I\'ve never had to interpret electrophoporation graphs and gels before and I was
ID: 151015 • Letter: I
Question
I've never had to interpret electrophoporation graphs and gels before and I was wondering if someone could help me understand the following results from a paper.This is the actal papers link: https://academic.oup.com/hmg/article/23/1/69/722169
and these are the graphs I need to be able to explain:
The caption under it is this : Figure 3 Inhibition of caspase-3 and -9 activities increases AChR density and improves ultrastructural changes of NMJ in mSCS. (A) Left: neuromuscular junctions of WT, mSCS and mSCS treated with API4, or C9DN immunolabeled with neurofilament-specific antibody (green) and TxR-BT (red) in WT, mSCS and treated mSCS muscle. Scale bar = 25 µm; n = 5. Scale bar = 25 µm; n = 5. Middle: images from A showing only AChR labeling with TxR-BT for AChR density area measurements. Right: upper: AChR density in WT, mSCS and treated mSCS (# = normalize to WT). Electroporation of AIP4 and C9DN to mSCS muscle increased AChR density to 0.78 ± 0.05 and 0.85 ± 0.06 of WT, respectively, whereas 0.49 ± 0.02 in mSCS and 0.51 ± 0.03 in mSCS control (SCS-pcDNA3; **P < 0.01, Mann–Whitney U-test). Lower: endplate area (m2) in WT, mSCS and treated mSCS. Endplate area was 598.36 ± 74.13 in WT, 460.09 ± 48.95 in SCS-API4 and 429.11.36 ± 79.22 in mSCS-C9DN, compared with 259.55 ± 46.11 in mSCS and 263.16 ± 61.23 in mSCS control (SCS-pcDNA3). n = 5; ***P < 0.001, Student's t-test. Number of end plates = 50/sample. (B) Ultrastructure of NMJs from WT, untreated control mSCS and treated mSCS muscle. NMJs of mSCS muscles exhibited ultrastructural changes as previously described (left). Degenerative changes in postsynaptic nuclei were improved in the treated mSCS muscles. The number of degenerative nuclei with apoptosis-like changes was quantified (right). 20.18% (23 of 114) of junctional nuclei in mSCS muscles showed degenerative changes (left, bottom row). WT muscle with normal subsynaptic nucleus is shown for comparison. API4-treated mSCS muscle showed improvement in ultrastructural abnormalities with a significant decrease in the number of degenerative nuclei to 2.27% (1 of 44). A similar trend was seen in the C9DN-treated mSCS animals with 9.09% degenerative nuclei (3 of 33). This trend did not reach statistical significance. The size bars correspond to 2 µm. The P-values for the comparison between control mSCS muscles and API4-treated mSCS muscles or between control muscles and C9DN-treated mSCS muscles, respectively, were calculated by 2 testing. *P < 0.005; xP < 0.14.
mSCS WT mSCS mSCS-C9DN 0.5 mSCS-C9DN mSCS AP14 WT mSCS mScs m3cS- mScS mscs
Explanation / Answer
The mentioned paper is aimed on studying the effect of caspase inhibition for the cure of slow channel syndrome (SCS). SCS is a condition in which acetyl choline (ACh) receptors have mutation because of which there is delay in closure of ACh mediated channels resulting in over load of calcium ions and hence damage to post synaptic region. This damage is caused by activation of caspases which are part of apoptosis.
The authors have tried inhibiting caspases so as to nullify their effect by electroporating the plasmids containing C9DN and API 4 genes and hence the effect of SCS in the diseased.
The Figure indicated in the question is the result in the form of size of end plate in neuromuscular junction, authors got by the effect of electroprated plasmids.
The figures shows the following results
Part A
It shows ulrastructures of Neuromuscular junction which is immunolabelled with . In first image there is wild type specimen ( i.e. without disease) followed by diseased untreated specimen mSCS) then those with C9DN and API4 treated specimens. The labelled area is more in treated ones in comparison to untreated which indicates that the introduced gene has inhibited the activity of caspases and hence the end plate size in diseased but treated ones are more than untreated ones and hence the effect of caspases has been nullified to some extent. The quantitative increase has been shown in the bar graph adjoining the images.
The specimen with plasmid containing C9DN and API4 genes were found to be twice and 1.8 times larger than untreated ones respectively. Both were however a bit smaller than that of wild type.
This indicated that the presence of these genes have decreased the activity of caspases.
Part B indicated the ultrastructures of neuromuscular junctions showing degenerated nuclei of in all four types. Those treated with plasmids had very less degenerated nuclei in comparison to untreated ones. Same has been indicated in the bar graph.
This observation confirmed that the inhibition of caspases has been braught about by these two plasmids as the cell death percentage has been found to decrease.
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