- 1 Week 10 - In situ programming of leukaemia-specific T cells using synthetic DNA nanocarriers
- 2 Week 9 - Trypanosoma brucei Parasites Occupy and Functionally Adapt to the Adipose Tissue in Mice
- 3 Week 8 - Heterogeneity and interplay of the extracellular vesicle small RNA transcriptome and proteome
- 4 Week 7 - Functional aspects of meningeal lymphatics in ageing and Alzheimer’s disease
- 5 Week 6 - Platelet-targeted dual pathway antithrombotic inhibits thrombosis with preserved hemostasis
- 6 Week 5 - Microbial signals drive pre-leukaemic myeloproliferation in a Tet2-deficient host
- 7 Week 4 - Parkin and PINK1 mitigate STING-induced inflammation
- 8 Week 3 - A liquid phase of synapsin and lipid vesicles
- 9 Week 2 - High speed of fork progression induces DNA replication stress and genomic instability
- 10 Week 1 - The Neuronal Gene Arc Encodes a Repurposed Retrotransposon Gag Protein that Mediates Intercellular RNA Transfer
Week 10 - In situ programming of leukaemia-specific T cells using synthetic DNA nanocarriers
Abstract: A new therapeutic approach is proposed to treat cancer which involves direct delivery of tumor targeting genes to chimeric antigen receptors (CAR) T cells that are endogenous to the patient. This proposed therapy is the next generation of the current therapy which involves isolation of patients T cells from a blood sample, growing in vitro and genetically engineering them, and then finally injecting the modified T cells back into a patient that is undergoing chemotherapy. The current process is expensive and time consuming. The new therapy uses biodegradable nanoparticles that carry genes to CAR T cells. Thus, reducing the cost and reducing the precious time in between diagnosis and treatment.
- They mention that the nanoparticle was internalized into the cytoplasm rapidly (120 min). How does this relate to other internalization rates because this seemed slow to me.
- How does CD3-mediated targeting work with relation to T cells?
Taylor: CD3 is the signaling molecule that sits right next to the TCR. When a TCR sees its cognate antigen, it induces signaling through CD3 so by stimulating or targeting CD3, you can activate the T cells
John: CD3 is also specific to T cells, which means your nanocarrier will be targeted to those cells specifically
- Is there enough space within a nanocarrier for DNA to allow a kill switch to be added?
- Where the non-targetted cells found in liver blood cells in circulation there or hepatocytes?
- I wonder how this compares to viral vector delivery of genes.
- They mentioned that the tumor targeting genes inserted into the T cells targeted the recognition of cell surface proteins that were "exclusively" on leukemia cells. Some cancers can relatively quickly change cell surface antigens to side step therapies. Do they see any resistance build up?
- First I should Say, Wow! What a paper!
- How they did analyzed characteristics of other cells in figure 3a? as I’m not seeing any explanation.
- What would be some disadvantage of non-targeting particles that they found in liver in figure 3c?
- For the presenters, I would like to hear about the imaging technique they used in figures 4b, 5, and etc.
- I second this.. I'm a bit confused about this imagining technique as well - Homma
- Is there any off-target risk associated with transfecting the hyperactive iPB7 transposase into target cells? Are the inverted repeats unique enough that other native sequences won't accidentally get moved?
- Could this method be used in sequence with traditional chemotherapy, if the CAR-T treatment does not fully eradicate the tumors?
- Could this technique be applied to other leukemias? Does tumor heterogeneity factor into the difficulty of applying this to other tumors?
- Eve: This review discusses using CAR T cells to target solid tumors - https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6235951/
- ...isn't this how I Am Legend starts?
- For the presenters, it would be nice to briefly cover how they generated these T cells prior to this paper.
- Why did they use the 4-1BB costimulatory domain instead of the CD28?
Taylor: CD28 signaling should not be necessary for effector and memory T cells and naive T cells should already express CD28.
- Are you able to use this method in conjugation with other drugs to create a better therapeutic outcome? What is the advantage or disadvantage to that method?
- Could the cancer in the host evolve and develop resistance to the use of these nanoparticles?
- What's the ultimate fate of these nanoparticles in an in vivo system?
- Are their toxicity tests thorough enough to say the nanoparticles are nontoxic? And would longer time frame (>24hr) tests necessary for this?
- Why might neutrophils, monocytes, and B cells be targetted by the nanoparticles? Could this really be called "non-specific" if they're binding to this degree?
- What pathways degrade the nanoparticles? Is there any concern about overloading the pathway?
- Could this drug-delivery method be used for a vast array of other diseases?
- Janelle: This is what I was thinking as well. I think in autoimmune diseases, or disease that have a mutation like CF, this could be an effective therapeutic approach once the price becomes affordable.
- It would be nice to have explanation on figure 5 especially Figure 5b , little confusing?
- how to prepare nanoparticle?
- What is zeta potential?
- How could this technique be applied to other blood/monocyte diseases?
- How "biodegradable" are these nanoparticles and does the metabolism of them have any toxic effect?
- This method has no specificity for effector/memory T cells compared to naive T cells. What happens when a transfected naive T cells sees CD19?
- What is the iPB7 transposase and how does it work?
- They claim that the T cells differentiate into long-live memory T cells, however I only really see data that the nanoparticle are taken up by existing memory T cells:
"Infused nanoparticles were taken up by all CD3-expressing T-cell subpopulations, including CD4+ and CD8+ naive, effector and memory cells..." Have they really shown that effector memory T cells differentiate into memory T cells, and how long do these persist? Maybe I missed some data relating to this...?
- The paper uses immune-competent mice, however, many cancer patients are immune compromised. How would this alter the outcome of using this as a treatment in such individuals?
