Self-Replication out-of-Equilibrium
Yang, Shuo
DOI:
10.33612/diss.171627402
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2021
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Yang, S. (2021). Self-Replication out-of-Equilibrium. University of Groningen.
https://doi.org/10.33612/diss.171627402
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Emergence of Competing Self-Replicators in
Dynamic Molecular Networks
Part of this work has been published as:
S. Yang, G. Schaeffer, E. Mattia, O. Markovitch, K. Liu, A. S. Hussain, J. Ottelé, A. Sood, S.
Otto. Angew. Chemie Int. Ed.2020, 60, 2-8.
S.O. supervised this chapter. S.Y. conceived and designed the study, performed the experiments and analyzed the data.
2.1 Introduction
A key process in Darwinian evolution is natural selection, where the species adapted best to the local environment are more likely to survive and replicate than their competitors. Understanding how natural selection may emerge in chemical systems represents a way toward unveiling the mystery of the origin of life. The emergence of complex life can be divided
into two phases1: a chemical phase where non-life transitions to simple life and a biological
phase where simple life evolve to complex life. Although Darwinian theory is of great importance in modern biology, it only addresses the biological phase of natural selection. However, the chemical phase is equally important as the biological phase because it bridges chemistry and biology. Thus, the extension of natural selection to the molecular level in synthetic systems represents an important research challenge.
In nature, mutations occur spontaneously and randomly as a result of the errors in DNA/RNA replication. Environmental factors then act on the existing heritable mutations ultimately leading to the survival of the fittest species and the extinction of the species that lost the competition. To investigate natural selection in abiotic systems, the creation of mutations in self-replicating systems capable of responding to the environment is a prerequisite.
In this chapter, we address the possibilities of implementing mutation in synthetic replicating systems. In Biology mutations refer to species carrying different genetic information. Similarly, in synthetic systems biomolecules or small molecules with similar but different structures capable of self-replicating can be considered as mutants when they cross-catalyze each
other’s formation. Seeman has reported a self-replicating system2 consisting of two
subpopulations (species1 and 2) based on DNA origami. The replication was controlled by
intra- and inter-molecular DNA triplex formation which was sensitive to a change in pH. As a
result, the replication rate of species 1 was higher than that of species 2 under acidic
conditions. In basic conditions the replication rates were reversed. When they were mixed together, the pH determined which species grew preferentially.
In Chapter 1 the peptide replicators developed by our group were introduced as a suitable
candidate to investigate evolutionary processes3. Previously, we have reported that changing
the structure of the peptide building blocks leads to the emergence of differently sized
self-replicators4. The ring size was inherited upon replication where information transfer was
mediated through the template effect. Different ring sizes can be regarded as mutants provided they cross-catalyze each other’s formation. It has been shown that the emergence of two differently sized replicators from the same building block occurred upon the modulation of
addition to cosolvent, salts7 and macromolecules8 can affect the secondary structures of
peptides and proteins, and we reasoned that they may influence the assembly of our peptide building blocks. To expand the diversity of mutations in replicating systems, we decided to change the environment by adding salts and tested its effect on the replicating peptide systems.
2.2 Results and Discussion
To study the effects of salts on our peptide building blocks, we focused on guanidinium chloride (GuHCl), which is known to be a strong denaturant in studies of protein stability and
folding7. We used building block1 (Figure 2.1A) to test the effects of salts because replicators
derived from it tend to assemble through forming β-sheets which should respond to denatures. The interaction formed by the hydrophobic phenylalanine moiety in the sequence should be sensitive to GuHCl.
Figure 2.1 Structure of building block 1 (A) and change in product distribution of DCLs (50 mM borate buffer pH 8.2)
made from 0.38 mM building block1 at 400 mM (B), 2.5 M (C), 3 M (D) and 4 M (E) of GuHCl. Summary of the
experiments in the presence of different concentrations of GuHCl (F). Mass spectra of all the species are displayed in the supporting informationFigure 2.S.2 to 2.S.6.
