Toxins
Man beskriver sin gärning enligt följande :
Toxins (ISSN 2072-6651; CODEN: TOXIB7) is an international peer-reviewed
open access journal which provides an advanced forum for studies
related to toxinology and all kinds of toxins (biotoxins) from animals,
microbes and plants. Toxins is published monthly online by MDPI.
The French Society on Toxinology and International Society for Mycotoxicology are affiliated with Toxins and their members receive a discount on the article processing charges.
Impact Factor:
3.273 (2017)
; 5-Year Impact Factor:
3.551 (2017)- Open Access - free for readers, with article processing charges (APC) paid by authors or their institutions.
- High visibility: indexed by the Science Citation Index Expanded (Web of Science), MEDLINE (PubMed) and other databases. Full-text available in PubMed Central.
- Rapid publication: manuscripts are peer-reviewed and a first decision provided to authors approximately 15.2 days after submission; acceptance to publication is undertaken in 4.8 days (median values for papers published in this journal in the second half of 2018).
- Recognition of reviewers: reviewers who provide timely, thorough peer-review reports receive vouchers entitling them to a discount on the APC of their next publication in any MDPI journal, in appreciation of the work done.
- Sections: published in six topical sections.
Till själva studien då benämnd :
Whole-Cell Multiparameter Assay for Ricin and Abrin Activity-Based Digital Holographic Microscopy
Received: 6 February 2019 / Accepted: 15 March 2019 / Published: 22 March 2019
Abstract
Ricin and abrin are ribosome-inactivating proteins
leading to inhibition of protein synthesis and cell death. These toxins
are considered some of the most potent and lethal toxins against which
there is no available antidote. Digital holographic microscopy (DHM) is a
time-lapse, label-free, and noninvasive imaging technique that can
provide phase information on morphological features of cells. In this
study, we employed DHM to evaluate the morphological changes of cell
lines during ricin and abrin intoxication. We showed that the effect of
these toxins is characterized by a decrease in cell confluence and
changes in morphological parameters such as cell area, perimeter,
irregularity, and roughness. In addition, changes in optical parameters
such as phase-shift, optical thickness, and effective-calculated volume
were observed. These effects were completely inhibited by specific
neutralizing antibodies. An enhanced intoxication effect was observed
for preadherent compared to adherent cells, as was detected in early
morphology changes and confirmed by annexin V/propidium iodide (PI)
apoptosis assay. Detection of the dynamic changes in cell morphology at
initial stages of cell intoxication by DHM emphasizes the highly
sensitive and rapid nature of this method, allowing the early detection
of active toxins.
Ur studien klipper jag in det som är PHI-relevant.
Digital holographic microscopy was performed using a Holomonitor M4
microscope (Phase Holographic Imaging AB, Lund, Sweden).
The Holomonitor
M4 is a label-free cell analyzer for adherent cells equipped with X20
magnification objective (NA-0.4), a 0.8 mW HeNe laser (633 nm, 100 µW/cm2,
exposure time 5 ms), and a motorized stage.
Digital holographic
microscopy, as a quantitative phase-contrast imaging method, is a form
of optical interferometry, which detects the phase delay related to the
light passing through the tested object.
Images were converted from
wavelength interaction to cells’ representation by a computer algorithm
(Hstudio 2.6, Phase Holographic Imaging AB, Lund, Sweden). This
computation enables to obtain information on various morphological and
optical parameters.
Two different threshold settings prior to
segmentation of cells were used for analyzing the experiments: Otsu
thresholding (OT) [56] and minimum error thresholding (MET) [57].
While the former results in a more accurate identification of cells, by
excluding focus debris, MET gave a more accurate cell outline. In our
study, the same trend was observed using the two threshold settings and
the DHM results presented in this study were analyzed according to MET
threshold. In general, cell lines (1–4 × 105) were seeded in
6-well plates at different degrees of confluence (10–30%) with or
without incubation for 4 to 6 h for cell adhesion. Following incubation,
cells were exposed to ricin or abrin at 1–100 ng/mL in a 3 mL volume.
The neutralizing effect of anti-ricin and anti-abrin antibodies was
assessed by incubating toxins with 20 μg MH1 or MH75 monoclonal
antibodies or 1:100 dilution of polyclonal antibodies. Cells were
monitored continuously, in time-lapse mode (every 10 min), in multiple
locations in each well using a high precision motorized stage.
Quantified parameters obtained from DHM in each frame were analyzed as
changes relative to time zero from each area and presented as mean ± SE
for all the monitored areas in each well. In this work we focused on
significant changes observed during intoxication in several parameters:
phase-shift (phase delay related to the light passing through the tested
object), confluence, area, irregularity (determines the deviation of
the cell region from the circular shape), perimeter, and roughness
(indication on the smoothness of the surface of the cell). As the
refractive index of the cell and the surrounding medium cannot be
measured, the presented cell thickness and volume values are expressed
as the optical thickness and effective-calculated volume (ECV).
Digital holographic microscopy (DHM) is a
time-lapse, label-free, and noninvasive imaging technique that can
provide both quantitative and qualitative phase information on
morphological features such as cellular area, shape, thickness, volume
and confluence.
This method does not require any media changes or addition of dyes,
simplifying high throughput screening. Thus, this technology is suitable
for sensitive measurements of various cellular events such as live cell
imaging, cell migration, proliferation, differentiation, and death.
Identifying and measuring specific morphological features by DHM during
early stages of exposure to toxic agents such as ricin and abrin, could
allow early cell death detection, thereby determining toxin activity.