- How do they decide on the material (in this case poly-beta-amino ester) when designing nanoparticles?
- Are these nanoparticles similar to the ones being used in other labs that are essentially artificial vessicles (Liposomes).
- Are there off target effects from the over expression of transposase in the targeted T-cells? Does this cause transposition of endogenous transposable elements in the T-cells potentially resulting in insertional mutagenesis?
- Why would CD3 target non-T-cells? Is the F(ab’)2 they used known to cross react? Is there a way to improve target specificity?
- Why does coating the particle with negative charges mitigate non-specific binding?
- What proportion of administered nanoparticles are successfully endocytosed by T cells? If not all nanoparticles are successful in being encoytosed, what is the consequence of their eventual degradation in the blood? These particles are only stable for 1-7hrs in aqueous conditions, how does that compare with the timescale of endocytosis following administration? what would be the consequence of degradation in the blood steam and subsequent release of DNA cargo?
- The authors stated that off-targets of these DNA-carrying nanoparticles are low and point to figure 2a. It shows 88.2 go to CD3 T-cells, but doesn't indicate were the 11-12% go. Shouldn't this be concerning? Do these nanoparticles just leave the body or go elsewhere?
- There is a phrase floating amongst scientists stating "a cell isn't what it is until other cells say what it is," reflecting that intercellular communication is important for the metabolic processes of the cells themselves, affecting gene/protein expression, which again produces signal cascade both in and out of the cell. There are also studies showing how cancer cells change its microenvironment by disrupting other cells' metabolism. Could we induce these DNA-carrying particles to essentially invade other cells around tumors?
Week 9 - Trypanosoma brucei Parasites Occupy and Functionally Adapt to the Adipose Tissue in Mice
Abstract: Trypanosoma brucei (T. brucei) is a parasite that can cause sleeping sickness. It is thought that the parasite rests in two areas: the blood then the brain after breaking the blood brain barrier. This paper shows that the parasite can also occupy fat cells, which could explain why infected individuals experience weigh loss. In the fat cells the parasite can replicate, and when a healthy individual is given the fat cells they become infected. The transcriptional activity of the parasites in the fat cells was different than activity of the parasites found in the blood. Further, they showed that the fat cell parasites could use fatty acids as an external carbon source. The researchers concluded that the fat cells is another location where the parasite lives in affected individuals.
Flic: here's a link to a nice and short preview article on this paper: https://www.sciencedirect.com/science/article/pii/S1931312816302189
- They mention that they do not see any difference between white and brown deposits. Would you expect there to be a difference? What is the physiological difference?
- The main conclusion is that the parasites can resided in the fat cells. How do you think the parasite might affect people of different BMI?
- What is the importance of the pentose-phosphate pathway?
- They showed that these parasites could breakdown fats for energy via RNA-seq and pulse chase, I am not convinced until they do a mutagenesis to show this ability. Could that be a future study?
- How many people in the world are affected by this parasite?
- could inhibition of beta oxidation in these parasites help to treat the disease?
- If the genes for fatty acid oxidation were already present in the genome, was another form expected/predicted prior to the study of this paper?
Mona: They mention in the intro that this was something puzzling. Essentially, the parasite has the genes for using fat as energy source but nobody could identify mechanism or evidence of that parasite actually is doing it.
- What is the benefit to completely shifting metabolic pathways (glucose vs. amino acids vs. fatty acids) instead of having all active but regulating level of expression?
- The article suggests that colonization of fatty regions happens early on (day 6) while parasitic load in other organs doesn’t increase until later on. What benefit does the colonization of fatty deposits provide that other organs don’t?
- How is T. brucei treated? The authors mention that its presence in fat could explain treatment failures. How?
Julia: Maybe it's something to do with the solubility of the drug? The current treatment appears to be effective in the earlier stages of infection when the parasite is in the blood, so my guess is that the drug is poorly soluble in hydrophobic media. The CDC has some good info about current therapeutics: CDC - African Trypanosomiasis
- How does T. brucei gene expression in the brain compare to blood and fat?
- How is kinetoplast DNA expression regulated? And what is the purpose of its structure?
- I'm wondering how they make the GFP::PAD1utr reporter parasite - specifically how are these "cultured"/grown (?) in the lab?
- The mice in this study were infect via intraperitoneal injection, however, this does not mimic the usual route of infection which is through the bite of an infected tsetse fly. Is there a reason the couldn't/opted not to infect via the subcutaneous route? I fell like this would be a better model.
Breanna: I felt kind of confused by that too, especially since they even mention that they don't know what this would be like in a subcutaneous route and that it is the typical route of infection. Why mention it in a paper rather than just do it in the first place? Is there a benefit to intraperitoneal injection in this case that I can't think of?
- They discuss differential gene expression in relation to cellular energy metabolism, for example upregulation of RBP42 in AFT parasites and say that it "could be involved in the metabolic rewiring when parasites enter the fat?" Is it likely that this is a necessary process for the parasite to enter fat, or rather a adaptation of metabolism, influence by what nutrients are available?
- How does quantifying parasite density via proxy gDNA work?
- Has anyone figured out the chemical basis for deuterium causing a peak shift on GC?
- What does the Pearson correlation really show in figure 5A? How does it shows that ATF parasites cluster separately from parasites isolated from the blood?
- I feel like while they did discover new things about Trypanosomes in this paper, I can't help but wonder how translatable this research is, because mice aren't the same as humans, and I wonder if virulence factors and mechanisms are different in humans, while still displaying a similar infection phenotype?
- Did they happen to test varying T. brucei strains to see if they all use this possible third reservoir? (AKA is this mechanism consistent in all strains? Would it matter?)
