenza, Mendel, Italy
Satoru Miyano The University of Tokyo, Japan
Andrzej Mizera Luxembourg Institute of Health and Luxembourg
Centre for Systems Biomedicine, Luxembourg
Pedro T. Monteiro Universidade de Lisboa, Portugal
Laura Nenzi Vienna University of Technology, Austria
Nicola Paoletti Stony Brook University, USA
Loïc Paulevé CNRS/LRI, France
Ion Petre Åbo Akademi University, Finland
Tatjana Petrov University of Konstanz, Germany
Carla Piazza University of Udine, Italy
Ovidiu Radulescu University of Montpellier 2, France
Olivier Roux IRCCyN, France
Guido Sanguinetti The University of Edinburgh, UK
Thomas Sauter University of Luxembourg, Luxembourg
Heike Siebert Freie Universität Berlin, Germany
Abhyudai Singh University of Delaware, USA
David Šafránek Masaryk University, Czech Republic
Carolyn Talcott SRI International, USA
Chris Thachuk California Institute of Technology, USA
P. S. Thiagarajan Harvard University, USA
Adelinde Uhrmacher University of Rostock, Germany
Verena Wolf Saarland University, Germany
Boyan Yordanov Microsoft Research, UK
Paolo Zuliani Newcastle University, UK

Tool Evaluation Committee

Giulio Caravagna The University of Edinburgh, UK
Matej Hajnal Masaryk University, Czech Republic
Juraj Kolčák LSV, Inria and ENS Paris-Saclay,
Université Paris-Saclay, France
Luca Laurenti University of Oxford, UK
Jiří Matyáš Brno University of Technology, Czech Republic
 Organization IX

Samuel Pastva Masaryk University, Czech Republic

Fedor Shmarov Newcastle University, UK
Max Whitby University of Oxford, UK

Additional Reviewers

Backenköhler, Michael Luisa Vissat, Ludovica

Chodak, Jacek Magnin, Morgan
Dague, Philippe Molyneux, Gareth
Demko, Martin Palaniappan, Sucheendra K.
Dreossi, Tommaso Pang, Jun
Gilbert, David Patanè, Andrea
Gyori, Benjamin Pires Pacheco, Maria
Hajnal, Matej Schnoerr, David
Hasani, Ramin M. Selvaggio, Gianluca
Helms, Tobias Shmarov, Fedor
Hemery, Mathieu Soliman, Sylvain
Kyriakopoulos, Charalampos Streck, Adam
Laurenti, Luca Troják, Matej
Lechner, Mathias Wijesuriya, Viraj Brian
Invited Talks
 What Time Is it?
Biological Oscillators to Robustly Anticipate Changes

Nicolas M. Schmelling and Ilka M. Axmann

Institute for Synthetic Microbiology, Cluster of Excellence on Plant Sciences

(CEPLAS), Heinrich Heine University Düsseldorf, Düsseldorf, Germany
ilka.axmann@hhu.de

Abstract. Even without looking at a watch, we have an inner feeling for time.
How do we measure time? Our body, in particular each of our cells, has an inner
clock enabling all our rhythmic biological activities like sleeping. Surprisingly,
prokaryotic cyanobacteria Synechococcus elongatus PCC 7942, which can
divide faster than ones a day, also use an inner timing system to foresee the
accompanying daily changes of light and temperature and regulate their phys-
iology and behavior in 24 hour cycles [4]. Their underlying biochemical
oscillator fulfills all criteria of a circadian clock though it is made of solely three
proteins, KaiC, KaiB, and KaiA [11, 12]. At its center is KaiC in its hexameric
form, which runs through a complete phosphorylation and dephosphorylation
cycle every 24 hours, even under fluctuating and continuous conditions. Fur-
thermore, the clock can be entrained by light, temperature, and nutrients.
Astonishingly, reconstituted from the purified protein components this
cyanobacterial protein clock can tick autonomously in a test tube for weeks [6].
This apparent simplicity has proven to be an ideal system for answering ques-
tions about the functionality of circadian clocks. Over the last decade various
parts of this circadian system were identified and described in detail through
computational modeling: The ordered phosphorylation of KaiC and temperature
compensation of the clock [1, 10], the stimulating interaction with the other core
factors and effects on gene expression [2, 5, 13], as well as the influence of
varying ATP/ADP ratios [9], which on the one hand entraining the clock, on the
other can cause misalignments due to rapid changes at certain times during the
period. In addition, the three-protein clock is embedded in a transcription
translation feedback loop, similar to eukaryotic clock systems [4]. A two-loop
transcriptional feedback mechanism could be identified in which only one
phosphorylation form of KaiC suppresses kaiBC expression while two other
forms activate its own expression [3]. Further, mathematical models have
identified different strategies for period robustness against internal and external
noise as well as uncoupling from the cell cycle that are used by cyanobacteria
[7, 8]. Overall the insights gained by studying the circadian clock of
cyanobacteria have substantial impact on the field of circadian computing,
which can be used for the design of synthetic switches, oscillators, and clocks
and the construction of new algorithms in parallel computing in the future.
XIV N. M. Schmelling and I. M. Axmann

References
1. Brettschneider, C., Rose, R.J., Hertel, S., Axmann, I.M., Heck, A.J.R., Kollmann, M.: A
sequestration feedback determines dynamics and temperature entrainment of the KaiABC
circadian clock. Mol. Syst. Biol. 6(1) (2010)
2. Clodong, S., Dühring, U., Kronk, L., Wilde, A., Axmann, I.M., Herzel, H., Kollmann, M.:
Functioning and robustness of a bacterial circadian clock. Mol. Syst. Biol. 3(1) (2007)
3. Hertel, S., Brettschneider, C., Axmann, I.M.: Revealing a two-loop transcriptional feedback
mechanism in the cyanobacterial circadian clock. PLoS Comput. Biol. 9(3), 1–16 (2013)
4. Ishiura, M., Kutsuna, S., Aoki, S., Iwasaki, H., Andersson, C.R., Tanabe, A., Golden, S.S.,
Johnson, C.H., Kondo, T.: Expression of a gene cluster kaiABC as a circadian feedback
process in cyanobacteria. Sci. 281(5382), 1519–1523 (1998)
5. Kurosawa, G., Aihara, K., Iwasa, Y.: A model for the circadian rhythm of cyanobacteria that
maintains oscillation without gene expression. Biophys. J. 91(6), 2015–2023 (2006)
6. Nakajima, M., Imai, K., Ito, H., Nishiwaki, T., Murayama, Y., Iwasaki, H., Oyama, T.,
Kondo, T.: Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation
in vitro. Sci. 308(5720), 414–415 (2005)
7. Paijmans, J., Bosman, M., ten Wolde, P.R., Lubensky, D.K.: Discrete gene replication events
drive coupling between the cell cycle and circadian clocks. Proc. Nat. Acad. Sci. 113(15),
4063–4068 (2016)
8. Paijmans, J., Lubensky, D.K., ten Wolde, P.R.: Period robustness and entrainability of the
kai system to changing nucleotide concentrations. Biophys. J. 113, 157–173 (2017)
9. Rust, M.J., Golden, S.S., O’Shea, E.K.: Light-driven changes in energy metabolism directly
entrain the cyanobacterial circadian oscillator. Sci. 331(6014), 220–223 (2011)
10. Rust, M.J., Markson, J.S., Lane, W.S., Fisher, D.S., O’Shea, E.K.: Ordered phosphorylation
governs oscillation of a three-protein circadian clock. Sci. 318(5851), 809–812 (2007)
11. Snijder, J., Schuller, J.M., Wiegard, A., Lössl, P., Schmelling, N.M., Axmann, I.M., Plitzko,
J.M., Förster, F., Heck, A.J.R.: Structures of the cyanobacterial circadian oscillator frozen in
a fully assembled state. Sci. 355(6330), 1181–1184 (2017)
12. Tseng, R., Goularte, N.F., Chavan, A., Luu, J., Cohen, S.E., Chang, Y.-G., Heisler, J., Li, S.,
Michael, A.K., Tripathi, S., Golden, S.S., LiWang, A., Partch, C.L.: Structural basis of the
day-night transition in a bacterial circadian clock. Sci. 355(6330), 1174–1180 (2017)
13. van Zon, J.S., Lubensky, D.K., Altena, P.R.H., ten Wolde, P.R.: An allosteric model of
circadian KaiC phosphorylation. Proc. Nat. Acad. Sci. 104(18), 7420–7425 (2007)
 Biomolecular Control Systems

Mustafa Khammash

ETH Zurich, Control Theory and Systems Biology Laboratory Basel,

Switzerland
mustafa.khammash@bsse.ethz.ch

Abstract. Humans have been influencing the DNA of plants and animals for
thousands of years through selective breeding. Yet it is only over the last three
decades or so that we have gained the ability to manipulate the DNA itself and
directly alter its sequences through the modern tools of genetic engineering. This
has revolutionized biotechnology and ushered in the era of synthetic biology. It
has also made it conceivable for the first time to engineer into living cells
genetic feedback control systems that automatically monitor and steer the cell’s
dynamic behavior. To realize the huge promise of such systems, new theory and
methodologies are needed for designing controllers that function in the special
and challenging environment of the cell. We refer to the resulting technology as
Cybergenetics—a modern realization of Norbert Wiener’s Cybernetics vision.
Here I will present our theoretical framework for the design and synthesis of
cybergenetic systems and discuss the main challenges in their implementation.
I will then introduce the first designer gene network that attains integral feed-
back in a living cell and will demonstrate its tunability and disturbance rejection
properties [1]. A growth control application shows the inherent capacity of this
genetic control system to deliver robustness and highlights its potential use as a
universal controller for regulation of biological variables in arbitrary networks
[2, 3]. I will end by exploring the potential impact of Cybergenetics in industrial
biotechnology and medical therapy.