We oxidized the dithiol building block1 by the oxygen present in the air in a series of borate
buffer solutions (50 mM, pH 8.2) containing different concentrations of GuHCl (0 to 4 M). We monitored the product distribution of the libraries over time using ultra performance liquid
chromatography- time-of-flight mass spectrometry (UPLC-TOF) analysis and macrocycles with
different sizes appeared when the concentrations of GuHCl were varied. Cyclic hexamer (16)
forms when the concentration of GuHCl is below 2 M, similar to the behavior in pure buffer
without adding any GuHCl (Figure 2.1B)4. When the concentration of GuHCl was 2.5 M,
interestingly, mixtures of cyclic pentamer (15) and trimer (13) were formed (Figure 2.1C). We
screened the concentrations of peptide building block and GuHCl very carefully, however15
and13always appeared simultaneously and pure15could not be obtained. Next,13became
the dominant species if the concentration of GuHCl was increased to 3 M (Figure 2.1D). We
observed fiber formation in the15and13mixture and in the sample dominated by13through
transmission electron microscopy (TEM) analysis (Figure 2.3). Furthermore, upon increasing
the concentration of GuHCl to 4 M the library end up forming predominantly13and14(Figure
2.1E) which turned out to be non-assembled species since no aggregation was observed
through TEM analysis. Note that UPLC peak areas can be used to quantify the relative
amounts of1 contained in the different replicators since the molar absorptivity of a unit of 1
was found to be independent of the ring in which it resides (supporting informationFigure
2.S.1). The behavior of building block 1 at different concentrations of guanidinium chloride is
summarized inFigure 2.1F.
Figure 2.2 Seeding-induced growth of macrocycles under conditions that favor their formation. (A) The library was
seeded with 10%15fibers in a 2.5 M GuHCl solution containing1 as food. (Note that the seed added also contains 5%
13fibers). (B) The library was seeded with 15%13in a 3.5 M GuHCl solution containing1 and unassembled 13and14
as food. Concentration of total building block with respect to1 is 0.38 mM. The experiments were performed in 50 mM
borate buffer (pH 8.2) and under shaking at 1200 rpm. The evolution of the product distribution of DCLs in full details is displayed in supporting informationFigure 2.S.7.
We performed seeding experiments to verify if the macrocycles (15 and 13) formed with
different concentrations of GuHCl are self-replicators (Figure 2.2 and supporting information
Figure 2.S.7). The results demonstrate that the addition of 15or13fibers enhanced their own
growth, indicating that both of them are self-replicators. We characterized the self-replicating
15and13by thioflavin T (ThT) fluorescence and circular dichroism experiments (Figure 2.3).
ThT is a fluorescent dye that is widely used for characterization of assembled peptide based
on β-sheets interactions9. The fluorescence of ThT is enhanced upon binding to amyloid
materials. The ThT results show that, compared to13,15has very limited β-sheets formation.
Circular dichroism (CD) is a light absorption technique that measures the difference in absorbance of left and right handed polarized light by a substance. It has been shown that
secondary structures can be analyzed through CD spectra in the far-UV region10. The CD
spectra of a15/13mixture and of13show the typical CD features for β-sheets with maximum
absorbance at 200 nm and minimum absorbance at 220 nm. The β-sheets formation of15is
somewhat less pronounced than that of 13 (as evident from the ThT data), yet the CD
absorbances of the15/13mixture and13are similar. More insight into the assembly of15will be
given in Chapter 5. Our previous work4shows that replicators of smaller ring size is favored as
the interactions between building blocks get stronger. We therefore speculated that the assembly of the nonpolar cores of the macrocycles into stacks was promoted due to the
enhanced hydrophobic interactions induced by GuHCl11, which drives the transition from16to
Figure 2.3 TEM micrographs of samples dominated by 15/13(A) and13(B). (C) Fluorescence emission spectra of
solutions of ThT when15/13and13is added. No peptide was added in the blank sample. (D) CD spectra of DCLs
dominated by15/13and13.