- Kathrina: nevermind, they answered this; they tested this on a few strains, and also compared between sexes
- Makes sense that they would use generic mouse model (C57BL/6J), but what is "pleomorphic clone AnTat 1.1"? What does it do?
- What is Linear Mixed-Effects Model (LME)? (pg
- While reading about how the parasite is essentially breaking down human Adipocyte Fatty Acids, I started to wonder if this could also be happening in other cells that are rich in Fatty Acids like Hepatocytes. I also wondered if essential fatty acids ingested by the host can be digested by the parasite and could be leading to other losses in the the host signaling molecules and molecules derived from those types of fatty acids.
- I would also be curious to know if the infected host adipocytes differ from uninfected cells beyond the fact that they are losing some mass, do they change in their hormone signaling to the rest of the body? Does the body respond to the fact that it is starving the same way it would in a calorically restricted diet?
- How is the parasite load quantified?
- How do the treatments for African sleeping sickness work?
- How do the trypanosomes evade the antibody response?
- What is kinetoplast DNA and what is the function?
- What is quorum sensing mechanism?
- GFP expression measured in blood and fat, as brain will be host, why they didn't look of the expression in brain?
- For presenters, I would really appreciate if you guys can go over immunohistochemistry method.
- I’m gonna play the devil’s advocate. HAT disease basically kills patients mostly because of disfunctions in brain. (https://www.dndi.org/diseases-projects/hat/) How does this paper, finding additional way that parasite is using fat, is relevant to combating a disease that is closely associated with CNS?
- I'm interested in learning more about the current therapeutics used to treat T. brucei infection. From what I was able to find online, the mechanism of action of the main drug used to treat these infections (pentamidine) is poorly understood. It is thought to impact nuclear metabolism (possibly transcription) in the parasite, so I'm curious how it does this without interfering with nuclear metabolism in the host.
- Are there other infectious diseases that involve adipocytes? The paper mentions that parasite localization to adipocytes may confer an evolutionary advantage because they are better able to "hide" from the immune system there. Why is that? Are there other infectious agents that exploit this feature?
- Is the form of the parasite infecting the mouse the same strain of the parasite that infects human?
- Trypanosoma brucei are extracellular parasites, what means are they using to stay localized within the adipose tissue?
- As extracellular parasites, how do they benefit from intracellular fat storage (seen in adipose cells)?
Week 8 - Heterogeneity and interplay of the extracellular vesicle small RNA transcriptome and proteome
Abstract: Extracellular vesicles (EVs) mediate cell-to-cell communication by delivering or displaying macromolecules to their recipient cells. While certain broad-spectrum EV effects reflect their protein cargo composition, others have been attributed to individual EV-loaded molecules such as specific miRNAs. In this work, we have investigated the contents of vesicular cargo using small RNA sequencing of cells and EVs from HEK293T, RD4, C2C12, Neuro2a and C17.2. The majority of RNA content in EVs (49–96%) corresponded to rRNA-, coding- and tRNA fragments, corroborating with our proteomic analysis of HEK293T and C2C12 EVs which showed an enrichment of ribosome and translation-related proteins. On the other hand, the overall proportion of vesicular small RNA was relatively low and variable (2-39%) and mostly comprised of miRNAs and sequences mapping to piRNA loci. Importantly, this is one of the few studies, which systematically links vesicular RNA and protein cargo of vesicles. Our data is particularly useful for future work in unravelling the biological mechanisms underlying vesicular RNA and protein sorting and serves as an important guide in developing EVs as carriers for RNA therapeutics.
- How did they choose the proteins used for analysis in the Western blot? Are these common proteins? What are their functions?
- What was the purpose of characterizing the EVs via Western blotting/nanoparticle analysis/TEM? The purpose of this study was to determine the contents of the EVs so I'm unclear on the purpose of characterizing their holistic structure since much is already known about the general shape/size of EVs.
Mona: I believe they just wanted to confirm that the vesicles from those cell lines are in fact the ones characterized before. In other words, they needed to show there are vesicles from the cell line. But, I agree that it was totally unnecessary.
- What is the meaning of "size-selection of libraries" (in the context of NGS)? I understand the length restriction employed in data processing, but not sure what they mean by this.
- "Again, it should be emphasized that the detected ‘rRNA’, ‘other RNA’ and certain subtypes of ‘smallRNA’ annotations (snRNA and snoRNA) correspond to fragments derived from these RNA species, since the data has undergone fltering, excluding reads outside of the 17–35 nucleotide size range" -- if this is the case, where are these fragments of rRNA coming from? The paper mentions apoptotic bodies could be a potential source of these in cells, but what about EVs? Is this just a result of RNA fragmentation during processing?
- They mention that the EVs have an enrichment of ALIX and TSG101. What are these and what might this finding suggest about how EV's work?
- What is the Panther Statistical Overrepresentation Test? What kind of information can we get from it? How reliable is it as a tool?
- What dictates the size of the EVs?
- How does the nanoparticle tracking analysis work?
- What are the future directions of this work?
- They mention at the end of their discussion that their short read length was problematic. What’s preventing them from using a longer read?
Janelle: I think they wanted to specifically look at small RNAs (specifically to look at miRNAs, piRNAs as well as snRNA- and snoRNA fragments), so looking at longer reads might not allow them to detect these small fragments. Both pose problems (having a longer or shorter read length), just based on their needs it was more important to have a short read length problem.
- Do anyone know why some miRNAs are more abundant in the EV's than the parent cells?
- Why are there no statistics presented in the main body of the paper? What does this say about the strength of the correlations they propose or the differences they see?
- The authors note that potential contamination with bovine serum derived miRNAs was observed at low levels... shouldn't this be alarming?