References
1. Briat, C., Zechner, C., Mustafa, K.: Design of a synthetic integral feedback circuit: dynamic
analysis and DNA implementation. ACS Synth. Biol. 5(10), 1108–1116 (2016)
2. Zechner, C., Seelig, G., Rullan, M., Mustafa, K.: Molecular circuits for dynamic noise fil-
tering. Proc. Nat. Acad. Sci. 113(17), 4729–4734 (2016)
3. Briat, C., Gupta, A., Khammash, M.: Antithetic integral feedback ensures robust perfect
adaptation in noisy biomolecular networks. Cell Syst. 2(1), 15–26 (2016)
 A Standard-Enabled Workflow
for Synthetic Biology

Chris J. Myers

University of Utah, Salt Lake City, USA

myers@ece.utah.edu

Abstract. A synthetic biology workflow is composed of data repositories that

provide information about genetic parts, sequence-level design tools to compose
these parts into circuits, visualization tools to depict these designs, genetic
design tools to select parts to create systems, and modeling and simulation tools
to evaluate alternative design choices. Data standards enable the ready exchange
of information within such a workflow, allowing repositories and tools to be
connected from a diversity of sources. This talk describes one such workflow
that utilizes the growing ecosystem of software tools that support the Synthetic
Biology Open Language (SBOL) to describe genetic designs, and the mature
ecosystem of tools that support the Systems Biology Markup Language (SBML)
to model these designs [1]. In particular, this presentation will demonstrate a
workflow using tools including SynBioHub, SBOLDesigner, and iBioSim.
SynBioHub (http://synbiohub.org) is a database designed for storing synthetic
biology designs captured using the SBOL data model, and it provides both a
RESTful API for computational access and a user-friendly Web-based frontend.
SBOLDesigner is a sequence editor that allows the designer to fetch parts from a
SynBioHub repository and compose them to construct larger designs. Finally,
iBioSim is genetic modeling, analysis, and design tool that provides a means to
construct SBML models for these designs that can be simulated and analyzed
using a variety of techniques [2]. Both SBOLDesigner and iBioSim also support
uploading these larger system designs back to the SynBioHub repository.
Finally, this talk will demonstrate how this workflow can be utilized to produce
a complete record of a genetic design facilitating reproducibility and reuse.

References
1. Zhang, M., McLaughlin, J.A., Wipat, A., Myers, C.J.: SBOLDesigner 2: an intuitive tool for
structural genetic design. ACS Synth. Biol. 6(7), 1150–1160 (2017)
2. Watanabe, L., Nguyen, T., Zhang, M., Zundel, Z., Zhang, Z., Madsen, C., Roehner, N.,
Myers, C.J.: iBioSim 3: a tool for model-based genetic circuit design. ACS Synth. Biol.
(2018)
 Modelling Biomimetic Structures
and Machinery Using DNA

Andrew J. Turberfield

University of Oxford, Department of Physics, Clarendon Laboratory,

Oxford, UK
a.turberfield@physics.ox.ac.uk

Abstract. Nucleic acids, archetypal biomolecules, can be used to model and

study natural biomolecular assembly processes and the operation of molecular
machinery. The programmability of DNA and RNA base pairing has enabled the
creation of a very wide range of synthetic nanostructures through control of the
interactions between molecular components. More sophisticated design tech-
niques allow control of the kinetics as well as the thermodynamics of these
interactions, creating the potential to study and control assembly pathways and
allowing the construction of both dynamic systems that process information and
of biomimetic molecular machinery. Techniques of simulation and verification
are important in understanding and designing these increasingly complex sys-
tems. I shall present a broad review of the rapidly developing research field of
dynamic DNA nanotechnology, with particular emphasis on our use of a
combination of experimental synthesis and computation to study DNA origami
assembly pathways [1, 2], kinetic control of strand displacement reactions [3–7],
synthetic molecular motors [8–12], and molecular machinery for the creation of
sequence-controlled polymers [13, 14].

References
1. Dunn, K. E., Dannenberg, F., Ouldridge, T.E., Kwiatkowska, M., Turberfield, A.J., Bath, J.:
Guiding the folding pathway of DNA origami. Nat. 525, 82–86 (2015)
2. Dannenberg, F., Dunn, K.E., Bath, J., Kwiatkowska, M., Turberfield, A.J., Ouldridge, T.E.:
Modelling DNA origami self-assembly at the domain level. J. Chem. Phys. 143, 165102
(2015)
3. Turberfield, A.J., Mitchell, J.C., Yurke, B., Mills, A.P., Jr., Blakey, M.I., Simmel, F.C.:
DNA Fuel for Free-Running Nanomachines. Phys. Rev. Lett. 90(11), 118102 (2003)
4. Genot, A.J., Zhang, D.Y., Bath, J., Turberfield, A.J.: The remote toehold, a mechanism for
flexible control of DNA hybridization kinetics. J. Am. Chem. Soc. 133, 2177–2182 (2011)
5. Genot, A.J., Bath, J., Turberfield, A.J.: Combinatorial displacement of DNA strands:
application to matrix multiplication and weighted sums. Angew. Chem. Int. Ed. 52,
1189–1192 (2013)
6. Machinek, R.R.F., Ouldridge, T.E., Haley, N.E.C., Bath, J., Turberfield, A.J.: Programmable
energy landscapes for kinetic control of DNA strand displacement. Nat. Commun. 5, 5324
(2014)
XVIII A. J. Turberfield

7. Haley, N.E.C. et al.: Mismatch Repair for Enhanced Kinetic Control of DNA Displacement
Reactions. Submitted
8. Green, S.J., Bath, J., Turberfield, A.J.: Coordinated chemomechanical cycles: a mechanism
for autonomous molecular motion. Phys. Rev. Lett. 101, 238101 (2008)
9. Muscat, R.A., Bath, J., Turberfield, A.J.: A programmable molecular robot. Nano Lett. 11,
982–987 (2011)
10. Wickham, S.F.J., Bath, J., Katsuda, Y., Endo, M., Hidaka, K., Sugiyama, H., Turberfield, A.J.:
A DNA-based molecular motor that can navigate a network of tracks. Nat. Nanotechnol. 7,
169–173 (2012)
11. Ouldridge, T.E., Hoare, R.L., Louis, A.A., Doye, J.P.K., Bath, J., Turberfield, A.J.: Opti-
mizing DNA nanotechnology through coarse-grained modeling: a two-footed DNA walker.
ACS Nano 7, 2479–2490 (2013)
12. Dannenberg, F., Kwiatkowska, M., Thachuk, C., Turberfield, A.J.: DNA walker circuits:
computational potential, design, and verification. Nat. Comput. 14, 195–211 (2015)
13. Meng, W., Muscat, R.A., McKee, M.L., Milnes, P.J., El-Sagheer, A.H., Bath, J., Davis, B.G.,
Brown, T., O’Reilly, R.K., Turberfield, A.J.: An autonomous molecular assembler for pro-
grammable chemical synthesis. Nat. Chem. 8, 542–548 (2016)
14. O’Reilly, R.K., Turberfield, A.J., Wilks, T.R.: The evolution of DNA-templated synthesis as
a tool for materials discovery. Acc. Chem. Res. 50, 2496–2509 (2017)
 Programming Languages for Molecular
and Genetic Devices

Andrew Phillips

Microsoft Research, Biological Computation Group, Cambridge, UK

andrew.phillips@microsoft.com

Abstract. Computational nucleic acid devices show great potential for enabling
a broad range of biotechnology applications, including smart probes for
molecular biology research, in vitro assembly of complex compounds,
high-precision in vitro disease diagnosis and, ultimately, computational thera-
nostics inside living cells. This diversity of applications is supported by a range
of implementation strategies, including nucleic acid strand displacement,
localisation to substrates, and the use of enzymes with polymerase, nickase and
exonuclease functionality. However, existing computational design tools are
unable to account for these different strategies in a unified manner. This talk
presents a programming language that allows a broad range of computational
nucleic acid systems to be designed and analysed [1, 2]. We also demonstrate
how similar approaches can be incorporated into a programming language for
designing genetic devices that are inserted into cells to reprogram their
behaviour. The language is used to characterise genetic components [4] for
programming populations of cells that communicate and self-organise into
spatial patterns [3]. More generally, we anticipate that languages for program-
ming molecular and genetic devices will accelerate the development of future
biotechnology applications.