Building block1 gave rise to three different replicators in different environments by varying the
concentration of GuHCl. We performed (cross-)seeding experiments to verify whether the ‘non-native’ replicators can replicate in environments different from the ones from which they emerged and to investigate the relations between ‘native’ and ‘non-native’ replicators, that is, to observe if the growth of the ‘native’ replicator in its own environment benefits from seeding
Figure 2.4 Seeding-induced growth of macrocycles under conditions that do not favor their formation. (A) growth of 16
when13was added as seed in 15 mM GuHCl. (B) growth of16when15/13was added as seed in 10 mM GuHCl. (C)
growth of15when16was added as seed in 2.5 M GuHCl. (D) growth of15when15/13or15/13+13was added as seed
in 2.5 M GuHCl. We repeated this seeding experiment and again it suggests small seeding effect (Figure 2.S.12). (E)
growth of13when16was added as seed in 3 M GuHCl. (F) growth of13when15/13or13was added as seed in 3 M
GuHCl. The complete time evolution of all the species in all the experiments is provided in the supporting information
Figure 2.S.8, 2.S.9 and 2.S.11. Concentration of total building block with respect to 1 is 0.38 mM.
In the case of native replicator16where concentration of guanidinium chloride is lower than 2
M (Figure 2.4A and 2.4B), the seeding with 15/13or13did not accelerate the growth the16. If
anything, the presence of non-native seed slightly reduced the replication rate of 16. The
complete traces (supporting informationFigure 2.S.8) show that the concentration of 15or13
was constant for the first few days before they were depleted, indicating that15/13 have a
limited replicating ability to replicate under these conditions, and are eventually converted into
16.
When15is considered as the native replicator in the presence of 2.5 M guanidinium chloride
(Figure 2.4C, 2.4D and supporting information Figure 2.S.9), the non-native seed 16has no
significant effect on the replication process. The consumption of 16 was relatively fast
indicating16is not stable in this environment (2.5 M GuHCl). When non-native13was used as
informationFigure 2.S.10). This result demonstrates that the energy barrier of nucleation of 15
is relatively high,so that adding13seed can direct the kinetic pathway to form13exclusively via
bypassing the barrier of nucleation of13. As we aimed to investigate the interaction between15
and13, we added15seed into both seeded and control samples to bypass the nucleation of15,
and added an extra amount of13fibers in the seeded sample. The data shows that13seed
slightly increases the growth rate of15.
In the case of native replicator13(in solution of 3 M guanidinium chloride), the non-native16
seed speeds up the formation of 13. When non-native seed 15 was added, there is no
significant effect on the growth of13 (Figure 2.4E, 2.4F and supporting information Figure
2.S.11). The non-native seed 15contains13as they form simultaneously, hence, we added the
same amount of13in the control sample (Figure 2.4F, blue solid triangle).
As the data shows, when placing ‘non-native’16in the environment that favors 13or when
placing ‘non-native’13in the environment that favors15, the ‘non-native’ replicator was capable
of cross-catalysis, albeit to a very limited extent. The other four sets of experiments all suggest that the ‘non-native’ replicators do not cross-catalyse the ‘native’ replicators. The results of the
entire set of experiments is summarized below (Figure 2.5).
Figure 2.5 Summary of the cross-seeding experiments where the arrows show the catalysis direction (green arrow:
modest cross-catalysis by non-native replicator; red arrow: no cross-catalysis by non-native replicator).
2.3 Conclusions
We reported a system consisting of three replicating species when a common peptide building block was subjected to different environments. The change of the environment was controlled through varying the concentration of guanidinium chloride, most likely resulting in different interacting strengths between the building blocks, which, in turn, determine the ring size of the replicators.
In the previously reported cosolvent system5 the cross-seeding experiments, where one
replicator was added as a seed under conditions that do not favor its formation, show that the two replicators were cross-catalytic. This feature hinders implementing the important biological process of extinction, since the two replicators can promote the growth of each other. While in
extent), which may allow implementing extinction at the molecular level.
The formation of the replicators in this chapter was either thermodynamically or kinetically controlled. However, a fueled out-of-equilibrium state is more relevant to living systems potentially allowing mutation and natural selection. The next step would be the implementation of the current system under out-of-equilibrium conditions, which will be described in Chapters 3 and 4.
2.4 Materials and methods
Peptide building block1 was synthesized by Cambridge Peptides Ltd (Birmingham, UK) from
3,5-bis(tritylthio)benzoic acid, which was prepared via a previously reported procedure3.
