För första ? gången har man använt HoloMonitor i syfte att förstå uppkomst av och senare bota infektioner i djurbesättningar, i detta fallet grisar som drabbas av PEDV (porcine epidemic diarrhea virus).
" Severe outbreaks of PEDV infections were reported in China in 2010 and in North America in 2013, leading to high mortality among infected piglets and huge economic losses."
Publikationen återfinns i senaste upplagan av det vetenskapliga organet cells.
Lack of LTβR Increases Susceptibility of IPEC-J2 Cells to Porcine Epidemic Diarrhea Virus
Received: 26 September 2018 / Accepted: 13 November 2018 / Published: 21 November 2018
Ur studien klipper jag in valda delar.
Abstract:
The essential requirement of the lymphotoxin
beta receptor (LTβR) in the development and maintenance of peripheral
lymphoid organs is well recognized. Evidence shows that LTβR is involved
in various cellular processes; however, whether it plays a role in
maintaining the cellular function of intestinal porcine enterocytes
(IPEC-J2), specifically during porcine epidemic diarrhea virus (PEDV)
infection, remains unknown. In this study, we generated LTβR null
IPEC-J2 cells using CRISPR/Cas9 to examine the importance of LTβR in
cell proliferation, apoptosis, and the response to PEDV infection. Our
results showed that the lack of LTβR leads to significantly decreased
cell proliferation, potentially due to S phase arrest in LTβR−/−
IPEC-J2 cells.
Label-free digital holographic microscopy was used to
record the three-dimensional morphology of both cell types for up to 72
hours and revealed significantly increased numbers of LTβR−/− cells undergoing apoptosis. Furthermore, we found that PEDV-infected LTβR−/−
null IPEC-J2 cells exhibited significant suppression of nuclear factor
kappa-light-chain-enhancer of activated B cells (NFκB) target genes
(interleukin (IL)-6 and IL-8) and mucosal barrier integrity-related
genes (vascular cell adhesion molecule 1 (VCAM1) and IL-22), which may
explain why LTβR−/− cells are more susceptible to PEDV
infection.
Collectively, our data not only demonstrate the key role of
LTβR in intestinal porcine enterocytes, but also provide data for the
improved understanding of the cellular response to PEDV infection.
Materials and Methods
2.8. Digital HoloMonitor Microscopy
HoloMonitor
M4 Microscopy (Phase Holographic Imaging AB, Lund, Sweden) is an
imaging time-lapse cytometer based on holographic microscopy, providing
imaging and quantification of unstained living cells directly in their
culture vessels.
LTβR+/+ and LTβR−/− cells were
seeded in 6-well plates and monitored for 72 h.
Apoptosis was analyzed
by Hstudio M4 Tracking software (Scheelevägen, Sweden).
3.4. LTβR Knockout Induces IPEC-J2 Cell Apoptosis
Digital
holographic microscopy offers an advantage in studying real-time
observations of critical events by exhibiting a continuous
two-dimensional (2D) and 3D visual picture of cellular activity in
second intervals.
A large portfolio of quantitative morphological parameters, including optical cell volume, thickness, area, irregularity, eccentricity, and single-cell tracking, can be recorded and analyzed.
A large portfolio of quantitative morphological parameters, including optical cell volume, thickness, area, irregularity, eccentricity, and single-cell tracking, can be recorded and analyzed.
Here, digital holographic microscopy was used to monitor dynamic activities and morphological changes of LTβR+/+ and LTβR−/−
cells in real-time for up to 72 hours.
The results showed that these
cells displayed distinct growth characteristics. Specifically, many more
LTβR null cells than LTβR+/+ cells exhibited increased cell volume (vertical axis) and decreased cell membrane thickness (horizontal axis) (Figure 4A, cells between the red lines). Figure 4B shows the 3D structures of observed living LTβR+/+ cells (Figure 4B, left) and LTβR−/− cells (Figure 4B, right). Clearly, LTβR−/−
cells with a white color are apoptotic because the flow of liquids
through apoptotic cell membranes (permeability malfunction) leads to
increased cell volume, and the lightened cell membranes therefore
reflect different colors.
Figure 4.
The effect of LTβR on IPEC-J2 apoptosis. LTβR+/+ and LTβR−/− cells were seeded into 6-well plates, and apoptosis was monitored for 72 h by HoloMonitor®M4 microscopy. (A) Dot plot of LTβR+/+ and LTβR−/−
IPEC-J2 cells after 72 h culture. The x-axis is the average thickness
(µM) of the cells, and the y-axis presents the surface area of the cells
(µM2). Apoptotic cells with lower thickness and higher
surface area are between the red lines. Note there are almost no
apoptotic cells between the red lines for the LTβR+/+ group (left), while significantly more apoptotic cells are found in the LTβR−/− cell population (right). (B) Three-dimensional structures of living LTβR+/+ (left) and LTβR−/− (right) cells after 72 h. The number of tall cells is noticeably increased in LTβR−/− cells. (C) Real-time PCR data of TNF superfamily member 10 (TNFSF10) and Caspase 3 (CASP3) in LTβR+/+ and LTβR−/− cells, * p < 0.05 and ** p < 0.01.
Min kommentar
Med denna studie visar kinesiska forskare med inriktning veterinär (forskning OM djur) sina kollegor världen över att det finns ett nytt användbart instrument som absolut går att applicera i djurforskningen.
Notera forskning OM djur, inte djurförsök.
De 7 kinesiska forskarna utgår från följande institut:
- Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China
- Department of Animal Science, Chinese Agricultural University, Beijing 100193, China
- State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
Behöver jag säga att användarmarknaden än en gång utökas?
Behöver jag säga att idag befintliga instrumenttillverkare riktade åt veterinärmarknaden nu kan räknas in bland kommande förvärvsintresserade bjässar?
Eller behöver jag säga att PHI har en ZooloMonitor M4 att kanske presentera för denna nya marknad?
Nix,det förstår man säkert själv.
Behöver jag säga att dagens aktiekurs är helt uppåt väggarna?
Ja,det behöver jag däremot säga för aktiemarknaden har helt missat vilken potential PHI sitter på.
Mvh the99
Tack för all info du gräver fram
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