References
1. Chatterjee, G., Dalchau, N., Muscat, R.A., Phillips, A., Seelig, G.: A spatially localized
architecture for fast and modular DNA computing. Nat. Nanotechnol. 12(9), 920–927(2017)
2. Chen, Y.-J., Dalchau, N., Srinivas, N., Phillips, A., Cardelli, L., Soloveichik, D., Seelig, G.:
Programmable chemical controllers made from DNA. Nat. Nanotechnol. 8(10), 755–762
(2013)
3. Grant, P.K., Dalchau, N., Brown, P.R., Federici, F., Rudge, T.J., Yordanov, B., Patange, O.,
Phillips, A., Haseloff, J.: Orthogonal intercellular signaling for programmed spatial behavior.
Mol. Syst. Biol. 12(1), 849–849 (2016)
4. Yordanov, B., Dalchau, N., Grant, P.K., Pedersen, M., Emmott, S., Haseloff, J., Phillips, A.:
A computational method for automated characterization of genetic components. ACS Synth.
Biol. 3(8), 578–588 (2014)
 Contents

Regular Papers

Modeling and Engineering Promoters with Pre-defined RNA Production

Dynamics in Escherichia Coli. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Samuel M. D. Oliveira, Mohamed N. M. Bahrudeen, Sofia Startceva,
Vinodh Kandavalli, and Andre S. Ribeiro

Deep Abstractions of Chemical Reaction Networks . . . . . . . . . . . . . . . . . . . 21

Luca Bortolussi and Luca Palmieri

Derivation of a Biomass Proxy for Dynamic Analysis of Whole

Genome Metabolic Models. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Timothy Self, David Gilbert, and Monika Heiner

Computing Diverse Boolean Networks from Phosphoproteomic

Time Series Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Misbah Razzaq, Roland Kaminski, Javier Romero, Torsten Schaub,
Jeremie Bourdon, and Carito Guziolowski

Characterization of the Experimentally Observed Clustering

of VEGF Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Emine Güven, Michael J. Wester, Bridget S. Wilson,
Jeremy S. Edwards, and Ádám M. Halász

Synthesis for Vesicle Traffic Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

Ashutosh Gupta, Somya Mani, and Ankit Shukla

Formal Analysis of Network Motifs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

Hillel Kugler, Sara-Jane Dunn, and Boyan Yordanov

Buffering Gene Expression Noise by MicroRNA Based

Feedforward Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Pavol Bokes, Michal Hojcka, and Abhyudai Singh

Stochastic Rate Parameter Inference Using the Cross-Entropy Method . . . . . . 146

Jeremy Revell and Paolo Zuliani

Experimental Biological Protocols with Formal Semantics . . . . . . . . . . . . . . 165

Alessandro Abate, Luca Cardelli, Marta Kwiatkowska, Luca Laurenti,
and Boyan Yordanov
XXII Contents

Robust Data-Driven Control of Artificial Pancreas Systems Using

Neural Networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Souradeep Dutta, Taisa Kushner, and Sriram Sankaranarayanan

Programming Substrate-Independent Kinetic Barriers with Thermodynamic

Binding Networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Keenan Breik, Cameron Chalk, David Doty, David Haley,
and David Soloveichik

A Trace Query Language for Rule-Based Models . . . . . . . . . . . . . . . . . . . . 220

Jonathan Laurent, Hector F. Medina-Abarca, Pierre Boutillier,
Jean Yang, and Walter Fontana

Inferring Mechanism of Action of an Unknown Compound from Time

Series Omics Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
Akos Vertes, Albert-Baskar Arul, Peter Avar, Andrew R. Korte, Hang Li,
Peter Nemes, Lida Parvin, Sylwia Stopka, Sunil Hwang, Ziad J. Sahab,
Linwen Zhang, Deborah I. Bunin, Merrill Knapp, Andrew Poggio,
Mark-Oliver Stehr, Carolyn L. Talcott, Brian M. Davis, Sean R. Dinn,
Christine A. Morton, Christopher J. Sevinsky, and Maria I. Zavodszky

Composable Rate-Independent Computation in Continuous Chemical

Reaction Networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
Cameron Chalk, Niels Kornerup, Wyatt Reeves, and David Soloveichik

Tool Papers

ASSA-PBN 3.0: Analysing Context-Sensitive Probabilistic

Boolean Networks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
Andrzej Mizera, Jun Pang, Hongyang Qu, and Qixia Yuan

KaSa: A Static Analyzer for Kappa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285

Pierre Boutillier, Ferdinanda Camporesi, Jean Coquet, Jérôme Feret,
Kim Quyên Lý, Nathalie Theret, and Pierre Vignet

On Robustness Computation and Optimization in BIOCHAM-4 . . . . . . . . . . 292

François Fages and Sylvain Soliman

LNA++: Linear Noise Approximation with First and Second

Order Sensitivities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
Justin Feigelman, Daniel Weindl, Fabian J. Theis, Carsten Marr,
and Jan Hasenauer
 Contents XXIII

Poster Abstracts

Reparametrizing the Sigmoid Model of Gene Regulation

for Bayesian Inference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
Martin Modrák

On the Full Control of Boolean Networks . . . . . . . . . . . . . . . . . . . . . . . . . 313

Soumya Paul, Jun Pang, and Cui Su

Systems Metagenomics: Applying Systems Biology Thinking to Human

Microbiome Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
Golestan Sally Radwan and Hugh Shanahan

List of Accepted Posters and Oral Presentations . . . . . . . . . . . . . . . . . . . . . 323

Author Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325

Regular Papers
 Modeling and Engineering Promoters
with Pre-defined RNA Production Dynamics
in Escherichia Coli

Samuel M. D. Oliveira , Mohamed N. M. Bahrudeen ,

Sofia Startceva , Vinodh Kandavalli , and Andre S. Ribeiro(&)

Laboratory of Biosystem Dynamics, BioMediTech Institute,

Tampere University of Technology, P.O. Box 553, 33101 Tampere, Finland
andre.ribeiro@tut.fi

Abstract. Recent developments in live-cell time-lapse microscopy and signal

processing methods for single-cell, single-RNA detection now allow charac-
terizing the in vivo dynamics of RNA production of Escherichia coli promoters
at the single event level. This dynamics is mostly controlled at the promoter
region, which can be engineered with single nucleotide precision. Based on
these developments, we propose a new strategy to engineer genes with prede-
fined transcription dynamics (mean and standard deviation of the distribution of
RNA numbers of a cell population). For this, we use stochastic modelling
followed by genetic engineering, to design synthetic promoters whose rate-
limiting steps kinetics allow achieving a desired RNA production kinetics. We
present an example where, from a pre-defined kinetics, a stochastic model is first
designed, from which a promoter is selected based on its rate-limiting steps
kinetics. Next, we engineer mutant promoters and select the one that best fits the
intended distribution of RNA numbers in a cell population. As the modelling
strategies and databases of models, genetic constructs, and information on these
constructs kinetics improve, we expect our strategy to be able to accommodate a
wide variety of pre-defined RNA production kinetics.

Keywords: Model of transcription initiation
Synthetic constructs

Rate-limiting steps
Gene engineering framework

1 Introduction

Several studies have determined that, in Escherichia coli, the main regulatory mech-
anisms of gene expression dynamics act at the stage of transcription initiation [1–9]. It
has recently become possible to combine time-lapse live cell microscopy with single
RNA detection techniques [6, 10–13], synthetic biology techniques for gene engi-
neering at the nucleotide level [4, 6, 9], stochastic models [14–18], and signal pro-
cessing methods [19–21] to study how the dynamics of gene expression in E. coli is
tuned by the kinetics of rate-limiting steps in transcription initiation [8, 9, 20, 22].
Using this, we propose a new strategy for, from the specification of the desired
dynamics of RNA production, mean and cell-to-cell variability in RNA numbers in
individual cells, and the use of detailed stochastic modeling of transcription initiation
© Springer Nature Switzerland AG 2018
M. Češka and D. Šafránek (Eds.): CMSB 2018, LNBI 11095, pp. 3–20, 2018.
https://doi.org/10.1007/978-3-319-99429-1_1
4 S. M. D. Oliveira et al.

[1, 14, 22], first, predict the necessary dynamics of the rate-limiting steps in tran-
scription initiation. Next, select an existing promoter that best fit these specifications.
Afterward, fine-tune the desired dynamics by single and double point mutations of the
selected promoter, so as to engineer a synthetic promoter whose RNA production
dynamics best fit the original specification. This fitting is analyzed at the single RNA
level, by making use of MS2-GFP probes for detection of RNA numbers at the single-
RNA level in live cells [9–11, 23, 24] and objective criteria to compare the dynamics of
synthetic promoters with that of the stochastic model, which, here, aside from tran-
scription dynamics, it also accommodates for RNA degradation and cell division.
The strategy has four main steps: (i) Design a stochastic model that best fits the
specifications using the modelling strategy proposed in [1, 13, 22]; (ii) Select the
promoter whose in vivo rate-limiting steps kinetics [23, 24] best fits the model
dynamics; (iii) Engineer mutant promoters of the selected one (previous step) and
probe their RNA production at the single-RNA, single-cell level [13]; and (iv) Select
the mutant promoter whose RNA numbers (mean and variability) best fit the model [1].
Here, we describe the strategy and the methods and present a case-study of the use
of this strategy to obtain promoters with pre-defined transcription dynamics.

