Journal of Experimental Marine Biology and Ecology 447 (2013) 93–99
Contents lists available at SciVerse ScienceDirect
Journal of Experimental Marine Biology and Ecology
journal homepage: www.elsevier.com/locate/jembe
Special issue: Cephalopod Biology
Octopus arm regeneration: Role of acetylcholinesterase during
morphological modification☆
Sara Maria Fossati a, 1, Francesca Carella b, 1, Gionata De Vico b, Fabio Benfenati a, Letizia Zullo a,⁎
a
b
Istituto Italiano di Tecnologia, Department of Neuroscience and Brain Technologies, Genoa, Italy
Federico II University of Naples, Department of Biological Science, Naples, Italy
a r t i c l e
i n f o
Article history:
Received 6 December 2011
Received in revised form 4 May 2012
Accepted 25 January 2013
Keywords:
Acetylcholinesterase
Cephalopods
Octopus
Proliferation markers
Regeneration
a b s t r a c t
The ability to regenerate whole-body structures has been long studied in both vertebrate and invertebrate
animal models. Due to this regeneration capability here we propose the use of the Cephalopod Octopus
vulgaris as a model of regeneration. We investigated the involvement of acetylcholinesterase (AChE) in the
octopus arm regeneration. AChE has been demonstrated to have non-cholinergic functions in various cell
types and to be involved in the regulation of cell proliferation, differentiation and apoptosis. In order to follow
cell replacement in the octopus arm, we first assessed the expression of specific markers involved in cellular
proliferation (AgNOR and PCNA). We showed that the activity of the enzyme AChE is related to the
proliferation stage of the arm regenerative process. In the very initial stages of regrowth when most of the
proliferation activity was at the level of the ‘blastema’ the cholinesterase activity was very low. AChE activity
climbed slowly during the subsequent phase of cellular multiplication and, by the onset of morphogenesis,
the activity rose sharply and active myogenesis was observed. AChE activity decreased then till reaching
basal level at the time when the process of histogenesis occurred and the reestablishment of all the structures
became evident. Interestingly AgNOR and AChE assay showed a similar trend in particular during the stages
when the morphogenesis was mostly dependent upon cell proliferation. We suggest that AChE protein may
have an important influence in the process of regeneration and that it could be considered as a potential
target to promote or regulate the regenerative process.
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
A regenerative process implies the renewal, restoration, and growth
of cells, tissues, and organs that have been physically or functionally
lost. Several vertebrates and invertebrates have been used as ‘model
system’ to study conserved and convergent mechanisms of plasticity
and regeneration pathway.
We are testing the hypothesis that acetylcholinesterase (AChE)
plays a major role in the regenerative process of the octopus arm. Several studies in both vertebrates and invertebrates have been pointing
toward the role of AChE in the regeneration process. The abundance
of acetylcholine within the regenerating tissue has been correlated inversely with the activity of the AChE (Ellman et al., 1961; Karnovsky
and Roots, 1964; Lenicque and Feral, 1976; Singer et al., 1960). The importance of AChE in regeneration lie in that during the early formative
phases of growth the development can be most dependent upon the
☆ This article is part of a special issue on Cephalopod Biology published under the
auspices of CephRes-ONLUS (www.cephalopodresearch.org).
⁎ Corresponding author. Tel.: +39 01071781559.
E-mail address: [email protected] (L. Zullo).
1
Equal contribution.
0022-0981/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jembe.2013.02.015
nerve. It seems that acetylcholine can be used by the nerve as an
agent to control the early events of regeneration (Singer et al., 1960).
In Aplysia AChE has been shown to promote neurite growth of adult
neurons (Srivatsan, 1999; Srivatsan and Peretz, 1997). In Planaria it
has been found to be connected with the regeneration stages
(Lenicque and Feral, 1976). Also in Triturus it has been found that the
activity of the enzyme changes in location and intensity during the
whole regeneration process (Singer et al., 1960). Recently it has been
proposed that AChE may contribute to various physiological processes
through its involvement in the regulation of cell proliferation, differentiation, apoptosis and survival. AChE was also found to be highly
expressed in proliferating myoblast during muscle regeneration. This
process is always accompanied by cell apoptosis and therefore it was
hypothesized that the AChE expression in myoblasts reflected the
development of the apoptotic apparatus (Pegan et al., 2010). AChE
participates in apoptosis in two ways: by promoting or suppressing
cell death. Both direct and indirect evidence reported the involvement of AChE in regulation of cell proliferation and apoptosis
(Bytyqi et al., 2004; Jiang and Zhang, 2008; Kehat et al., 2007;
Robitzki et al., 1998; Soreq et al., 1994).