Doubly distilled water was used in all experiments. Hydrogen chloride, sodium hydroxide, and the buffer ingredients boric acid and potassium hydroxide were obtained from Merck Chemicals. Guanidinium chloride and sodium perborate (oxidant) were purchased from Sigma Aldrich. UPLC analysis: water (ULC/MS grade), acetonitrile (ULC/MS grade) and trifluoroacetic acid (TFA) were obtained from Biosolve BV. Replicator experiments were conducted in UPLC vials (12 × 32 mm) with a teflon-lined snap cap. Samples were placed in an Eppendorf Thermomixer Comfort and shaken at 1200 rpm at 25 °C.
Library preparation
Building block1 was dissolved to a concentration of 0.38 mM in borate buffer (50 mM in boron
atoms, pH 8.2) for16formation. The same borate buffer containing 2.5 M or 3 M guanidinium
chloride was used for15/13or13formation, respectively. If needed, 1.0 M KOH solution was
added to adjust the pH of the solution to 8.2. The volume of each library was 1.0 mL. The replicators were the dominant products after 1 week of shaking the solutions at 1200 rpm in the presence of oxygen from the air.
Seeding experiments
A solution of building block1 (0.38 mM in borate buffer) was oxidized by sodium perborate to
give a library containing1 and unassembled 13 and14. The resulting solution was split into
the design. One sample was not seeded as control. The concentration of guanidinium chloride is 2.5 M or 3.5 M depending on the experiments. The samples were shaken at 1200 rpm and monitored by UPLC over time.
Cross seeding experiments
A solution of building block1 (0.38 mM in borate buffer) was oxidized by sodium perborate to
give a library containing1 and unassembled 13 and14. The resulting solution was split into
multiple samples: different amounts of pre-formed16,15/13or13fibers were added depending
on the design. One sample was not seeded as control, unless stated otherwise. The concentration of guanidinium chloride ranged from 10 mM to 3 M depending on the experiments. The samples were shaken at 1200 rpm and monitored by UPLC over time.
UPLC-MS analysis
UPLC analyses were performed using a Waters Acquity UPLC H-class system with a reversed-phase UPLC column (Phenomenex Aeris Peptide, 2.1 × 150 mm; 1.7 μm). The column temperature was 35 °C and UV absorbance was monitored at 254 nm. Injection volumes were 2-5 μL (1:15 dilution in water with 0.6% TFA, where TFA was used to quench the disulfide exchange) and the eluent flow rate was 0.3 mL/min.
Eluent A: UPLC grade water with 0.1% trifluoroacetic acid added Eluent B: UPLC grade acetonitrile with 0.1% trifluoroacetic acid added
The eluent gradient used for UPLC analysis is shown inTable 2.1 below.
Table 2.1. UPLC method for the analysis of DCLs.
Time (min) % water + 0.1% TFA % MeCN + 0.1% TFA
0 90 10 1 90 10 1.3 75 25 3 72 28 11 60 40 11.5 5 95 12 5 95 12.5 90 10
UPLC-MS analyses were performed on a Waters Xevo G2 UPLC/TOF. Electro-spray ionization was used to acquire positive-ion mass spectra. The capillary, sampling cone and extraction cone voltages were set at 2.5 kV, 30 kV and 4 V, respectively. Nitrogen was used as cone and desolvation gas with flow rates of 5 L/h and 500 L/h, respectively. The temperatures of source and desolvation were 150°C and 500°C, respectively.
Transmission electron microscopy
A droplet (5 μL) of sample was deposited on a copper grid (400 mesh) covered with a carbon film (Agar Scientific). The droplet was blotted on filter paper after 30 s and the sample was stained twice (4 μL each time) with a 2% uranyl acetate solution (deposited on the grid and blotted on filter paper after 30 s each time). The grids were observed in a cryo-electron microscope at 120 kV (Philips CM120). Images were recorded by CCD camera.
Thioflavine T (ThT) fluorescence
Libraries of replicators was diluted to a concentration of 76 μM with respect to building block1
in potassium borate buffer (50 mM, pH 8.2). Then the diluted sample (3.2 μL) was added to potassium borate buffer (50 mM, pH 8.2) containing 22 μM ThT. The resulting solution was incubated for 5 minutes and transferred into a quartz cuvette (HELMA 10X2 mm). A JASCO FP6200 spectrophotometer was used to measure the fluorescence (Ex 440 nm, Em 460 to 700 nm).