2 Methods

2.1 Stochastic Model of Transcription and Cell Division

The stochastic model transcription used here is based on multiple studies of tran-
scription dynamics of individual genes [1, 9, 25]. The values set for each parameter
were obtained from empirical data [9, 26–30].
The multi-step transcription process of an active promoter, PON, is modeled by
reaction (1) [31] and its repression mechanism by reaction (2).
In reaction (1), the closed complex (RPc) is formed once an RNA polymerase
(RNAp) binds to a free, active promoter [32]. Subsequent rate-limiting steps follow to
form the open complex (RPo) [31, 32].
Finally, elongation starts [33], clearing the promoter. Elongation is not explicitly
modeled since its time-length is much smaller than that of the rate-limiting steps in
initiation [9]. Further, this process only affects noise in RNA production (mildly), not
its mean rate [18]. In the end, an RNA is produced and the RNAp is released.
In the multi-step reaction (1), k1 is the rate at which an RNAp finds and binds to
promoter P, k−1 is the rate of reversibility of the closed complex, k2 is the rate of open
complex formation, and k3 is the rate of promoter escape (expected to be much higher
than all other rates, and thus assumed to be ‘negligible’ [9]):

ð1Þ

The reaction in (1) should not be interpreted as elementary transitions. Namely,

they represent the effective rates of the rate-limiting steps in the process, which is what
defines the promoter strength [9]. Next, reaction (2) models the changes in the state of
 Modeling and Engineering Promoters with Pre-defined RNA Production Dynamics 5

the promoter, from repressed (POFF.Rep) to free for transcription, i.e. active, due to the
binding/unbinding of repressor proteins (Rep) to the promoter region:

ð2Þ

In general, under full induction, the cell contains sufficient inducers to render all
repressors “inactive” at all times (which can be modeled by having no Repressors in the
cell). Finally, the single-step reaction (3) models RNA degradation [34]:

ð3Þ

We note that in this model, as our RNA probes (MS2-GFP coating of the target
RNA, see Sect. 2.4) cause the RNA to be non-degradable for a time longer than the
observation time [9–11, 23, 24], reaction (3) is not included in the model.
Finally, we assume that our gene of interest is integrated into a single-copy plasmid
(not anchored to the cell membrane). Thus, we assume that the accumulation of super-
coiling caused by topological constraints is negligible [35].
Given this model, we define sprior as the mean expected time for a successfully
closed complex formation, which depends on the mean-time and number of attempts to
initiate an open complex formation (which depends on the RNAp intracellular con-
centration). Meanwhile, the remaining time to produce an RNA, safter, includes the
steps following commitment to open complex formation (e.g. isomerization [36]), and
prior to transcription elongation. The mean time interval between consecutive RNA
productions (Dtactive) of a fully active promoter is thus given by:

Dtactive ¼ sprior þ safter ð4Þ

Relevantly, safter does not depend on the RNAp intracellular concentration [36].
This is of significance in that, e.g., changes in this concentration will only affect sprior
and thus, will only partially affect Dtactive. Based on this, we simplify the model, so as
to be in accordance with the sensitivity of the measurements of rate-limiting steps (see
below), as follows. From (1), we assume the following approximate model:

ð5Þ

where: k1 ¼ s1 1

prior , k2 ¼ safter , and k3 = fast (i.e. ‘negligible’ in that it does not act as a
rate-limiting step in RNA production [9].
In addition, aside from transcription, note that cell division has a major effect on
RNA numbers due to ‘dilution’, as the RNAs are partitioned in the two daughter cells.
Here, we assume a near-perfect process of partitioning [37] as the RNAs are expected
to be randomly distributed in the cytoplasm. Namely, we assume that, when the
number of RNAs is even, each daughter cell receives half of them. If the number is
odd, one daughter cell receives (half + 0.5) and the other receives (half − 0.5).
6 S. M. D. Oliveira et al.

This model assumes only one copy of the promoter in each cell at any given time.
This approximation is made possible by the slow division time of the bacteria strain
used here (see Sect. 2.3). Specifically, in our measurement conditions, it was estab-
lished that these cells spend no more than 11 ± 1.2% of their lifetime with two copies
of the target promoter (in agreement with previous measurements [9]).

2.2 Stochastic Simulations

Simulations are performed by SGNS [15], a simulator of chemical reaction systems
based on the Stochastic Simulation Algorithm [38] and the Delay Stochastic Simulation
Algorithm [22]. It thus allows simulating multi-delayed reactions within hierarchical,
interlinked compartments that can be created, destroyed and divided at runtime. During
cell division, molecules are near-evenly segregated into the daughter cells. Each model
cell consists of reaction (5) along with the rate constants values (see Results section)
and the initial number of each of its component molecules.
Our model (described in Sect. 2.1, reaction 5), uses the following parameter
values: k1 ¼ 3901 s1 , k2 ¼ 2101 s1 . Given these, we expect Dt ¼ 600 s, and
safter =Dt ¼ 0:35. We begin simulations with 300 cells containing no RNAs. Each cell
contains 1 promoter and 1 RNAp molecule. These numbers were shown to be able to
reproduce realistically the RNA production kinetics of PLac-Ara-1 in [9]. Also, we set a
mean cell division time of 1200 s (for simplicity assumed to be constant), and we
analyze the RNA numbers in the cells at the end of the simulation time (3600 s).
Finally, the partitioning of RNA molecules in cell division is performed as described in
Sect. 2.1.

2.3 Strain, Cell Growth, and Stress Conditions

E.coli strain used is DH5a-PRO (identical to DH5a-Z1) [39], and its genotype is: deoR,
endA1, gyrA96, hsdR17(rK− mK+), recA1, relA1, supE44, thi−1, D(lacZYA-argF)U169,
U80dlacZDM15, F−, k−, PN25/tetR, PlacIq/lacI, and SpR. Plasmids construction and
transformation were done by using standard molecular cloning techniques (see
Sect. 2.4). From single colonies on LB agar plates, cells were cultured in LB medium
with the appropriate concentration of antibiotics and incubated overnight at 30 °C and
250 rpm. The overnight cultures were then diluted to an initial optical density (OD600)
of 0.05 in fresh LB medium, with a culture volume of 5 ml supplemented with the
antibiotics, kanamycin for the reporter gene, and chloramphenicol for the target gene
(Sigma-Aldrich, USA). Cells along with antibiotics were then incubated at 37 °C with
a 250 rpm agitation until reaching an OD600 of *0.3.
Next, to induce the expression of the reporter MS2-GFP proteins, 100 ng/ml of aTc
(Sigma-Aldrich, USA) was added and cells were incubated at 37 °C for 30 min with
250 rpm agitation. Then, cells were incubated at 37 °C (Innova® 40 incubator, New
Brunswick Scientific, USA) for 15 min with agitation, before activating the target gene.
Following full induction of the target gene (1 mM IPTG and 0.1% L-arabinose, Sigma-
Aldrich, USA), cells were incubated for 1 additional hour at 37 °C, prior to image
 Modeling and Engineering Promoters with Pre-defined RNA Production Dynamics 7

acquisition. Partial induction of the target gene was achieved by adding to the media
either only 1 mM IPTG or 0.1% L-arabinose. Finally, oxidative and acidic stresses
were induced by adding, respectively, 0.6 mM of H2O2 and 150 mM of MES to the
culture for 1 h along with the induction of target gene during cell exponential phase, as
described in [40].

2.4 Single-RNA Detection System in a Single-Copy F-plasmid

We detect individual RNA molecules using a fluorescent MS2 tagging system that,
using confocal microscopy, allow sensing integer-valued RNA numbers in individual
cells, as soon as they are produced [10, 11]. From these numbers in individual cells
over time, we characterize the dynamics of transcription initiation of the promoter of
interest [9, 23, 24]. For this, we obtain intervals between consecutive RNA production
events in individual cells (here defined as ‘Δt’). Also, we obtain the distribution of
RNA numbers, and from them, calculate the mean (M) and coefficient of variation
(CV) of RNA numbers in individual cells [4, 26].
This technique uses an RNA coding sequence of multiple MS2 binding sites [10,
11]. Here, we engineered an RNA with 48 MS2 binding sites, with unique restriction
enzymes, validated by sequencing. The construction of the single-RNA, single-protein
fluorescent probe was done into two steps. First, a promoter region, a coding region for
a fluorescent protein (mCherry), and the RNA with binding sites for MS2d-GFP
proteins were independently synthesized de novo (GeneScript, USA). Second, using
GenEZ™ molecular cloning, these sequences were ligated until forming the sequence
of the ‘target gene’. Next, they were cloned into a single-copy F-plasmid (GeneScript,
USA). This probe informs on the kinetics of RNA production, at the single cell level
(Fig. 1).
In our case-study, the ‘target gene’ is controlled by a PLac-Ara-1 promoter controlling
the expression of a mCherry fluorescent protein, followed by an array of 48 binding
sites for MS2d-GFP. Figure 1 shows the complete single-RNA detection system,
composed of the ‘target gene’ and the ‘reporter gene’. The latter is on a multi-copy
plasmid carrying the PL-tetO1 promoter controlling the expression of the fused
fluorescent protein ‘MS2d-GFP’. This protein rapidly binds to the MS2 binding sites of
the target RNA, making it visible as a fluorescent ‘spot’ under fluorescence microscopy
in less than 1 min, provided sufficient MS2d-GFP proteins in the cell (Fig. 3). In one of
the strains engineered, the target promoter is PtetA. In this case, we replaced the PL-tetO1
promoter controlling the expression of MS2d-GFP by the PBAD promoter.
By combining again de novo synthesis of DNA fragments with common molecular
cloning and DNA assembling techniques, this new fluorescent probe can be further
modified in the promoter region, RBS, and in regions between arrays of 12 MS2
binding sites, since pre-defined ‘cutting points’ were inserted on those regions.
8 S. M. D. Oliveira et al.

Fig. 1. Schematics of the engineered strain DH5a-PRO with the ‘target gene’ and its RNA
tagging system, along with the intake system of one of the inducers of the ‘target gene’, IPTG.
When in the cytoplasm, IPTG neutralizes the overexpressed LacI repressors by forming inducer-
repressor complexes (LacI-IPTG). This allows the PLac-ara-1 promoter to express RNAs that
include the array of 48 MS2d-binding sites. Meanwhile, MS2d-GFP expression is controlled by a
PL-tetO-1 promoter, which is regulated by TetR repressor, produced by its native promoter in
E. coli’s chromosome, and the inducer anhydrotetracycline (aTc). Once an individual RNA
molecule is produced, multiple tagging MS2d-GFP proteins (referred to as G) rapidly bind to it
forming a visible bright spot under a confocal microscope [6, 23]. The tagging of MS2d-GFP
molecules provides the RNA a long lifetime, with constant fluorescence, beyond the observation
time of the measurement (see Fig. 3) [4].