Regeneration of arms in cephalopods and in particular in Octopods
has been the subject of several studies due to their high regenerative
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S.M. Fossati et al. / Journal of Experimental Marine Biology and Ecology 447 (2013) 93–99
power (Callan, 1940; Dingerkus and Santoro, 1981; Feral, 1988;
Lange, 1920; May, 1933; Voight, 1992). However, the morphological
modification and molecular pathway of the regenerative process in
the octopus arm remain to be elucidated.
Detailed information on the proliferative activities of cells relevant
to events of regeneration is important to understand the mechanisms
underlying regeneration. In this study immunohistochemical staining
for proliferating cell nuclear antigen (PCNA) and argyrophilic
nucleolar organizer regions (AgNORs) have been utilized to detect
proliferating cells. Reported literature show that AgNOR expression
is strictly related to the cell duplication rate in both cancer (De
Vico et al., 1994, 1996; Madewell, 2001) and regenerative tissues
(Tuccari et al., 1999) and it is believed to be a useful method for examination of nucleolar structure and variations in nucleolar activity
(Egan and Crocker, 1992; Sirri et al., 2000). PCNA is a nuclear protein
which serves as a cofactor for DNA polymerase-delta. It is involved in
the coordination of cell cycle progression and DNA replication (Hall
et al., 1990; Jónsson et al., 1998).
Due to the octopus typical cholinergic innervation here we are
testing the hypothesis that AChE plays a major role in the arm regenerative process (Barlow, 1971; D'Este et al., 2008; Florey, 1963; Florey
and Winesdorfer, 1968; Loe and Florey, 1966; Messenger, 1996, 2001;
Rohrbach and Schmidtberg, 2006; Talesa et al., 1995; Welsch and
Dettbarn, 1972). The identification of differences in the activity and
localization of AChE activity during regeneration can provide useful
information on latent basic pathways which could be unlocked to
promote regeneration. In this context, AgNOR and PCNA can be helpful instruments to evaluate cell types involved in the regenerative
process.
2. Materials and methods
2.1. Animals and treatment
Specimens of Octopus vulgaris of both sexes were collected from
the Ligurian coast during the spring/summer period and placed in
80 × 50 × 45 cm marine aquaria. The tanks were filled with artificial
sea water (ASW) and kept at a temperature of 18 °C at 12 h light/
dark cycle. Water cleaning and oxygenation were assured by a
pump-filter and aeration system which continuously circulated the
water through biological filters. All relevant chemo/physical water
parameters were constantly checked to prevent the occurrence of
unhealthy or stressing conditions for the animals. Animals were left
to adapt to captivity for at least 10 days before experimentation.
The experimental animals were selected on the basis of the following
criteria: healthy shape (all the arms and body parts had to be intact,
the animal showed normal reflexes and voluntary movements such
as arm extension, walking, etc.), regular eating and motivation to attack a prey (for a description of the behavior of O. vulgaris in captivity
see for example: Boycott, 1954; Hochner, 2008).
Experimental animals (6 females) of around the same body
weight (500–800 g) were used. Animals were anaesthetized in 2%
ethanol in ASW (Andrews and Tansey, 1981; Boyle, 1981; Crook
and Walters, 2011; O'Dor et al., 1984) for about 5 min, until clear
change in body patterns confirmed that the cephalopod underwent
the physiological process of anesthesia. Small portions of the arm
tip (1–2 cm) were cut with fine scissors from all the arms (ranging
between 35 and 38 cm), in a transverse plane perpendicular to the
longitudinal axis of the arm and immediately fixed in 4% formaldehyde in ASW. Following surgery, each animal was placed in the
experimental tank where it slowly (about 2–5 min) recovered from
anesthesia. The animal did not display behavioral modification after
the operation, and in all the animals the amputated arms regenerated
as in the natural environment (Crook and Walters, 2011). Arm samples from intact arms were used both for anatomical description of
non-regenerating arms and as controls for the detection of specific
markers involved in the regeneration process. The regenerating arm
tips (which include the entire regenerating arm portion plus a small
piece of the intact arm) were collected at different days of regeneration and further processed for microscopical investigations. At the
Day 134 all the animals were sacrificed by immersion in 2% ethanol
until respiration ceased and the heart stopped pumping. We focused
on 3, 11, 17, 21, 28, 42 and 55, 108 and 134 days of regeneration
when, substantial changes in the arm morphology could be observed
(Lange, 1920).