As a label-free technique, DHM does not require pipetting steps which
can affect the cell and even induce strong bias as some detached dead
cell can be washed out.
We therefore set out to develop an activity
assay for ricin and abrin based on DHM evaluation of cells following
intoxication.
In this study, we present the
feasibility of a cell-based assay for ricin and abrin intoxication using
DHM.
In contrast to current methodologies, DHM is noninvasive, rapid
and able to monitor changes in cell morphology dynamically, allowing
early detection of low dose intoxication. The method described herein
may be applied for early and sensitive detection of these as well as
other toxins and substances.
2. Results
2.1. Morphological Changes in Cell Lines Observed by DHM Following Ricin Exposure
In
order to characterize morphological changes mediated by ricin exposure,
HeLa cells were seeded in six-well plates at different confluence
levels (10–30%) and incubated for 4–6 h for cell adhesion. Following
incubation, cells were exposed to ricin at 100 ng/mL and digital
holograms of four different areas in each well were recorded at 10 min
time intervals for 20 h.
The resulting digital
holographic images were used to measure and define changes in various
morphological and optical features of treated versus untreated cells.
Images of treated cells depicted in Figure 1A
show a representative 3D holographic images of HeLa cells treated with
100 ng/mL ricin. A visual inspection of the images shows a decrease in
cell numbers and confluence within 7 to 11 h and increased cell
thickness and roundness within 11 h post-ricin exposure compared to
untreated cells. Increased cell thickness and roundness following ricin
exposure, indicates that the cells undergo apoptosis or detachment from
the plate. These differences between treated and untreated cells were
quantified by the DHM. Results (Figure 1B)
show that significant changes occur within 3 to 6 h post-ricin exposure
in confluence, irregularity, and optical cell thickness. Moreover, DHM
enables the monitoring of additional parameters including phase-shift,
cell area, perimeter, roughness, and effective-calculated volume (ECV)
which could not be observed visually. In addition, the relative changes
in cell counts measured by DHM support the visual inspection indicating a
marked decrease in cell divisions (Figure S1). In general, significant changes were observed within 4–7 h post-ricin intoxication.
 |
| Figure 1. Morphology features of ricin intoxication detected by digital holographic microscopy (DHM). Effect of ricin treatment on HeLa cells was tested using DHM. Cells were subjected to ricin (100 ng/mL), and digital holograms of four different areas in each well were recorded for 20 h at 10 min intervals. Untreated cells were used as a control. (A) Representative three-dimensional Images of treated and untreated cells as captured at 0, 3, 7, 11, and 17 h. Marker for optical thickness depicted on the right side. (B) Quantification of the relative changes in various morphological parameters was performed in parallel in the presence of neutralizing antibodies MH1 (monoclonal anti-subunit A), MH75 (monoclonal anti-subunit B), and polyclonal antibodies. * indicate p < 0.05 of intoxicated vs. untreated cells according to 2-tailed Student’s t-test. |
Early Morphological Changes Detected by DHM Are Attributed to Live Cells
Thus
far, we demonstrated that DHM allows for morphological analysis that
can identify significant changes induced by ricin/abrin intoxication by
analyzing the entire field of view. The cell populations analyzed are
comprised of cells undergoing all stages of ricin intoxication,
including early and late apoptotic cells. As a consequence, each
parameter is an average of a heterogenic cell population. Using the
annexin V and PI apoptotic assay we could evaluate the early and late
apoptotic cell populations reaches ~15% within 8 h post 100 ng/mL ricin
exposure (Figure 4A and Figure S2A).
The apoptotic cells, exhibiting annexin V+/PI+ double staining, reflect
late apoptotic stages. These cells show also significant changes in
size and granularity detected by the forward and side scatter (FSC/SSC)
parameters (Figure S2B).
In an attempt to focus on the morphological changes of the live cell
fraction (undergoing early intoxication stages), we analyzed individual
cells in each frame by using the ’tracking cells’ application. This
allows the exclusion of presumed dead cells defined by parameters such
as roundness and thickness (Figure 4B). We presume that these cells are the late apoptotic cells defined by flow cytometry in Figure 4A and Figure S2.
Next, we analyzed the remaining cell population (live cells and early
apoptotic cells) for morphological changes. Analysis of these cells
during the first 11 h post-ricin exposure shows the same trend in
morphological parameters as previously described, starting 5 h and
increasing with time up to 11 h after intoxication (Figure 4C).
These results show that morphological changes after intoxication are
manifested also by cells in early stages of intoxication and are not
fully related or biased by the late stage of intoxicated cells.
Interestingly, the roughness feature was unaffected in treated tracked
cells, suggesting roughness is in fact a specific indicator of late
apoptotic outcome. Detection of morphological changes in live cells upon
early stages of intoxication emphasizes the high sensitivity of this
method for assessing toxin activity.
 |
| Figure 4. Intoxication evaluation of live cells by DHM. HeLa cells were exposed to ricin 10–100 ng/mL. (A) Percentage of apoptotic cells (mean ± SE) during 8 h of ricin intoxication was determined using annexin V and PI apoptosis assays. (B) Live cell segmentation was performed using DHM cell tracking application according to roundness and thickness parameters in each frame. In each frame, 50–60 cells were tracked and analyzed. Treated and untreated tracked live cells were analyzed for morphological changes during 11 h post-ricin exposure (100 ng/mL). Shown are representative results out of three independent experiments (mean of 4 different areas in each well ± SE). (C) A representative DHM 2D image for presumed live cell segmentation analysis (cell margins are highlighted by colored lines). |
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