2.5 Relative RNAp Quantification

To achieve different RNAp concentrations in cells, we altered their growth conditions
as in [5]. For this, we used modified LB media which differed in the concentrations of
some of their components. The media used are denoted as m x, where the composition
per 100 ml are: m g tryptone, m/2 g yeast extract and 1 g NaCl (pH = 7.0). E.g. 0.25x
media has 0.25 g tryptone and 0.125 g yeast extract per 100 ml.
Relative RNAp concentrations were measured using E. coli RL1314 cells with
fluorescently-tagged b’ subunits. These were grown overnight in the respective media.
A pre-culture was prepared by diluting cells to an OD600 of 0.1 with a fresh specific
medium and grown to an OD600 of 0.5 at 37 °C at 250 rpm. Cells were pelleted by
centrifugation and re-suspended in saline. Fluorescence from the cell population was
measured using a fluorescent plate-reader (Thermo Scientific Fluoroskan Ascent
Microplate Fluorometer). As a control, we also measured the relative RNAp concen-
trations in RL1314 cells under a confocal microscope (see Sect. 2.7). Relative RNAp
concentrations were estimated from the mean fluorescence of cells growing in each
media. We found no differences using either method.
 Modeling and Engineering Promoters with Pre-defined RNA Production Dynamics 9

2.6 Mutant Target Promoters

We engineered 4 mutant target promoters from the original PLac-Ara-1 (referred to as
‘control’). Figure 2 shows these sequences, including the control. As also shown in the
Results section, single- and double-point mutations in the −35 and −10 promoter
elements can affect the transcription initiation rate-limiting steps kinetics [2, 36].

Fig. 2. Schematic representation of the target promoter’s sequences: The −35 and −10 promoter
elements are shown in black boxes. The transcription start site (+1 TSS) are marked in orange.
Operator sites are marked as cyan and blue. In the mutants, specific nucleotide changes in the -35
and -10 regions are marked as red circles. (Color figure online)

2.7 Microscopy and Image Analysis

Cells with the target and reporter genes were grown as above. After, cells were pelleted
and re-suspended in *100 µl of the remaining media. 3 ml of cells were placed on a
2% agarose gel pad of LB medium and kept in between the microscope slide and a
coverslip. Cells were visualized by a Nikon Eclipse (Ti-E, Nikon) inverted microscope
with a 100x Apo TIRF (1.49 NA, oil) objective. Confocal images were taken by a C2+
(Nikon) confocal laser-scanning system. MS2-GFP-RNA fluorescent spots and RNAp-
GFP were visualized by a 488 nm laser (Melles-Griot) and an HQ514/30 emission filter
(Nikon). Phase contrast images were taken by an external phase contrast system and
DS-Fi2 CCD camera (Nikon). Phase contrast and confocal images were taken once and
simultaneously by Nis-Elements software (Nikon).
For time series imaging, a peristaltic pump provided a continuous flow of fresh LB
media (supplemented with inducers for the target and reporter genes and chemicals for
stresses, at appropriate concentrations) to the cells, at the rate of 0.3 ml/min, through
Another random document with
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 For the purpose of histological examination it is essential that
portions of the brain, spinal cord, liver, kidney, and intestine, should
be examined microscopically. The nervous tissue should be placed
in formalin and Müller’s fluid, and a portion in alcohol for the
examination of the fibres. The liver, kidney, etc., should be placed in
5 per cent. formaldehyde. The tissues are then treated by the
ordinary histological methods, and sections prepared. With nervous
tissue it is essential that those prepared for the examination of the
cells should be made by the celloidin method; the others may be
treated by imbedding in paraffin. The points to be sought for in the
tissues are sufficiently indicated in the chapter on Pathology and
Symptomatology, but may be briefly recapitulated:
In the brain, as well as in all the tissues, careful search should be
made for minute microscopical hæmorrhages, and for evidences of
old hæmorrhages in the form of small masses of fibrous tissue, etc.
Parenchymatous degeneration, chromatolysis of nuclei, etc., nerve
degeneration.
The arteries and veins should also receive close scrutiny, as the
presence or absence of arteritis should be noted.
In the kidney particularly, search should be made for both
interstitial and parenchymatous nephritis.
The liver frequently shows signs of microscopic hæmorrhage, and
it is as well, in taking a portion of tissue for examination, to choose
those portions which appear to be specially congested.
In the brain and spinal cord and nervous tissue, search is to be
made for the same hæmorrhages as already noted. In addition, the
condition of the nerve fibres should be noted, the presence or
absence of periaxial neuritis, as well as degeneration of the axis
cells, and the various ganglion cells both in the brain and spinal cord
should be closely examined for chromatolysis and nuclear atrophy.
No evidence is afforded by micro-chemical tests of any of the
sections thus obtained, except those of the lung. It may be possible
in the case of the lung to determine the presence of lead granules in
the alveolar cells, and attention should be paid to this. It is possible
also that some evidence may be afforded by examination
microscopically of the red bone-marrow.
 The intestinal walls should be examined for evidence of lead
particles.
If any dark staining, deep or superficial, be found in the intestine, it
should be removed for chemical analysis. Necrotic areas of the
intestinal wall should be sought for.
Hæmatology.—For all practical purposes, the best stain for
detection of basophile granules in the erythrocytes is Wright’s
modification of Romanowski’s stain. This stain may be obtained in
appropriate tablets, and may be prepared immediately before use,
although a stain which has been standing for ten days or a fortnight
gives much better results than a quite new stain. The stain consists
of a solution of polychrome methylene blue, together with eosin in
methyl alcohol, and the method of procedure is as follows:
Blood is obtained by a small puncture, and slides smeared and
allowed to dry. Immediately on drying the slip is flooded with the
stain, and allowed to remain for two minutes. This causes fixation. At
the end of the two minutes the stain is diluted with an equal volume
of distilled water, and allowed to remain on for a further three
minutes. At the end of this time the stain is poured off, and the slip
washed in distilled water for another three minutes, or until the
characteristic purple-violet appearance is produced. It is better to
examine such films with an oil-immersion lens, the oil being placed
directly upon the films, and not covered with a cover-slip, as the
action of Canada balsam tends to decolorize the blue. If such
specimens are required to be kept, the oil may be washed off with
xylol. It is possible to observe basophile staining with a good sixth,
but an oil-immersion lens gives much the best result. The typical
staining produced by this means gives darkish bodies scattered
about the red corpuscles, staining sometimes deeply as the nuclei of
the white corpuscles. In other cases the appearance is like that of
fine dust scattered throughout the cell. In addition to these two
forms, the whole red cell may take on a slight generalized lilac tint,
the normal cells remaining free from granules, and stained red by the
eosin. Search of 100 fields of the microscope should be made, and if
no basophile granules are found in such fields it is unlikely that they
will be found.
 Basophile staining is not more pathognomonic of lead poisoning
than of any other form of anæmia, but may be regarded as a highly
important confirmatory diagnostic sign.
A differential count of the leucocytes present may be also made on
the same film in which basophile staining is observed; 300 should be
counted at least. In a typical case of lead poisoning it is found that
diminution in the polymorphonuclear leucocytes has taken place with
a corresponding increase of the lymphocytes, and possibly also the
large mononuclears, and probably a slight increase in the number of
eosinophiles.
This hæmatological method of diagnosis is of the utmost
importance in lead poisoning. A differential count such as is given on
p. 137, showing a large diminution in the polymorphonuclears, an
increase in the lymphocytes, evidence of changes in the red cells,
consisting of basophile staining, alteration in the shape of individual
cells, poikilocytosis, with vacuolation, is strong presumptive evidence
of lead absorption.
To complete the hæmatological examination, the hæmoglobin
should be estimated. This is best performed with Haldane’s
instrument—an exceedingly simple one to use. The estimation of the
number of red cells and white cells present is useful, but does not by
any means give such valuable information as does the differential
count and search for basophile granules.
Blood-Pressure.—Several methods are available for the
estimation of the blood-pressure. The pressure may be roughly
estimated as too high or too low by means of the finger. The
presence of thickening of the arteries may be also estimated in this
way, but for determining the absolute blood-pressure it is necessary
to use one or other of the instruments on the market. The estimation
of blood-pressure is an important point in relation to the suspected
presence of arterio-sclerosis, and should be performed wherever
possible. Sphygmographic tracings may also be taken. Such a
tracing in a case of typical poisoning gives a peculiar form of curve,
which, however, may be present in alcoholism and heavy work, and
arterio-sclerosis of many types.
Urine Examination.—In suspected cases of lead poisoning the
examination of the urine may reveal the presence of lead. In
addition, albumin is frequently present, especially in the early stages
of kidney inflammation. The ordinary tests for albumin should be
carried out, and it is also advisable to examine the urine
spectroscopically, as at times hæmoglobin, methæmoglobin,
hæmatoporphyrin, may be present in small quantities, each of which
can be detected by means of spectroscopic examination. Blood is
not common in the urine of lead-poisoned persons, although
microscopically hæmorrhages undoubtedly take place in the kidney.
These hæmorrhages are interstitial, and as a rule do not cause any
blood-pigment to be passed in a quantity that can be determined. It
is as well, however, to centrifugalize the urine, and examine the
deposit for red blood-cells.
The presence of hæmatoporphyrin, as suggested by Steinberg[10],
is probably due to hæmorrhages in the intestine, and its presence in
the urine should be regarded with suspicion in a lead-worker.
Where a lead-worker is suffering from continued absorption of
lead, even without the manifestation of other symptoms, a change
has been noted in the acidity of the blood—namely, a loss of normal
alkalinity. The estimation of the alkalinity or acidity of the blood direct
is an exceedingly difficult process, but much information may be
obtained by careful estimation of the acidity of the urine, and of the
acidity of the urine in relation particularly to the phosphates.
Joulie[11] has pointed out the extreme value which may be
obtained from a knowledge of the urinary constituents by the means
of estimation of the acidity with suchrate of chalk. The reagent is
made by slaking lime in such a way that the resulting compound is
practically dry. A quantity of this—about 25 grammes—is then
thoroughly shaken up with 10 per cent. solution of cane-sugar,
allowed to stand, and the solution titrated against decinormal acid
until it is of one-twentieth normal. The urine is then estimated
directly, the suchrate is run into the 25 c.c. of urine until a faint white
flocculent precipitate appears. The number of c.c. of the solution of
suchrate is then noted, and multiplied by the factor of the solution.
This gives the acidity related to the phosphate and other organic acid
contents, and is similar to the method used to determine the acidity
of wines.
 The second estimation consists of estimating the phosphates
present by means of a standard solution of uranium nitrate, using
either potassium ferrocyanide or cochineal as an indicator. The
specific gravity of the urine is also determined. The result is then
expressed in terms of this specific gravity, or, rather, in the terms of
the density of the urine in relation to distilled water, and the whole
answer returned per litre. By this method it is not necessary to obtain
a twenty-four hours sample of the urine, the urine passed first thing
in the morning being taken for examination.
By using this density figure the quantity of acid and phosphate is
expressed in relation to the density, the equation being—
The observed acidity
= Acidity per litre.
The density per litre
The phosphate content is expressed in the same manner, while
the ratio of phosphate to acidity gives the ratio of excretion of
phosphate to acidity.
There is in lead-workers a considerable diminution in the amount
of phosphate excreted, and, as has been pointed out by Garrod and
others, lead apparently produces alteration in the solubility of the uric
acid content of the blood, and may therefore allow of its
decomposition. Probably lead as a urate is stored up in the tissues.
For further particulars of this method of the estimation of the urine,
the reader is referred to “Urologie Pratique et Thérapeutique
Nouvelle,” by H. Joulie.
An examination of the fæces of persons suspected of lead
poisoning may often give definite results both of the presence of lead
and hæmatoporphyrin. If small hæmorrhages have occurred high up
in the intestine, the presence of hæmatoporphyrin in the fæces may
result. The substance may be easily determined by means of the
characteristic absorption bands. A quantity of fæces is taken and
extracted with acid alcohol, and the filtrate examined
spectroscopically. Urobilin bands are commonly present, and,
particularly, where much constipation exists these bands are very
well marked. There is no difficulty whatever, however, in
distinguishing them from the characteristic bands of acid
hæmatoporphyrin.
 Examination of the Fæces for Lead.—The moist method or
chemical examination
given above is the best one to apply for the determination of lead in
the fæces. As has already been pointed out, lead is commonly
excreted in the fæces, and, if only about 2 milligrammes per diem
are being excreted by the fæces in a lead-worker, the quantity
cannot be regarded as indicative of poisoning. One of us (K. W. G.)
has at times found as much as 8 to 10 milligrammes of lead excreted
in the fæces of persons engaged in a lead factory, and exhibiting no
signs or symptoms whatever of lead poisoning. If, however, the
quantity of lead in the fæces rises to anything above 6 milligrammes
per diem, there is definite evidence of an increased absorption of
lead, and if at the same time clinical symptoms be present,
suggesting lead poisoning, such a chemical determination is of the
first importance.
In estimating the presence of lead in fæces, it may be necessary
to deal with the separation of iron, which may be precipitated as
phosphate and filtered off, the quantitative estimation being
proceeded with in the filtrate.
Lead is much more commonly present in the fæces of lead-
workers than in the urine, and it is better to examine the fæces rather
than the urine in suspected cases.