Circular Dichroism (CD)
Spectra were obtained at 20 °C using a JASCO J715 spectrophotometer (range = 190–400 nm, pitch = 2 nm, bandwidth = 5 nm, response = 2 s, speed = 50 nm/min, continuous scanning). Solvent spectra were subtracted from all spectra. All spectra were measured using samples diluted to 8 μM (with respect to building block) in HELMA 10 × 2 mm quartz cuvettes.
2.5 Supporting information
Figure 2.S.1 (A) Representative UPLC chromatogram of a mixture of macrocycles prepared from building block 1. (B)
Representative UPLC chromatograms of mixtures with different amounts of13and16showing a comparable total
peak area, indicating that the molar absorptivity of the building block is independent of the macrocycle in which it resides. Thus, relative peak areas can be used to quantify the amount of these replicators.
Figure 2.S.2 Mass spectra of monomer 1 from the LC-MS analysis of a DCL made from 1. M/z calculated: 760.35
Method_ XGLKFK28-Aug-2016 m/z 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 % 0 100
flow-2 1164 (9.994) Cm (1155:1189) 1: TOF MS ES+
2.21e4 758.9985 758.6601 759.6642 760.0029 1138.0167 1137.5057 760.3416 760.6804 761.0079 1138.5280 1139.0116 1139.5231 1140.0070
Figure 2.S.3 Mass spectra of 13from the LC-MS analysis of a DCL made from1. M/z calculated: 1137.50 [M+2H]2+,
759.01 [M+3H]3+; m/z observed: 1137.50 [M+2H]2+, 758.66 [M+3H]3+. Method_ XGLKFK28-Aug-2016 m/z 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 % 0 100
flow-2 1020 (8.760) Cm (1012:1033) 1: TOF MS ES+
1.95e3 1011.5690 1011.2304 759.1565 758.9139 738.9725 759.6699 760.1553 760.3811 992.0499 804.6034 928.6902 1011.8816 1012.2203 1012.2985 1012.8848 1012.9761 1013.2889 1517.3671 1516.8727 1013.5757 1518.8508 1688.8723
Figure 2.S.4 Mass spectra of 14 from the LC-MS analysis of a DCL made from 1. M/z calculated: 1516.67
[(M+2)+2H]2+, 1011.45 [(M+2)+3H]3+, 758.59 [(M+1)+4H]4+ ; m/z observed: 1516.87 [(M+2)+2H]2+, 1011.23
Method_ XGLKFK m/z 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 % 0 100
638059_SY_20151102-F(D)-53 809 (6.951) Cm (809:817) 1: TOF MS ES+
3.30e3 948.15 947.90 947.87 947.64 947.63 758.71 758.49 632.59 632.43632.91 758.32 759.11 759.32 759.50 1263.89 1263.88 948.40 1263.85 948.41 1263.53 948.90 949.16 1263.21 949.17 949.40 949.55 949.66 949.90 1264.21 1264.54 1264.87 1264.89 1265.20 1265.52 1271.51 1271.86 1282.17 1289.84
Figure 2.S.5 Mass spectra of 15 from the LC-MS analysis of a DCL made from 1. M/z calculated: 1263.67
[(M+3)+3H]3+, 948.75 [(M+3)+4H]4+, 758.80 [(M+5)+5H]5+, 632.00 [(M+4)+6H]6+;m/z observed: 1263.89 [(M+3)+3H]3+, 948.15 [(M+3)+4H]4+, 758.71 [(M+5)+5H]5+, 632.59 [(M+4)+6H]6+. Method_ XGLKFK28-Aug-2016 m/z 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 % 0 100
flow-2 1059 (9.094) Cm (1055:1080) 1: TOF MS ES+
8.14e3 1138.2517 1138.0167 1137.7476 1137.5126 911.1966 910.6034 759.6868 758.9816760.6691761.1774 911.3820 911.7900 948.8461 1138.5142 1138.7491 1517.6862 1138.9978 1517.0322 1139.1913 1516.6814 1139.4331 1518.0211 1518.3721 1518.6753 1518.8668 1519.3616
Figure 2.S.6 Mass spectra of 16 from the LC-MS analysis of a DCL made from 1. M/z calculated: 1516.67
[(M+3)+3H]3+, 1137.75 [(M+3)+4H]4+, 910.80 [(M+5)+5H]5+, 759.00 [(M+4)+6H]6+;m/z observed: 1516.68 [(M+3)+3H]3+,
Figure 2.S.7 Evolution of the product distribution of DCLs in full detail. (A) Libraries seeded with and without 15/13in
2.5 M GuHCl. (B) Libraries seeded with and without13in 3.5 M GuHCl. (Total concentration 0.38 mM building block1
in 50 mM borate buffer, pH 8.2).
