Nchow-pcoleman: "We are here - Chow and Coleman"
Peternelson: "Hi there. I'm Pete Nelson. I guess most people haven't arrived yet. Maybe we could wait a minute or two before intros and discussion begins."
Nchow-pcoleman: "Fine by us. BTW Pete, we just e-mailed our responses to your comments to June K. She suggested we do it this way, so she could append them to
your comments. If you wish to discuss any of the points you raised on-line here, let's do so."
Peternelson: "Maybe in around two minutes we should start, okay?"
Nchow-pcoleman: "OK. Questions? Points? Answers?"
Peternelson: "Perhaps we should begin with some introductions. My name is Peter Nelson. I'm a MD/PhD finishing my MD at U.Chicago. I'm interested in AD pathobiology."
Kieran_breen: "Hi, I'm Kieran Breen, a lecturer from Dundee in Scotland." Peternelson: "Hi Dr. Breen"
{PRIVATE} Peternelson tells Nchow-pcoleman "Would it be bad for me to ask publicly about results you have achieved beyond the substance of this paper with your technique?"
Kieran_breen: "I was very interested in the paper. One question that sprung to mind was if you are going to use this technique to look at gene expression in "damaged" cells, is it possible that only robust cells will survive the initial cell separation."
Nchow-pcoleman: "Hi, I'm Nienwen Chow, a Ph.D.. I am working in Paul Coleman's group."
Peternelson: "I had the same concern. Certainly, there'll be SOME greater frailty in injured cells, by definition."
Nchow-pcoleman: "A number of questions have now been raised. With regard to the issue of frailty in "sick" cells, we are not sure what aspect of the procedure you are referring to. There are other methods of picking cells that are more recently available, such as laser capture micro dissection, that may get around your concerns."
Peternelson: "But the point is, you're comparing cells of different healthfulness, in re: their RNA. Just by being unhealthy, certain RNAs might be degraded artifactually in a way that has not actual bearing on AD."
Nchow-pcoleman: "There also was a private message about asking about more recent, unpublished results. That's OK too."
Peternelson: "Well? Have you found anything interesting?"
Nchow-pcoleman: "Let us first address the issue of cells of different health. Is this not an issue with any method that looks at diseased vs. healthy tissue? We'll wait for an answer to this before going on to more recent results."
Peternelson: "Well, especially one that so directly and dramatically manipulates the cells before analyzing them for something as fragile as RNA."
Kieran_breen: "Of course it is, and it is one of the main problems in this research area. However, a technique such as IHC or ISH looks at the cells as they are, while single cell dissection may enrich for a given sub population, thus giving spurious results." Nchow-pcoleman: " True, that is why we are exploring other methods of cell isolation. However, we would also emphasize that for selected messages we also confirm results by in situ hybridization."
Peternelson: "Does it pretty much check out, in relation to ISH? Are there discrepancies?"
Kieran_breen: "Another question - do you think that it would be possible to differentiate between "sick" and ordinary cells following dissection? Do they maintain any sort of morphological changes (e.g. do tangle-bearing cells still have tangles?)."
Nchow-pcoleman: "For example, our aRNA data suggested no change of NF-M expression in AD, contrary to what has been previously demonstrated using Northern blotting. The result of no change in AD was confirmed with in situ, and we now suggest that the earlier studies showing decreased NF-M expression were a consequence of decreased numbers of neurons."
Nchow-pcoleman: "Tangled cells do still have tangles after picking. We emphasize that any cell property that can be revealed by IHC or any other procedure, such as Congo red, etc., that leaves the RNA intact can be applied to this technique."
Peternelson: "Of course, this last point is of considerable interest..."
Kieran_breen: "This is good, because even if you enrich slightly for more healthy cells, you will, at the end of the day, be choosing the particular cell in which you are interested and you can confirm the cell by its morphology."
Peternelson: "...doesn't mean that it's not changed systematically and artifactually, though, Dr. Breen."