REFERENCES.
[1] Gautier: Meillère’s Le Saturnisme, p. 74.
[2] Marsden and Abram: The Lancet, vol. i., p. 164, 1897.
[3] Shufflebotham and Mellor: Ibid., vol. ii., p. 746, 1903.
[4] Hebert: Comptes Rendus, tome cxxxvi., p. 1205, 1903.
[5] Fresenius and von Babo: Liebig’s Annalen, vol. xlix., p. 287, 1884.
[6] Glaister: Medical Jurisprudence and Toxicology. 1910.
[7] Dixon Mann: Forensic Medicine and Toxicology, p. 496.
[8] Vernon Harcourt, A.: A Method for the Approximate Estimation of
Small Quantities of Lead—Transactions of the Chemical Society, vol. cxvii.,
1910.
[9] King Alcock, S.: Brit. Med. Journ., vol. i., p. 1371, June 24, 1905.
[10] Steinberg: International Congress Industrial Hygiene. Brussels, 1910.
[11] Joulie, H.: Urologie Pratique et Thérapeutique Nouvelle.
 CHAPTER XI
TREATMENT
In laying down the general lines of treatment for both lead
poisoning and lead absorption, it is essential in the first place to
distinguish carefully between the two states; for although lead
absorption may gradually drift into definite lead intoxication and lead
poisoning, with all the classical symptoms associated with the
saturnine cachexia, a large number of cases, particularly those in
industrial processes, do not and should not progress beyond the
early symptoms of lead absorption. The treatment, therefore, will
depend in the first place on whether the case be one so constantly
met with in industrial processes, where generalized symptoms of
lead absorption are manifest without any definite and disabling
symptoms traceable and sufficiently pronounced to enable a
diagnosis of lead poisoning to be made.
The facts given in the chapter on Pathology, on the methods of
entrance of lead, on the toxic manifestations, and the blood-
changes, and, above all, the facts relating to microscopical
hæmorrhages and other profound changes in the bloodvessels, point
clearly to the lines along which the general treatment for
amelioration, prevention, or cure of poisoning should be undertaken.
The treatment of the so-called “presaturnine state,” or what is
preferably termed the “state of lead absorption,” is one that the
appointed surgeon or certifying surgeon in lead factories or other
processes in which lead is manufactured or used, is constantly
called upon to treat. Lead poisoning is a definite entity as a disabling
disease, whereas lead absorption, although the prodromal stage of
such disease, cannot be defined as actual lead poisoning, as in
many instances persons may show signs of continued lead
absorption, but their powers of elimination can be maintained at such
a level that the ratio of absorption to elimination remains in
equilibrium.
 With the preventive treatment of lead poisoning we have dealt in
another place (see p. 199). What is particularly required here is the
medicinal treatment, which may be helpful in preventing lead
absorption passing on to definite lead poisoning.
For many years it has been customary in the treatment of men
employed in lead works to give occasional purgatives, and it is,
moreover, a common and proper precaution to keep a stock of some
simple aperient medicine, preferably saline composed of sodium
sulphate and magnesium sulphate, at the works in charge of the
foreman, so that any man who so desires may obtain a dose of an
ordinary aperient mixture. We have seen from the pathological
evidence that the largest proportion of lead is excreted by the bowel,
and that, therefore, the sweeping away of the bowel contents—
particularly where constipation is set up—will naturally tend to
remove from the body a good deal of the lead which has been
already excreted into the intestine and which may presumably
become reabsorbed unless it be swept away. In a large electric
accumulator factory Epsom salts in the form of the granular
effervescing preparation is much appreciated. In winter 50 per cent.,
and in summer 90 per cent. of the men are said to take a daily dose.
In an important white-lead works chocolate tablets containing hypo-
(thio-)sulphite of sodium are supplied to the workers.
Another medicine made use of in lead works is the sulphuric acid
lemonade, this being acidulated with sulphuric acid and flavoured
with lemon. It is very questionable whether this substance has any
definite effect in the special direction in which it is supposed to work
—namely, that of forming an insoluble sulphate of lead in the
stomach and so preventing its absorption. The use of this drug was
suggested on the presumption that lead poisoning as a rule took its
origin from the dust swallowed and converted into a soluble form in
the stomach. As we have seen, there is very little evidence that this
entrance of lead is of much importance, although it does
occasionally take place. Furthermore, from the experiments of one of
us [K. W. G.[1]], it has been found that the sulphate of lead is at any
rate as soluble as other lead salts, such as white lead or litharge,
when acted upon by normal gastric juice.
 With regard to the drinks supplied to workers in lead factories, it is
highly important that some form of fluid should be supplied which the
men may drink without harm, particularly in the more laborious forms
of employment, and, above all, in the factories where smelting,
desilverizing, etc., of lead is carried on. In these factories the use of
some type of lemonade containing sodium citrate is to be
recommended, as it has been shown that one of the pathological
effects of lead absorption is to produce an increased viscosity of the
blood, and the use of such drugs tends to some extent to diminish
this. A drink containing a few grains of sodium citrate to the ounce
and flavoured with lemon is freely drunk by workmen engaged in the
laborious processes.
Finally, as a general routine treatment, it is advisable to keep at
the factory some form of mixture containing iron, which may be given
to those persons who are showing signs of slight anæmia, generally
associated with some degree of constipation, and it is therefore
better to use a form of iron cathartic. This medicine should also be
kept in the care of the foreman, who will see that it is administered to
the men properly. In this way any persons who at the weekly
examination exhibit signs of anæmia may be promptly treated, and
what is more, the surgeon is assured that the workmen in question
actually obtain the medicine prescribed regularly.
During the routine weekly or monthly examination, or at whatever
intervals the medical examination takes place, particular attention
should be paid to the records kept of the state of health of the
various persons, and whenever possible alteration of employment
should always be enjoined when early signs of anæmia make their
appearance.
The surgeon should spare no pains to determine if any of the
workmen are confirmed alcoholics, and such persons should be
removed from work in dangerous processes, while at the same time
care should also be taken to eliminate any persons suffering from
those diseases which are known to be predisposing causes of lead
poisoning. The card system of registration of any symptoms noted or
treatment given facilitates supervision of the health of the men.
In times of stress where some particularly dangerous process is in
operation, as, for instance, where portions of a building which has
become thoroughly impregnated with lead dust is being pulled down,
or where machines are being altered, removed, or rebuilt, especial
care should be exercised with the workmen so employed, and it is
advisable in such cases to adopt preventive measures on the
supposition—generally correct—that such persons are absorbing a
larger quantity of lead owing to their peculiarly dusty employment
than they were under normal circumstances. At such times, also, it
may be advisable to administer some form of mild iron cathartic to all
persons employed in the factory for, say, a week at a time. It must
not be supposed, however, that these methods of treatment in any
way supersede the precautions for the prevention of lead poisoning
by mechanical and hygienic means; they are merely additional
precautions which may be put in force under special circumstances.
The Treatment of Lead Poisoning.—The treatment of definite
lead poisoning, as the treatment of lead absorption, is directed
towards the elimination of the poison, the promotion of repair to the
damaged tissues, and special treatment directed towards those
special organs which suffer mostly in lead poisoning. At the same
time, special treatment of urgent symptoms may be called for; but in
the treatment of the urgent symptoms the fact of the general
elimination of the poison must not be lost sight of.
We have already seen that the channel through which the poison
leaves the body is mainly the fæces. Treatment must therefore be
directed, as in the former instance (lead absorption), towards
eliminating the poison by this means as much as possible, both by
the use of enemata, and later the use of sulphate of magnesia,
which may be added to the ordinary fluid enema; and it is far better
in obstinate cases of constipation and colic to give enemata than to
continue with the huge doses of salines or other aperients, such as
croton-oil, elaterinum, or castor-oil.
Colic.—Lead colic may be simple, acute, recurrent, or chronic
and continued. In whatever form colic appears pain is invariably
referred to the lower part of the abdomen, frequently into the groins,
and occasionally to the umbilicus. The pain has to be distinguished
particularly from acute gastritis, and occasionally from appendicitis,
and sometimes from that of typhoid fever. Acute colitis—not common
in this country—and dysentery, may, to some extent, simulate the
pain of lead colic, but John Hunter’s[2] original definition of “dry
bellyache” conveys very vividly the type of pain. Occasionally
diarrhœa may be met with, but as a rule obstinate constipation is
present. In continued colic, or chronic colic, sometimes lasting for
several months, obstinate constipation is the rule. In the simple
acute colic the pain passes off in the course of five or six days,
generally disappearing about four days after the lower intestine has
been thoroughly cleared.