- Kathrina: I know! I read that too, and it's super weird. Bovine serum is a commonly used serum for cell culture growth, so perhaps the authors are assuming even with little contamination, it is overall viable. I could see the authors argue that all in vitro cells would be similarly affected in any experiment.
- Do you think that the EV characteristics of cell lines may differ from the characteristics observed if this type of study was done in a different model?
- what are piRNAs?
- Colin: Piwi-interacting RNAs; they're the largest group of non-coding RNAs and they interact with a class of proteins called Piwi proteins (a whole other bag of worms) and have epigenetic functions
- What is the biological purpose of having RNA in extracellular vesicles besides targeted gene delivery?
- Can someone explain the Panther Statistical Overrepresentation Test?
- Why is the piRNA enriched in Neuro2a EVs?
- They mention that the rRNAs have a similar pattern in the EV and cell except for the mitochondrial rRNA. Why would this be?
- Why did they focus on the Overrepresentation Test rather than the Panther Statistical Enrichment Analysis?
- So, authors mention that they are characterizing exosome-enriched vesicles, but what did they do to make sure they purified in fact exosomes not the other types of vesicles?
- For presenters, it would be nice if we can talk about the potential applications of using EVs in therapeutic strategies.
- Why didn't they look at primary cells that produce EVs?
- What is Vault and Y RNA?
- Since mouse cells had different RNA cargo than the human immortalized cell lines, wouldn't a mouse model not be a viable option for studying EVs?
- In figure two, why is there such large enrichment for small RNAs in the cells - shouldn't rRNA be most abundant? Does this have to do with the method for RNA extraction?
- Number of reads for miRNA was relatively constant across cell lines except for Neuro2A - anyone want to hazard a guess why they would have less miRNA and more piRNA?
- Fig 4 mentions that "other RNA" includes a large number of particular tRNA + anticodons but the authors don't discuss this further - why would cells traffic particular anticodons?
- what is Nanoparticle tracking analysis?
- What is the specific Therapeutic or diagnosis potential does EV has?
- What Vault RNA ?
- Colin: Vaults are large ribonucleoprotein particles in eukaryotic cytoplasm, usually associated with the nuclear membrane and thought to do with nuclear transport. Vault RNAs are small, non-translated RNAs found in these vaults and thought to play some functional role
- Do EVs carry the machinery to generate some of their cargo (e.g. the various RNAs, proteins)? Or does their cargo remain static after budding from the parent cell?
- Why does it make sense to focus on the HEK and C2C12 cells for the EV proteome experiments?
- How much might the proteome vary from one EV to another? Might the lower % abundance proteins be mistakenly budded?
- How long can an EV last before degradation? How would degradation be initiated outside of a cell?
- Does release of RNA ever occur into the extracellular space, and could this activate an immune response, for example via TLR3/7/8?
- Is the fusion of EVs with "target cells" directed in anyway, or can fusion occur with any membrane in close proximity? How diverse is this? Are there "homing" ligands and receptors...?
- Could pathogens hijack EVs and evade the immune response by avoiding presentation via MHC? They may not be able to enter pre-formed EVs for lack of receptors, but could they somehow "make their way" to EVs as they are being formed?
Flic: Probably not - EVs may not be big enough to house a pathogen. John: Maybe. EVs vary in size between 30 - 2000 nm, viruses vary from 20 - 400 nm. This may be best done in apoptotic vesicles which I think are the larger of the EVs. I don't know, but this would be interesting to look into. Flic: I think this is super interesting too! Perhaps the EVs could house smaller molecules from pathogens such as decoy receptors/molecular mimicry that allow pathogens to evade the immune response, which could in turn be a potential route for infection-induced autoimmunity?
- What are piRNAs, Y RNAs, and Vault RNAs?
- Could the presenters discuss the nanoparticle tracking test they did?
- Could pathogens use this to bypass the immune system?
- Is delivery cell type specific?
- Can cells make multiple distinct types of EVs?
Week 7 - Functional aspects of meningeal lymphatics in ageing and Alzheimer’s disease
Layman's Abstract: The lymphatic system runs in parallel to the blood circulatory system and plays an important role in draining toxic waste products and cellular debris away from tissues. An important part of this system, is the draining of these waste product to local lymph nodes, where critical priming of the immune system against potential pathogens takes place. Whilst the brain parenchyma (the pink squishy part) lacks a vascular structure, the meninges (the surrounding membranes) has recently been “re-discovered” to have such a vascular system. In this paper from Da Mesquita et al., the authors successfully tackle the challenge of impairing drainage from the lymphatics vessels in the meninges to the cervical lymph node (CLN), using several approaches which they visualise using fluorescent tracers. Mice with ablated lymphatics showed altered behaviour traits and changes in gene expression in the brain. The authors then showed that delivering the VEGF-C gene – implicated in growth of lymphatic vessels – into the brains of “old mice” restored drainage of the tracer into the CLN, after which, an improvement in cognitive behaviour was observed. The authors went on to investigate a mouse model for Alzheimer’s, showing that ablation of lymphatic vessels resulted in accelerated accumulation of amyloid-beta deposition. Whilst techniques for studying the lymphatic system of the meninges requires some fine-tuning, this paper presents multiple approaches for studying this interesting new field and data that has implications for human neurodegenerative diseases such as Alzheimer’s.
- For people interested, this review came out recently and is a fabulous summary of the current state of the field :)
Also useful and a very short read: https://www.nature.com/articles/d41586-018-05763-0
- Did they choose AD because it has something unique about it among neurological plaque diseases, or was it an arbitrary start point that could later extend to other diseases?