2.2. Histology and immunohistochemistry
Three arms from three different animals for each regenerating day
(3–55) were employed. Samples were fixed for 4 h in 4% formaldehyde in ASW at 4 °C and then embedded either in paraffin wax or
OCT compound, serially sectioned at 5–10 μm and collected
on Superfrost slides (Bio-Optica). For basic anatomical description
longitudinal sections were processed using a common hematoxylin
and eosin staining to characterize the morphology of normal and
regenerating tissue. Sections were examined and photographed
with bright field microscopy (Nikon eclipse 80i upright microscope).
Image acquisitions and analysis were performed with the Nis
Elements (Nikon) and with ImageJ software. All the quantification
analyses were performed by two people “blinded” to the stage of
regeneration at which samples were taken.
2.2.1. AgNOR and PCNA assay
Longitudinal sections of control and regenerated arms from 3 animals
were serially sectioned (5 μm) from the paraffin wax block, dewaxed in
several baths of xylene and hydrated through graded alcohols to
ultrapure water. Sections were processed for AgNOR (Ploton et al.,
1986) and PCNA (proliferating cell nuclear antigen) evaluation.
Microscopical assessment of sections treated for both AgNORs and
PCNA was performed at about 100 μm from the base of the regrowth
(proximal part) and at about 100 μm from the arm tip (distal part).
The specific activity of muscular cells was also evaluated. The number
of dots of AgNORs (Days 3–55) per nucleus was assessed in all the
exanimated animals on one focal plane with a 100× objective lens in at
least 100 nuclei per specimen, as recommended by Crocker et al.
(1989). The enumeration of each silver stained dot per cell counted at
the microscope was performed carefully focusing through the section
thickness at very high magnification (100×). On each slide, two operators independently defined the mean AgNOR number. According to the
recommendations of Crocker et al. (1989) and De Vico et al. (1994),
when a dot aggregation could not be resolved in individual NORs by
focusing, the cluster was considered as one discrete AgNOR.
PCNA positive nuclei were scored assessing PCNA positivity per
100 cells (PCNA Index). Values were expressed as Mean ± SD.
2.2.1.1. AgNOR. The staining solution was obtained instantly by rapidly
mixing one part of solution A (2% gelatine solution dissolved in
ultrapure water, to which formic acid is then added to make a final
1% solution) with two parts of solution B (50% silver nitrate solution
in ultrapure water). Re-hydrated sections were immersed in 0.01 M
sodium-citrate monohydrate buffer pH 6.0 and autoclaved at 120 °C
for 20 min. Subsequently the sections were allowed to cool down to
room temperature and then treated with silver staining for 25 min
at room temperature in a dark place.
2.2.1.2. PCNA. After dewaxing of the tissue sections, endogenous peroxidase was blocked by incubation in H2O2–methanol (4:1) for
20 min. Sections were hydrated and incubated with sodium citrate
buffer (pH 6.0) in a microwave for 5 min at 575 W, in order to unmask the antigens and epitopes. Subsequently the sections were
allowed to cool down to room temperature and washed several
times in PBS. Sections were then treated with 10% goat serum,
S.M. Fossati et al. / Journal of Experimental Marine Biology and Ecology 447 (2013) 93–99
95
permeabilized with PBS-T/BSA 1% and incubated overnight at 4 °C
with the primary antibody (mouse monoclonal [PC10] to PCNA,
Sigma). Sections were rinsed in PBS-T and treated with the corresponding biotinylated secondary antibody for 20–30 min at room
temperature. Sections were then rinsed in PBS-T and the detection
was performed via DAB (Dako, Italy).The sections were counterstained with hematoxylin for 5 min at room temperature, dehydrated
through graded alcohols and mounted for microscopical examination.