The pain of lead colic is relieved by pressure upon the abdomen,
whereas that of gastritis and most other forms of abdominal pain
may be generally elicited along the descending colon and splenic
flexure; mucus is commonly found in the stools, especially the first
evacuation, after obstinate constipation occasionally of several days’
duration associated with an ordinary attack of lead colic. Blood may
be passed, but this symptom is not common. The pain in the acute
form is paroxysmal; it is rarely persistent, being typically intermittent.
During the paroxysm distinct slowing of the pulse-rate with an
increased blood-pressure takes place, and the administration of
vaso-dilators—such, for instance, as amyl nitrite—during a paroxysm
rapidly relieves the pain and lowers the blood-pressure, and in this
way distinguishes acute colic of lead poisoning from, say, subacute
appendicitis.
Vomiting may or may not be present, though the patient usually
complains of feeling sick, but there may be at times vomiting of a
frothy mucus.
It is unusual for a patient to die from acute colic, but acute
paroxysms have been recorded in which yielding of the blood-
vessels of the brain has occurred.
Recurrent colic is as a rule less severe than the simple acute form,
but may last for several weeks, clearing up for three or four days at a
time and then recurring with little diminution in violence from the first
attack. Such cases are probably due to the gradual excretion of lead
by the intestine, and should be treated on this supposition.
In the continued or chronic colic the pain may persist for as long
as two months, during the whole of which time the patient complains
of uneasiness and even constant pain in the lower part of the
abdomen, which becomes considerably worse after each
evacuation, and almost invariably is associated with exceedingly
obstinate constipation. It is this type of case that olive-oil or liquid
paraffin relieves, while in the acuter forms drastic purgatives such as
castor-oil, croton-oil, or pulv. jalapæ comp. may be administered.
For the treatment of pain in colic one of the various vaso-dilators
should be used, as, in addition to the spasm of the intestine, a very
considerable vaso-constriction of the whole of the vessels in the
mesenteric area occurs. Amyl nitrite gives immediate relief, but the
effect passes off somewhat rapidly, whilst scopolamine, although
taking somewhat longer to act, is better for continuous use, as its
action is longer maintained. Sodium nitrite, liquor trinitrini, and
antipyrin are also of use. Atropin may be used, but it is perhaps
better given in conjunction with magnesium sulphate.
Whatever form of purgative is given, some form of anodyne should
be combined. Drissole and Tanquerel[3] are said to have obtained
excellent results with croton-oil; one drop is given, followed seven or
eight hours later by another, and then by an enema of 2 pints of
normal saline. After two or three days the croton-oil may be again
given, one drop at a time each day. In addition, Tanquerel made use
of belladonna and opium together, finding that their combined action
was better than that of opium alone, as the physiological effect of
belladonna probably assists in preventing the intestinal cramp.
Hoffmann[4] recommends the use of olive-oil and opium, giving 3
to 4 ounces of olive-oil. He says that this relieves the spasm of the
pylorus, and is of particular use where severe vomiting is associated
with the colic. This use of olive-oil, first suggested by Hoffmann in
1760, and revived by Weill and Duplant[5] in 1902, is somewhat
interesting, in view of the modern tendency to administer paraffinum
liquidum in the treatment of chronic constipation.
Briquet[6] recommends 4 grammes of alum and 4 grammes of
dilute sulphuric acid three times daily, with the addition of 0·05
gramme of pulv. opii at night. Briquet says that although the
purgative method rapidly diminishes the colic, the elimination of the
poison does not take place as rapidly as by means of the treatment
he recommends, though it is open to doubt whether the use of either
of these two drugs is likely to produce any further neutralization or
excretion of absorbed lead than sulphate of magnesia. It is quite
certain that the magnesium sulphate does not act as a neutralizer of
the poison, as in a factory where sulphate of lead is manufactured
some cases of definite lead poisoning occurred, in which at least half
must have been due to the inhalation of lead sulphate dust. Under
these circumstances it seems hardly worth while to attempt to form a
sulphate of lead in the body. The action of magnesium sulphate and
other salines, however, in promoting the flow of fluid towards the
intestines, and rapidly diluting and washing out the contents, tend to
eliminate such lead as has already been excreted into the bowel.
A number of other drugs have been given from time to time for the
purpose of forming an insoluble compound with the metal in the
intestine, such, for instance, as sulphur in many forms, which is still
much used in French hospitals. Peyrow[7] advises sulphide of soda,
whilst Meillère prefers potassium sulphide as being less irritating. He
considers sulphuretted hydrogen a proper prophylactic against
reabsorption. Both experimental work and clinical observation show
that a change to sulphide does take place in the lower bowel, and
that staining of this part of the intestine is due to lead sulphide; but
as the figure on Plate II. shows, the lead may exist in the form of
granules of a dark nature, deeply embedded in the intestinal wall,
besides being situated in the exterior.
Stevens[8] suggests the use of ¹⁄₂-grain doses of calcium
permanganate thrice daily to relieve pain.
A certain number of other drugs may be also made use of from the
point of view of diminishing the pain, and one French observer
advocates the hypodermic injection of cocaine, but it is doubtful
whether any good would follow from such a procedure. Hypodermic
injections of morphia should be given whenever the pain is great,
and diaphoretics as well as diuretics should also be given, such, for
instance, as ammonium acetate, citrate of potash, or soda.
Chloroform water and chloral and bromine water may be also used,
and when no other drug is at hand, the inhalation of chloroform will
rapidly relieve the acute vaso-motor spasms associated with colic.
During the attack of colic, and for at least a day subsequent to its
disappearance, the patient should be kept on a fluid diet; milk is
best, and 10 grains of sodium citrate should be added to each glass
of milk. After the colic has subsided, a light farinaceous diet should
be given, and it is better not to give meat until at least a week has
elapsed. Alcohol is to be avoided.
The Anæmia of Lead Poisoning.—As has been pointed out in
Chapter VIII (p. 135), the anæmia of lead poisoning is one due to the
destruction of the red blood-cells. This is evidenced not only by the
curious sallow complexion, by the occasional presence of
hæmatoporphyrin in the fæces and urine, and often by the curious
yellow of the sclerotics, but also by an increase in the viscosity of the
blood itself. Moreover, the urine of persons suffering from lead
poisoning is invariably highly coloured, and may even show the
presence of methæmoglobin. As the anæmia is generally a symptom
of continued lead absorption for a long period, and does not
necessarily occur with every case of colic—in fact, acute colic may
often supervene without any symptoms of continued anæmia—the
persons suffering from lead anæmia should be removed from their
direct contact with the dangerous processes, and should be given, if
possible, work in the open air. Iron and arsenic may be used,
preferably in combination, whilst the iodide of iron often gives good
results. Whatever preparation of iron is given, care should always be
exercised in avoiding any constipating effect, and the free action of
the bowel should be maintained, together with a liberal supply of
milk. Potassium iodide may be also given.
With regard to the action of potassium iodide, there is division of
opinion amongst various physicians as to the efficacy of the drug in
the elimination of lead from the body. At the same time a very large
number of persons hold that the administration of fairly large doses
of potassium iodide in the case of a person suffering from chronic
lead absorption may at times be associated with sudden
exacerbation of the disease, and that the drug apparently may
determine the production of acute symptoms, such as
encephalopathy or paralysis, when these have not been previous
features of the case. Our experience supports this statement, and on
more than one occasion one of us (K. W. G.) has seen a distinct
increase of symptoms follow the administration of large doses of
potassium iodide. From a comparison with other cases it seems that
these symptoms would have been unlikely to make their appearance
without some secondary cause. Against this point of view must be
quoted further experiments already referred to by Zinn[9], who found
that when lead iodide was administered to experimental animals
iodine alone was found in the urine; but it must be pointed out that
no estimations were made of the fæces, and it is possible that a
certain amount of lead was eliminated in this way. What exactly is
the action of iodide on the solubility of lead in the body it is difficult to
say; yet the use of iodine compounds has been followed with
considerable success in a number of chronic inflammatory diseases,
and it is possible that it may have the action of splitting off the
particular lead compound from its organic association with the
tissues, especially as it is well known that iodine plays a very
important rôle in the process of cell metabolism. Another point which
tends to support the use of iodine is the fact that the other two
halogens, bromide and chloride, both of which enter largely into cell
metabolism, also have a slightly beneficial effect on the excretion of
lead. The dose of the iodine given should not be large to commence
with, 3 grains three times a day is sufficient, the dose being run up to
some 30 or 40 grains per diem, the symptoms meanwhile being
carefully watched.