- I am curious what about buildup of plaques leads to neurological disease.
- Does plaque formation maybe prevent cells from being able to communicate properly?
- This may sound a comment that a reviewer will make, but…the authors mention they used a software developed by themselves to evaluate their MRI images, did they try to validate by using another software and compare the results?
- How did they actually performed experiments mentioned in figure 1j?
-these are standard behavior tests in neuroscience studies. we will go over morris water maze because thats where all the data comes from. it assesses learning and memory. -Haley
- I should say, I very liked the comment paper made about the lack of a model in which amyloid development is related to aging in Alzheimer’s disease.
- For the presenters, I would like to see some explanation about MRI methods, specifically the ones they performed with patients.
- For the experiments in figure 1, they use two different controls (having the injection of vehicle followed by photoconversion and injection of visudyne without the photoconversion step). Why are both necessary as controls to perform this experiment?
- When taking the intracranial pressure measurements, they drilled holes into the skull of the mice and inserted a pressure sensor catheter. Do you think this could affect their measurements since the hole (even if covered) could affect the pressure? Or, since they are only measuring a change in pressure, do you think this approach is sufficient?
- What is the difference between J20 transgenic mice and 5xFAD mice?
- J20 seems to be a mouse model that overexpresses human APP with two mutations linked to familial Alzheimer's disease. 5xFAD mice rapidly develop severe amyloid pathology and the plaques spread through the hippocampus and the cortex. - Homma
- Do we know where amyloid-beta is coming from?
- Is it possible that some of the neurological defects seen, come from inflammation due to the physical act of vessel ablation?
- Are there B cells found in the brain under noninflammatory conditions?
- How is the skull thinning procedure performed?
- What is Bonoferroni's post hoc test (Figure 1)? Why did they need to run that test in their analysis? (sorry I don't know statistics, I've never heard of this before)
- Does the amount of MRI's that they take on an animal lead to brain damage? Does that skew some of their results?
- Why did they use AAV1?
easy to transduce cells. the standard for in vivo transduction. -Haley
- Mouse models of AD don’t develop tangles. Neurofibrillary tangles, resulting from tau aggregation, are associated with cognitive impairment in Alzheimer's patients. How are AD mouse models chosen? Why did they use a model with overexpression of APP and no models with hTau?
- In the mouse models of Alzheimer’s disease, is there brain atrophy? Or is tangle development necessary for the atrophy seen in Alzheimer’s patients?
- When performing MRI acquisition, the authors only found significant differences between the test group and one of the control groups. Shouldn't they have expected to see a difference between the test group and both controls? Do they address this or give an explanation?
- The article mentions that an in house program called Lymph4D was used to assess the MRI data. Why not an already developed program? Are there any concerns with them using their own software to analyze their data?
- J20 mice did not show improvement when treated with a vector for increasing mVEGF-C expression. What does this say about Alzheimers pathology in general? What does this imply about the role of meningeal lymphatics in Alzheimers?
- What was the previous model for influx and efflux? Was there one?
- Is seven days long enough to confirm off-target effects? (In regards to method: "The use of this method resulted in effective ablation of meningeal lymphatic vessels (Fig. 1b, c), without any detectable off-target effects in the coverage of meningeal blood vasculature seven days after the procedure".)
- Have there been similar tests to see improvement lewy body diseases such as dementia?
- In the discussion, they mention that transgenic mouse models of AD have nearly all the same pathology hallmarks except the mice don't get deposition of a-beta in the dura mater. What might be the significance of this difference, and is it worth considering when assessing the translation potential of this research to human AD?
- Is my understanding accurate that they claim to notice significant changes in hippocampal gene expression in ablated mice only following MWM, compared to controls? Isn't that a fairly substantial finding, that ablation impacts gene expression induced by environmental cues (e.g. having to find a platform), or is that to be expected and they just mention it to further fill out their paper?
- What is the mechanism by which amyloid plaques increase upon AQP4 deletion? Is clearance reduced, gross amount of plaques increased, etc.?
- Why might AQP4 expression be reduced in vivo? Is there differential expression in Alzherimer's (or other relevant disease) patients?
- For posthumous samples (mouse, human), is there concern of degradation and to what degree? RNA degredation, etc.?
- in fig 1j how the behavioral test relevant to impaired meningeal lymphatic drainage?
- What are examples of CSF macromolecules that cross to the BBB to enter the brain?
- The paper says there were no off-target effects with regard to the visudyne-directed ablation of meningeal lymphatic vessels, but I’m really surprised that there would be no inflammation in response to this? Are the cells making up the vessels not dying, are therefore would there not be any DAMPs? How does this work?
- Is it simply increased circulation of CSF/ISF that facilitates extracellular beta-amyloid plaque removal or are there factors involved that are more frequently at the site of amyloids as a result of that increased circulation?
- How is the skull thinning performed? Where is it thinned and how does this facilitate VEGF-C intracranial injection? Are they injecting through the skull?
- The discussion mentions antibody-based treatments for Alzheimer's which may be impacted by declining lymphatic function. The intro of the paper also mentions the brain as being an immune privileged organ -- how can an antibody-based treatment be effective on an organ that is immune privileged and relatively isolated from the rest of the body?
- What is a Hamilton syringe? Are they typically used for certain types of injections (i.e. I.P.)?
- Do Alzheimer's patients have abnormal amyloid-Beta levels in their CSF?
- Do we know if a beta amyloid plaque is causative or caused by the cause of alzheimer's disease?
- Are there other indicators of alzheimer's disease and were they exacerbated by decreased lymphatic draining?