2.2.2. Enzymatic assay
Samples from 3 animals were fixed for 4 h in 4% formaldehyde in
ASW at 4 °C, embedded in paraffin wax, serially sectioned at 10 μm
and collected on Superfrost slides (Bio-Optica). Sections were hydrated in PBS for 5 min at room temperature and then incubated in a solution containing 1 mg/ml of acetylthiocholine iodide (Sigma), 0.1 M
NaH2P04, 0.1 M Na-citrate, 30 mM CUS04, 5 mM K-ferricyanide and
distilled water for 4 hr at 4 °C. Three different concentrations of substrate (1, 2 and 5 mg/ml of acetylthiocholine iodide) were tested in
order to get the concentration at which the activity of the enzyme
AChE was revealed as deep brown precipitates against a white background. All three concentrations produced a color of the same intensity and localized at the site of the enzymatic activity. Therefore
we decided to perform all the enzymatic assays using 1 mg/ml of
substrate (Karnovsky and Roots, 1964, modified). Slides were then
Fig. 2. Relationship between days of regeneration and length of the regenerating arm
portion used for AChE assay.
mounted for microscopical examination. The presence of the enzyme
was revealed as deep brown precipitates.
The evaluation of the AChE activity was performed using ImageJ
software. A specific region of interest (ROI) was selected and measurements of volumes (calculated by multiplying the area of each section
Fig. 1. Macro-microscopical observation of regenerating arms. A: Macro images, control, Days 3–55. Scale bar control: 300 µm; Day 3: 400 µm; Day 17: 1 mm; Day 21: 1 mm; Day 28:
300 µm; Day 55: 300 µm. B: Longitudinal section in control arm: note at tip level a well-developed nervous system. Scale bar: 50 µm C: Longitudinal section in arm at Day 17: ‘Blastema’
appearance (arrowheads) accompanied by intense vascular component (V). Scale bar: 50 µm Ep: epithelia; De: derma, NS: nervous system. Hematoxylin and eosin staining.
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S.M. Fossati et al. / Journal of Experimental Marine Biology and Ecology 447 (2013) 93–99
Fig. 3. Acetylcholinesterase assay. A: Control arm after reaction for AChE detection; B–H: Regenerating arms after reaction for AChE detection (B: Day 11; C: Day 17; D: Day 20; E: Day 28;
F: Day 42; G: Day 108; H: Day 134). Scale bar: 500 µm. Note that pictures F, G and H report only a small portion of the regenerated arm (about 3 mm). MS: muscle system; NS: nervous
system; S: sucker.
for its thickness) and the average intensity of AChE were collected from
all sections (100 sections per sample) from both control and regenerating
arms. It is important to point out that in control arms the ROI correspond
to the entire arm, while in regenerating samples only the regenerated tip
was analyzed. The average intensity was measured as the sum of the intensity values of all the pixels within the ROI divided by the number of
pixels. Measure of the intensity over the total volume (intensity mean
value) is reported in order to get an average AChE intensity respectively
for control and regenerating arms at different days of regeneration. AChE
activity was calculated as the ratio between activity in regenerating and
control arms at each day of regeneration.
3. Results
3.1. Macroscopical observation
Macroscopically, in regenerating arms 3 days after injury, a little
knob was evident at the cutting point. At Day 11 we could observe a
protrusion and later a hook-like structure appeared (Day 17). At
Day 55 a complete structure was visible with the restoration of all
the missing components (Fig. 1A). From histological analysis at
knob level a very thin layer of undifferentiated cells was evident.
Subsequently (Day 11) a typical morphological feature of ‘blastema’
at the arm tip was visible together with a semi-circular structure of
mesenchymal cells accompanied by an intense vascular component
(Fig. 1B, C). This configuration disappeared at Day 28.
3.2. Acetylcholinesterase detection
In normal non-regenerating tissue AChE activity was located in
the axial nerve cord (cell layer and neuropil) and restricted to nerve
fibers in the intrinsic muscles of the arm and in the sucker nerves.
In regenerating arms AChE activity and localization changed together with the modification of the tissue structures (Figs. 2, 3, 4A).