Nchow-pcoleman: "With regard to more recent results, we do have a manuscript submitted in which we use the aRNA method, and our picking approach to distinguish pyramidal neurons from hippocampus as a function of disease state. Using a multi variate canonical analysis we are able to distinguish brains as to disease state very nicely."
Peternelson: "Neat! Using the tangle/non-tangle criteria? Or AD vs. PD vs. control &c.?"
Hpotter enters.
Nchow-pcoleman: "It was AD vs. "control". We put "control" in parens because although these were clinically control cases ( one lady living at home, paying bills, etc.) on neuropath exam, they were early Braak stages."
Nchow-pcoleman: "Hi Hunt"
Nchow-pcoleman nods solemnly.
{PRIVATE} Peternelson tells Hpotter "Hi, Hunt!"
Hpotter: "Hi. This is my first chat so I will probably listen for awhile"
Nchow-pcoleman: "Please joins as you see fit."
Peternelson: "Would it be gauche to ask for a preview of how the AD cases differed from controls in their hippocampal neuron RNAs?"
Nchow-pcoleman: "Long pause while we consider this."
Hpotter: "One question is whether you are surprised to see ACT in neurons and if you also checked it in astrocytes"
Nchow-pcoleman: "First, I would emphasize that the answers one gets depend on the panel of cDNAs used as probes. Given that, we find that the genes that contribute most heavily to the distinction include cyclin D1, an HSP, ACT, wee1."
{PRIVATE} Peternelson tells Hpotter "I'm not sure if you remember me--I came to Harvard when Clif Saper moved there. I hope that all goes well."
Kieran_breen: "Sorry, have to run. Thanks for the interesting discussion."
Peternelson: "Cyclin D1 is a very interesting molecule, viz. Esiri's recent work." Peternelson: "Bye Dr. Breen!"
Jhenry enters.
Nchow-pcoleman: "Nice to chat with you. Welcome Jhenry."
Peternelson: "Did you distinguish tangles from non-tangles in your more recent work?" Nchow-pcoleman: "Not in what we have submitted. This is on-going now."
Peternelson: "Is there anything you'd like to say about that later work?"
Nchow-pcoleman: "We agree that cyclin D1 is interesting, especially in view of the data of Steve Estus indicating its expression induced by APP. The Eseri work also do tie in here. The question is why is cyclin D1 being expressed."
Peternelson: "What is the variability, from brain to brain, in how good the neurons are for this technique?"
Peternelson: "Oh, in re: the Esiri work, and the reason for the expression of cyclin D1. Perhaps your technique could be used to find the earliest signs of a diseased state, which causes other things to go haywire secondarily."
Nchow-pcoleman: "Statistical analysis shows that the % contribution to variability are: 16% due to cell-to-cell, 4.4% brain to brain and 17.6% to disease."
Peternelson: "...And all the cells are definitely hippocampal pyramidal neurons of the same Sommer's Sector?"
Nchow-pcoleman: "Agree with Nelson last comment - that is one of the aims we have." Hpotter: "I think that I got lost someplace and had to come back in. One further question I have is whether wee 1 is a common mRNA in the brain and whether it is restricted to neurons or glia"
Nchow-pcoleman: "We believe that our cell sample included both CA1 and subiculum pyramids.. It is of note that end stage AD brains all clustered very tightly."
Peternelson: "Dear Mssrs Coleman, Chow, and Potter: it has been an interesting discussion, but I must go to a class. Thanks very much."
Hpotter: "By the way. Hi Pete. Yes I remember you and your sheep eps."
Nchow-pcoleman: "With regard to Hunt PotterŐs question, we see wee1 in both AD and "control" neurons. We have not looked at glia yet."
Peternelson: "Thanks, Hunt!"
Nchow-pcoleman: "Good to meet you Peternelson"
Peternelson: "Bye! Thanks!"
Hpotter: "curious. One thing Carmela and I saw some time ago was that ACT antibodies sometimes stained neuronal cell bodies."
Nchow-pcoleman: "Well, I guess that our data confirm that."
Hpotter: "No I am happy good luck with the new exps. over and out"
Nchow-pcoleman: "Goodbye"
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