Week 6 - Platelet-targeted dual pathway antithrombotic inhibits thrombosis with preserved hemostasis
- My understanding is that the significance of this therapeutic strategy is that a primary wound plug is still able to form without creating a large clot (since the FXa and GPiib/iiia cascades are effectively blocked, the plug cannot recruit more platelets/fibrinogen so it doesn’t grow.) In theory this represents a therapy that would prevent thrombosis without substantially increasing the risk of bleeding since it does not affect primary plug formation. But there has to be a tradeoff, right? Would a single layer primary wound plug really be enough to prevent increased bleeding? Would this affect the repair/healing mechanisms involved in healing a wound?
- Is this intended as a long term therapeutic, or for more acute usage (following surgery, for example.) I’m wondering if there are possible long term effects of this drug, i.e. since it is a protein it’s possible it could elicit an immune response after extended usage? Have they looked at any long term models?
- The authors mentioned that "immunogenicity presents a potential limitation to the application of TAP". Might you expect an immune response? why or why not? What could preliminary next steps could be taken to explore this potential issue?
- Other bifunctional recombinant biologicals of promise are also mentioned in the article. What makes this treatment a better alternative?
- The authors also mention active debate in the field over the best "step" to inhibit in the thrombotic pathway. Is FXa really the best target or is there a better step out there? What are some of the reasons FXa might be best and what are some of it's downsides?
- How is thrombus formation quantified?
Flic: They use something called ImageJ. It "calculates area and pixel value statistics of user-defined selections".
- In what way would this therapy be administered in the clinic?
- How is this an improvement over existing therapies?
- They mentioned that "to characterize targeting, SCE5-TAP and MUT-TAP were incubated with anti-HIs AF488 to label construct His tags." Why did they specifically tag Histidines instead of another residue in both of these constructs?
I believe they're just referring to a histidine tag. They're commonly used to label proteins for purifications, but in this case, they're using it as a target for their labeling method. - Daniel
- Is it possible to treat thrombotic diseases without having to use triple therapy? Is it possible to just create one drug that can encapsulate the effector functions of 3 different drugs?
- Are there some drugs that would work better in combination than the ones that they used? Did they have experiments beforehand that determined that these drugs work effectively together?
- What are the potential off-target effects of this type of treatment? Could the localized anticoagulant effects lead to worse outcomes?
- What is the likelihood of this reaching clinical trial stages? What are the factors promoting or inhibiting this next step toward development?
- What made S2 cells a better choice for growing the fusion protein (over bacteria or a different eukaryotic cell line)?
- How did the multi-domain fusion protein stop the TAP domain from inhibiting FXa in the absence of SCE5:activated platelet binding (how did it keep from inhibiting hemostasis)? Is this not dependent on thrombin at all, or is just that there is sufficient soluble FXa to turnover enough prothrombin to maintain hemostasis?
- What happened with the LIBS-TAP antibody reported in 2004? Did that every become clinically relevant? What advantages does SCE5-TAP have over that older construct?
- How do they make this conformationally specific?
- This seems like a pretty solid method, where/where could they look for potential improvements?
- What activates GPIIb/IIIa?
- This may sound not really related, but why did they make the recombinant protein in Drosophila not bacteria?
- What is an electrolytic inferior vena cava model (EIM)? What does it tell you?
- Eve: I had the same question. I found this JOVE article helpful: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3196180/.
- It says that all P values were determined by ANOVA and Bonferroni’s multiple comparison test. Are there other ways to interpret the data, and could that affect the significance of their conclusions?
- In this study, they are trying to balance increasing the effectiveness of antithrombotic therapy while reducing the risk of major bleeding. Do you think it is fair to generalize these results to a population? Like a higher dose might work with some people but adversely affect others. How could you personalize this dosage?
- How as SCE5--the single chain antibody--become a good use to target activated platelets? Plenty in the literature have shown that many antibodies are not as specific as they are. So how was this validated?
- This has been tested on carotid and inferior vena cava thrombosis; have they tested this on other artery/vein thrombosis? Did they test it on those sites because those are the most common areas of thrombosis?
- What possible side effects are there for SCE5-TAP? And how long does SCE5-TAP stay in an organism system?
- Julia: I was wondering this as well. Some possible side effects I'd imagine would be an immune response against the complex, although since it's a rather small protein it might not be highly immunogenic. I also wonder if there are possible consequences related to bleeding/wound healing. This therapeutic seems to be better at mitigating bleeding issues caused by other antithrombotics, but with only a single layer plug forming at a wound site, I wonder how well that would "hold up" so to speak?
- How does the flow chamber adhesion assay work?
- With this therapy did they follow-up to see whether there was an anti-antibody (anti-drug) response?
- For the presenters: Id really like to see a diagram and explanation of the pathway/mechanism of these clotting factors.
- How does Ferric Chloride induce arterial thrombosis?
- What is upstream of GP11b/IIla? What normally controls this?
- What is factor X? how is this activated/inactivated?
Breanna: Factor X is a clotting factor that activates to become a serine protease to initate coagulation. I found this breakdown of it really helpful: https://www.sciencedirect.com/topics/neuroscience/factor-x
- Are there any compensation mechanisms that they should have accounted for?
- Have other works tried to target different aspects of the coagulation cascade? For example, why not target thrombin directly instead of an activator?
- Is laser or FeCl3 treatment to induce thrombosis relevant to thrombosis in the heart? Do we need to be concerned about the other effects of corrosion that FeCl3 would cause in the model?
- Can the SCE5-TAP be selectively targeted to the heart clinically?
- The authors mention two small molecules, one isolated from grasshopper and the other from Agkistrodon acutus venom, that can inhibit platelet aggregation and FXa. How does the mechanism of action of these small molecules differ from the SCE5-TAP fusion protein?