During the first days after injury when the arm goes into the wound
healing process the activity of AChE dropped down the basal level
Fig. 4. Evaluation of acetylcholinesterase and AgNOR during regeneration. A: AChE detection; a specific region of interest (ROI) was selected and measurements of volumes and the
average intensity of AChE were collected. AChE activity is reported as the ratio between activity in regenerating arm and control arm (see inset) at each stage and is plotted against
the corresponding day of regeneration (N = 3 animals). Measurements of the intensity over the total volume (intensity mean value) are reported as average AChE intensity, respectively for control and regenerating arms at different days of regeneration. B: AgNOR detection: Mean values of AgNOR are reported in relation to the days of regeneration. Solid line:
Counting of AgNOR proximal (basal) to the regenerating region, dashed line: counting of AgNOR distal (apical) to the regeneration region.
S.M. Fossati et al. / Journal of Experimental Marine Biology and Ecology 447 (2013) 93–99
(Days 3–17; Figs. 3B, C, 4A). In this period the first sign of regeneration is represented by a little knob on the arm edge which later will
develop into a small process (a hook like structure). At this stage
the AChE enzymatic activity became one third of the initial value
and was restricted to the axial nerve cord.
After three/four weeks from operation (Days 20–28) the AChE
activity rose above basal level and was localized into the axial nerve
cord, in the nerve fibers of the intrinsic arm muscles and into nerves
of the suckers (Fig. 3D, E). The morphological changes were mostly
related to the proximal arm region which represented the area of
active regeneration of missing structures. At this stage the muscles
and the nervous system regenerated, and both the suckers and the
first chromatophores appeared.
At Day 42 the activity of AChE began to decrease (Figs. 3F, 4A).
Around this stage all the tissue components of the new arm were
visible and the enzymatic activity was distributed in nerve cells,
nerve fibers and neuromuscular junctions in the arm as observed in
the intact arm. At Day 130 the AChE activity was almost back to
basal level and the overall morphology of the regenerating arm was
restored (Figs. 2, 3G, H, 4A).
3.3. Histological and immunohistochemical assay
On the histological sections stained for the AgNORs, well-defined
dark-brown to black silver-stained dots were observed in all nuclei.
AgNORs were distributed as one or more cluster or occurred as an
individual large dot at neuron level (Fig. 5A, B). In regenerating
arms, interestingly, AgNOR showed a parallel increase in number in
97
Table 1
AgNORs (no/cell) and PCNA (Index%) evaluation in controls and regenerating arms as
N and ±SD.
Sample
Control
Day 3
Day 11
Day 17
Day 21
Day 28
Day 43
Day 55
Proximal
Distal
Proximal
Distal
Proximal
Distal
Proximal
Distal
Proximal
Distal
Proximal
Distal
Proximal
Distal
Proximal
Distal
AgNOR (no/cell)
AgNOR muscle
PCNA Index %
0.92 ± 0.01
1.02 ± 0.2
3.02 ± 0.01
3.21 ± 0.52
2.85 ± 0.75
3.12 ± 1.76
1.32 ± 0.51
1.46 ± 0.64
2.06 ± 0.62
1.53 ± 0.58
2.08 ± 0.81
1.53 ± 0.58
1.81 ± 0.72
2.12 ± 1.09
2.01 ± 0.22
1.87 ± 1.07
1.12 ± 0.01
0
0
75
100
70
65
35
90
55
27
64
32
25
100
22
19
2.06 ± 0.2
2.25 ± 1.32
1.65 ± 2.03
1.81 ± 0.72
2.44 ± 1.23
2.15 ± 2.21
1.91 ± 0.89
both proximal and distal region during the first two weeks of regeneration (Days 3–11). Subsequently, an inverse trend with an increase
cell activity first at proximal and subsequently at the distal arm region
was recorded (Table 1; Fig. 4B). This examination also showed that
muscular components were highly active in the regenerative process,
with AgNORs maximum number of 2.25 ± 1.32 at Day 11 (Table 1).
During Days 3, 17 and 43 PCNA showed a similar trend with a 100%
of cells in S phase localized in the blastema (Table 1; Fig. 5C, D).