Figure 2.S.8 Evolution of the product distribution of DCLs in an environment (15 mM GuHCl) that favors formation of 16when monomer food1 is seeded with non-native seeds (0.38 mM building block 1 in 50 mM borate buffer, pH 8.2).
Figure 2.S.9 Evolution of the product distribution of DCLs in an environment (2.5 M GuHCl) that favors formation of 15
when monomer food1 is seeded with non-native seeds (0.38 mM building block 1 in 50 mM borate buffer, pH 8.2). The
growth of15without (A) and with (B)16as seed. The growth of15with15/13(C) and with15/13+13(D) as seed. Note
that15is also added in (C) and (D), otherwise15was not obtained (seeFigure 2.S.10).
Figure 2.S.10 Evolution of the product distribution of a under conditions DCL (2.5 M GuHCl) that favor formation of 15
Figure 2.S.11 Evolution of the product distribution of DCLs in an environment (3 M GuHCl) that favors formation of 13
when un-assembled food13and14is seeded with non-native seeds (0.38 mM building block1 in 50 mM borate buffer,
pH 8.2). The growth of13without (A) and with (B)16as seed. The growth of13with13(C) and with (D)15/13as seed.
Figure 2.S.12 (A) Growth of 15when15/13or15/13+13was added as seed in an environment (2.5 M GuHCl) that
favors formation of15(0.38 mM building block1 in 50 mM borate buffer, pH 8.2). The complete evolution of the
2.6 References
1. Pross, A. Toward a general theory of evolution: extending Darwinian theory to
inanimate matter. J. Syst. Chem.2, 1 (2011).
2. He, X. et al. Exponential growth and selection in self-replicating materials from DNA
origami rafts. Nat. Mater.16, 993–997 (2017).
3. Carnall, J. M. A. et al. Mechanosensitive self-replication driven by self-organization.
Science327, 1502–1506 (2010).
4. Malakoutikhah, M. et al. Uncovering the selection criteria for the emergence of
multi-building-block replicators from dynamic combinatorial libraries. J. Am. Chem. Soc.
135, 18406–18417 (2013).
5. Leonetti, G. & Otto, S. Solvent composition dictates emergence in dynamic molecular
networks containing competing replicators. J. Am. Chem. Soc.137, 2067–2072 (2015).
6. Roccatano, D., Colombo, G., Fioroni, M. & Mark, A. E. Mechanism by which 2, 2,
2-trifluoroethanol/water mixtures stabilize secondary-structure formation in peptides: a
molecular dynamics study. Proc. Natl. Acad. Sci.99, 12179–12184 (2002).
7. Arakawa, T. & Timasheff, S. N. Protein stabilization and destabilization by guanidinium
salts. Biochemistry23, 5924–5929 (1984).
8. Mukherjee, S., Waegele, M. M., Chowdhury, P., Guo, L. & Gai, F. Effect of
macromolecular crowding on protein folding dynamics at the secondary structure level.
J. Mol. Biol.393, 227–236 (2009).
9. Levine III, H. Thioflavine T interaction with synthetic Alzheimer’s disease β‐amyloid
peptides: Detection of amyloid aggregation in solution. Protein Sci.2, 404–410 (1993).
10. Greenfield, N. J. Using circular dichroism spectra to estimate protein secondary
structure. Nat. Protoc.1, 2876 (2006).
11. Fujita, T., Watanabe, H. & Tanaka, S. Effects of salt addition on strength and dynamics