- Why is SCE5-TAP more potent than LIBS-TAP?
- Do other rodent model systems have cardiovascular systems more in line with the human system?
- How does this compound interact with aspirin or other pills a patient might end up taking without thinking about it?
Week 5 - Microbial signals drive pre-leukaemic myeloproliferation in a Tet2-deficient host
Layman's Abstract: Leukemia is a form of cancer that originates in the blood, and is the most common form of cancer in children. The cause of leukemia is believed to be multi-factorial, with both familial and environmental influences playing a role in its realization. Some of the familial factors that we inherit from our ancestors are reoccurring factors that are found in many patients with leukemia, suggesting that these factors are a predisposition to leukemia. However, these predisposing factors alone are not sufficient by themselves to cause leukemia. Researchers interested in identifying other factors associated with the onset of leukemia examined the roles of the predisposing factors in our guts. The researchers were able to elucidate a complex pathway by which the predisposing factors allow the digestive bacteria in our gut to migrate to places they are not meant to be, causing a defensive response from our bodies. The researchers suggest that the predisposing factors in combination with the response from our bodies to the migrating digestive bacteria help elucidate one pathway by which leukemia can emerge.
- Can dextran cross endothelial cells into the blood stream? How is “intestinal permeability” assayed?
- What is the significance of bacteria residing in particular portions of the intestines in promoting PMP? Do bacteria in other sections of the intestine also promote PMP?
- What other cytokines are bacteria induced that promote PMP?
- How did they control for the possible resistance that the bacteria could have acquired after antibiotic treatment? Could we assume that the mice were germ-free after antibiotics? Could the varying level of resistant bacterial populations have a residual effect on preventing and reversing the PMP in Tet2 -/- mice?
- Hypothetically, could the type of bacterial infection influence the degree of leukemia one is diagnosed with? They primarily look at Lactobacillus in this study, a primarily non-pathogenic bacteria. Why didn't they look into pathogenic bacteria infections and see how that influences hematopoietic malignancies?
Mona: I think they looked at Lactobacillus since it was the highest hit when they analyzed 16S. Although it's not pathogenic, they showed it can cause elevation of IL-6 signaling and in turn promote PMP. I was looking at other articles, and interestingly there is a positive correlation between being infected with pathogens and onset of leukemia.
- In Figure 1a, they say that the re-plating efficiencies of both the symptom-free Tet2 -/- mice and the Tet2 -/- mice with PMP are above that of the litter controls. I just want to confirm, are the blue bars meant to be the controls? It is not explicitly stated, and they looked at both bone marrow and splenic haematopoietic progenitors for all three types of mice so I was just unsure what the blue bars represented.
Flic: in other figures (Fig 4. and in the main text they have stated that blue indicates littermate controls, whilst red indicated Tet2 -/-. This is also stated in the key in Fig. 1b. I would presume that they used the same colour scheme throughout...?
- What is in vivo FITC–dextran intestinal permeability assay? How can they measure increased intestinal permeability in vivo?
- Homma: Dextran is a group of glucose polymers made by certain bacteria. I'm assuming that this assay has fluorescently-labelled molecules that bind to dextran polymers that are made by these bacteria when they permeate out of the intestine.
- Why do they use so many different types of mice? They use Tet2f/fVavcre, Tet2f/fVillincre, Tet2f/fLysMcre, and Tet2f/fVavcre. I was confused why they looked at so many different types.
- Eve: I was also confused by this because they don't really explain it fully. I believe they used the VavCre (hematopoietic cells and progenitors), VillinCre (epithelial cells, small and large intestines) and LysMCre (monocytes, mature macrophages and granulocytes) strains to distinguish which cell types were responsible for the increase in IL-6.
- Lauren: The different CREs are expressed in the different cell types Eve listed above so TET2 (which is floxed) was knocked out in only those tissue types
- Eve: I was also confused by this because they don't really explain it fully. I believe they used the VavCre (hematopoietic cells and progenitors), VillinCre (epithelial cells, small and large intestines) and LysMCre (monocytes, mature macrophages and granulocytes) strains to distinguish which cell types were responsible for the increase in IL-6.
- The group use CD11b+Gr1+ cells as a biomarker that is predictive of disease, but what actually are these antigens and what function do they have?
Gabriel: CD11b+Gr1+ are two cell surface molecules that they used to identify what lineage (myeloid or lymphoid) the cell is in. Above 16% of the entire cell population that displayed these markers were classified as PMP - meaning over proliferation in the myeloid lineage
- In Figure 2b and c, the authors suggest that the differences in morphologies observed between the Tet2-/- and littermate controls is indicative of increased intestinal permeability. These “holes” looks really circular and BIG compared to the size of the epithelial cells. If these cells aren’t dying of apoptosis – what might be another mechanism for this increased intestinal permeability, and the cause of these "hole" looking things? Perhaps it's extrusion of epithelial cells?
- I’m a bit confused about the role of the intestinal barrier disruption. The paper suggests that a combination of deficiencies in Tet2 and certain bacterial infections may lead to PMP, and their results show that altering the intestinal barrier with DSS resulted in excessive myeloproliferation. However, in their mouse model they also show that Tet2-/- mice have live bacteria present in the MLN and spleen. Did they do anything to disrupt the intestinal barrier in these Tet2-/- mice? Or are they suggesting that deficiencies in Tet2 may result in disruption of the intestinal barrier?
- People with later stages HIV may suffer from disruption of the intestinal barrier resulting in bacterial dissemination; are they at higher risk of AML?
- What is the specific technical difference between germ-free conditions and specific-pathogen-free conditions for mice care?