Fig. 5. AgNORs and PCNA detection. A: AgNOR detail at ‘blastema’ level (Day 11), note that more than one dot per cell is visible; scale bar: 100 μm. B: AgNORs in the nervous and
muscular tissue at Day 28; note that single dots per cell are visible. Scale bar: 100 μm. C–D: PCNA at proximal (C) and distal (D) region (blastema—arrowheads); scale bar: 100 μm.
V: vascular component, NS: nervous system, Ne: neuron, M: muscle.
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S.M. Fossati et al. / Journal of Experimental Marine Biology and Ecology 447 (2013) 93–99
4. Discussion
Acknowledgments
This study represents the first identification of pattern and distribution of proliferating cell types together with the evaluation of the AChE
activity during the arm morphogenesis.
Emerging evidences are now pointing to non-cholinergic functions of
AChE isoforms. These isoforms are important contributors in various cell
types to the regulation of cell proliferation, differentiation and apoptosis.
The process of tissue regeneration is always accompanied by cell
apoptosis and AChE has been shown to contribute to apoptosis by
promoting or suppressing cell death (Jiang and Zhang, 2008; Xie et al.,
2011). The mechanisms underlying the involvement of AChE in the cell
cycle are not fully understood. A model where an enhanced AChE variant
expression influences the expression of other group genes, including
those involved in apoptosis has been proposed. In this model the level
of cellular stress response would play an important role in determining
the proportion of the variants changes (Ben Ari et al., 2006). Another
model proposes the formation of a 55 kDa AChE protein from the cleavage of 68 kDa AChE during apoptosis and the level of the cleaved protein
positively correlated with cellular apoptotic levels (Xie et al., 2011). Also
in a recent study Pegan and collaborators found AChE to be highly
expressed in proliferating myoblast during muscle regeneration. Given
that this process is always accompanied by cell apoptosis it was hypothesized that the AChE expression in myoblasts reflected the development
of the apoptotic apparatus (Pegan et al., 2010).
Cell activity during regeneration was here estimated with AgNOR
protein quantity assay and PCNA immunoreaction. PCNA is associated
with cell duplication and thus represents a valuable parameter of cell
kinetics (Derenzini et al., 1990, 1995; Ofner et al., 1992). It has been
shown that in proliferating cells the number of AgNORs in the
interphase progressively increases from early G1 phase, reaches a
maximum value at the end of S-phase and remains constant up to
the late G2 phase, parameter usually used to evaluate cancer cell
activity (Derenzini and Ploton, 1991).
We showed that in the very early stages of regrowth (Days 3–17)
when the process of wound healing occurs most of the proliferation
activity, estimated with AgNOR and PCNA, was found at the level of
the ‘blastema’ a region distal to the wound tissues formed by the
accumulation of mesenchymal cells. At this stage the blastema region
is relatively devoid of the AChE enzyme and the cholinesterase activity was very low compared to the basal level of non-regenerating tissue. During the subsequent phase (Day 20) characterized by process
of cellular multiplication a significant cell proliferation was found at
the level of mesenchymal tissue at the arm tip (distal to the wound)
and in some cases in the region proximal to the wound as demonstrated by the positive PCNA reaction and by the AgNOR assay. In
this stage AChE activity climbed up slowly. At about the time of
onset of morphogenesis (Day 28) a significantly increased number
of AgNORs in the muscle elements in comparison to control arms
was observed and active myogenesis was observed. At this stage the
AChE activity rises sharply. At around Days 42–134 when the process
of histogenesis occurred and the reestablishment of all the structures
became evident the process of cell proliferation was still active mostly
at the arm tip and nervous cells were clearly discernible. At this stage
the AChE activity decreased till reaching basal level.
Here we showed that the level of AChE in the arm tissue varies
during regeneration and it is strictly related to the proliferation
stage of the regenerative process. Interestingly during the stages
when the morphogenesis is mostly dependent upon cell proliferation
AgNOR and AChE assay showed a similar trend (Days 11–30; compare
Fig. 4A and B). We suggest that AChE protein may have an important
influence in the process of arm regeneration and that it could be considered as a potential target to promote or regulate the regenerative
process. By targeting the AChE activity at single specific regeneration
stage it will be possible to study the regenerative process in its
proceeding and regulate single phases of the reparative pathway.
The authors want to thank Dr. Grazia Villari for the technical assistance. This work was supported in part by the EU-FP7 OCTOPUS project
n.: 231608. [SS]
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