Other symptoms often associated with the anæmia of lead
poisoning are—
Rheumatic Pains.—These pains are suggestive of muscular
affection, and are possibly due to minute hæmorrhages occurring in
the muscle tissue, which have been discovered in the muscles of
experimentally poisoned animals. For the rheumatic pains
diaphoretics and citrates of soda and potassium may be given.
Lumbago.—The lumbago constantly complained of in chronic
lead poisoning and even in the early stage of lead absorption, is very
generally related to chronic constipation rather than to a definite
affection of the lumbo-sacral joints.
Nephritis.—Affections of the kidney associated with lead
poisoning are almost entirely confined to sclerosis. The presence of
albumin in the urine is not a very common symptom. As has been
pointed out already, the presence of lead in the urine is by no means
a regular feature of lead poisoning, though it may at times be
present, and the urine should always be examined for changes in the
kidneys; but as a number of cases of chronic lead poisoning are
associated with alcohol poisoning, the changes in the kidney cell are
almost certain to be present. On p. 95 the illustration showing the
disease in the kidney produced by experimental dosage with lead,
and the kidney of a fatal case of lead poisoning in a man who at the
same time had a strong alcoholic history, shows fairly definitely the
difference between these two points.
Acute nephritis occurs so rarely in the course of industrial lead
poisoning that it cannot be considered to be a disease due to lead.
In chronic nephritis treatment should be along the ordinary lines
and the same remark applies to enlargement of the liver.
Heart.—Symptoms due directly to disease of the heart are rarely
caused by lead alone. The heart muscle may suffer in the same way
as the other muscles of the body, and in lead poisoning in animals
distinct hæmorrhages are found between the muscular fibres in the
heart muscle, and it is therefore probable that a form of myocarditis
may exist in lead poisoning. This, together with the increased arterial
tension, may cause dilatation, but the symptoms are those related
more to the general condition of arterio-sclerosis than to any direct
heart lesion, and as a rule these symptoms do not call for any
special treatment.
Treatment of Nervous Manifestations in Lead Poisoning.—
With one or two exceptions, the diseases of the nervous system
associated with lead intoxication only appear when actual lead
poisoning is established. Certain evidences of affection of the
nervous system are occasionally seen in the prodromal stage, or
stage of lead absorption. These may be merely temporary and
disappear often under treatment, by change of employment and
reduction in the quantity of lead absorbed. Thus, dilatation of the
pupils—the reaction to light being extremely sluggish or absent—is
often a feature of the later stages of the condition of lead absorption.
Tremor may also be a symptom, the outstretched hands exhibiting a
fine undulatory movement, often increased on attempting to perform
some act such as touching the nose, or touching the two fingers
together, and when these symptoms occur they must always be
regarded as of somewhat grave import. But it must be remembered
that tremor may occur as a common complication of alcoholic cases,
and further, follows excessively hard manual work, though there is
usually little difficulty in distinguishing between the various forms.
The symptomology of nervous diseases associated with lead
poisoning has already been carefully set out in Chapter IX., and the
pathological changes underlying these symptoms in Chapter V.
Of the general treatment, little needs to be added to what has
already been stated for the treatment of lead anæmia and general
lead intoxication. Iron and arsenic (not strychnine, especially in
presence of colic), and other similar drugs, should be employed
together with iodides either as potassium iodide or as an injection in
the form of an organic compound, of which there are several
varieties on the market.
The injection of normal serum has been advised, as well as saline
injections, and in some instances venesection has been practised,
but it is doubtful whether anything is to be gained by this form of
treatment.
Further, it has been stated that some lead is excreted through the
skin, and for this reason sulphur baths, bathing in sulphuretted
hydrogen water, etc., have been recommended to neutralize any
lead that has gained access to the skin. Serafini[10] has claimed that
by means of electrolytic baths a certain amount of lead can be found
present in the water after continuous passing of a current, and it has
been supposed by these observers that the lead has been actually
driven out of the body under the action of the electric current. It is, of
course, possible that such lead as is discoverable in the water was
merely that which had already become incorporated with the
patient’s skin through mechanical contact.
Whatever form of treatment be adopted of a general type, the
patient must certainly be removed from the chance of any further
lead absorption; a person who is suffering from wrist-drop or other
form of paresis should not be employed in any portion of a lead
works where he may come into contact with any form of lead or its
compounds for at least a year after the paresis has disappeared, and
even then it is inadvisable for such a person to return to any form of
dangerous lead work.
The electrical treatment of the injured nerves and muscles should
be undertaken energetically; both the galvanic or faradic currents
may be used. Probably the best form is the galvanic. A small
medicinal battery may be utilized, the method of application being as
follows: One pole of the battery should be placed over the affected
muscle, and the other pole placed in a basin of water into which the
patient’s hand is dipped. The current should then be passed. It is
better not to use a current of too great intensity, particularly at the
start, although it is found in practice that a much greater current can
be borne in the early stages of the treatment than when the muscles
and nerves commence to recover. As a rule the patient experiences
no inconvenience whatever from a considerable current during the
first week of his affection, but at the end of a fortnight or three weeks
less than one-third of the initial current can be borne. The current
should not be passed continuously, but should be used for a short
time and then shut off, being again switched on for five or six
minutes, and then again shut off. The applications may also be
modified by placing one hand in the vessel of water and stroking the
affected muscle and nerve with the free electrode. The application of
the current should be for not more than half an hour at a time, and
may be applied twice in the twenty-four hours. It is quite easy to
instruct the patient to perform the electrical treatment for himself in
this manner when the paresis is affecting either the upper or lower
extremity.
With the faradic current the circuit should be closed while the
current is at a minimum, and then the quantity of current raised to
some 15 to 20 milliampères.
For affections of the lower extremity the application may be made
by means of one of the usual baths in which the foot is immersed,
the other electrode being placed on the back or other suitable
position. If both the lower extremities are involved, then both feet
should be placed in a bath into each of which the source of electricity
is connected.
Ionization by means of the faradic current may also be made use
of. For this purpose one of the halogens, preferably iodine or
chlorine, should be used, it being remembered that chlorine and
iodine ions enter from the negative pole, so that in such a case the
bath in which the affected limb is placed must be connected with the
negative pole of the battery.
 Subsequently, with either form of electrical treatment, the part
should be well rubbed, and passive movements as well as massage
are an advantage in promoting the return of normal function. As the
muscles gradually return towards their normal state, graduated
muscular exercises should be used.
When treated in the first week or two of the onset, lead paresis
frequently recovers, and in a person suffering from lead palsy for the
first time, confined only to the hands or to a group of muscles in the
shoulder, prognosis is good. The prognosis of palsy of the lower
limbs is not so good.
Paralysis of the facial nerve is occasionally seen in lead poisoning,
and where this occurs it should be treated as previously
recommended, by means of iodides in association with localized
electrical treatment. One pole of the battery should be placed below
the external auditory meatus, and the other one passed over the
face on the affected side.
In long-standing cases where no attempt has been made at
treatment in the early stages of the disease, and where considerable
muscle degeneration has already taken place, the prognosis as a
rule is very bad. Efforts should always be made in an early case by
passive movements and massage of the affected muscles to
improve their nutrition as far as possible. The diet should be light,
and alcohol should not be given at any time.
Affections of the Central Nervous System.—The typical form
of affection of the central cerebral nervous system caused by lead, is
lead encephalopathy. The disease may be insidious in its onset, and
may be preceded by a long stage of chronic headache with slight or
total remissions. Headaches may last for several months before the
actual acute stage of the disease is reached. In the examination of
several brains of persons who have died from lead encephalitis,
microscopic sections of the brain have shown signs of hæmorrhages
which must have taken place some considerable time prior to death,
and were no doubt associated with the headache that had been
complained of for some time previously, before the onset of the fatal
illness. (See Plate III.) Persistent headache occurring in a lead-
worker should always be regarded with grave suspicion, and such a
case should be treated on the assumption that it is an early case of