- Lauren: specific pathogen free just excludes particular bacteria/parasites from a facility, usually accomplished by periodic pathogen testing and treatment with antibiotics or antiparasitics if a mouse tests positive
- Are there other susceptible barriers for bacterial spread that should be looking into, in light of this paper?
- Are other inflammatory cytokines, e.g. IL-8, TNF-a, that are elevated in PNP states?
- What are the background levels of bacteria in the relevant parts of our bodies?
- What are some other blood conditions that are a predisposition for leukemia?
- Are the inflammatory cytokines specific to environmental influences and pathogen influences?
- Does the term "bacterial translocation" simply refer to the presence of bacterial DNA within host cell cytoplasm or is it actually incorporated into the genome?
- Why are the myeloid cells not further phenotyped past their status of CD11b and Ly6C/G?
- How is the TLR2 agonist Pam3CSK4 inducing myeloproliferation without affecting intestinal barrier integrity?
Mona: TLR2 is a component of bacterial cell wall. The authors used it to show that bacterial signaling is important for development of PMP in TET2 deficient mice. However, intestinal barrier integrity is not effected by the presence of bacterial signaling; it's the TET2 deficiency that affects the intestinal barrier function as they showed by transcriptome analysis.
- What is the difference between GF and SPF. Why are the number of CD11b+Gr1+ cells in Tet2-/- GF and SPF in blood and spleen and why isn’t Tet2+/+ SPF data shown?
Taylor: GF refers to germ free mice that lack most of the microbiome. SPF refers to specifically pathogen free mice that are simply kept in relatively clean environments but otherwise have a normal microbiome
- Why did they only monitor IL-6? Did they check levels of other ILs?
- Could IL-6 blockers be used to prevent PMP?
- Can anyone explain the Dextran permiability assay?
- What (type)antibiotic used for reversed PMP?
Mona: I believe they didn't use only one antibiotics, it was a combination of different ones like kanamycin, gentamicin, and etc.
- What is FITC-Dextran Intestinal Permeability assay?
- What mouse mutants were used for this study? Why/what was the importance of the mice models used?
- Why was permeability only seen in the jejunum? Why not other areas?
Gabriel: That is a super interesting observation that they didn't look into in this paper. I suspect TET2 plays an epigenetic role in the small intestine - and disrupting the epigenome causes an increase in permeability. But a pathway way study would have to be conducted to deduce that
- Is there a known mechanism for why antibiotic treatment actually caused reversal and not just stalling?
- Does disrupting other parts of the TET2 pathway (maybe before TET2) also develop hematopoietic malignancies?
- Would microbial treatment from a healthy individual *but* from a different region of the world be good or bad treatment?
Gabriel: I don't believe that would help with treating the disease. The patients (and mice) microbiome are 'normal' - it's the translocation of that bacteria into the blood that causes the phenotype, not necessarily the type of bacteria itself
- By what mechanism does TET2 knockout lead to the increase in intestinal permeability?
- What do the results of this study mean for other leukemia research done using germ free mice?
- Would a TLR2 deficient bacterial strain also cause PMP in TET2-deficient mice? Do any other TLRs show a similar effect?
- Mice tend to have naive immune systems, as they are kept from pathogen exposure in breeding colonies. As such, this may be the mouse's first immune response. Would responses other than the first response still yield this result?
- What size of dextran oligomer was utilized for the FITC dextran assay? Was it bacterial sized or much smaller?
- Intestinal permeability was shown to increase, which allowed bacterial entry. Is failure to clear the bacteria normal, or is the myeloid line knockout of TET1 affecting immune response efficiency as well?
- Could other pathogens (respiratory viruses for example) cause this response as well?
- Could somatic TET1 -/- in a subpopulation of cells cause this phenotype, or does it need to be germline inheritance?
- what pathogenic factors are responsible for the PMP response seen in mice with bacterial translocation? Does the identity of the bacterium matter? Or is it more related to the host inflammation/immune response?
Week 4 - Parkin and PINK1 mitigate STING-induced inflammation
Layman's Abstract: Two proteins called PINK1 and Parkin work together to help cells maintain healthy mitochondria, which is vital to maintaining a functional cell. When mitochondria are damaged, Parkin and PINK1 work together to eliminate them from the cell. Failure to eliminate damaged mitochondria can lead to a host of conditions, including Parkinson's disease. Parkinson's is characterized by a loss of motor function, uncontrollable shaking and trembling, and neurodegeneration. When Parkin and PINK1 are not present in the cell, damaged mitochondria cannot be removed. In this study, mice which did not have the genes for Parkin and PINK1 were bred and observed following heavy exercise, which stresses the mitochondria. In a normal mouse, PINK1 and Parkin would help fix the stressed mitochondria state. Since these mice had neither PINK1 or Parkin, they could not fix the mitochondria damage, and they showed symptoms very similar to Parkinson's (poor motor function and brain function.) They also showed a type of inflammation typically caused by an immune response that takes place by the STING pathway. To determine the role of the STING response in these mice, they were bred to create animals lacking PINK1/Parkin and STING. In these animals, the symptoms were reversed, even though they never regained the function of PINK1 or Parkin. Thus, the condition was effectively reversed, which indicates that the ill effects of losing PINK1 and Parkin are a result of overactive STING response. This is an important finding in understanding how Parkinson's disease progresses, and how it may be managed.
- What is the pole test?
Answer by Breanna: they put a mouse trained to descend a pole nose down and put it on a pole and measure how long it takes them to turn around and descend
- What are tandem ubiquitin binding entities?
- Was measuring their serum inflammatory cytokines their only measure for neuroinflammation?
- For figure 1, the authors did the experiment with Pink1-/- mice and not the Prkn-/-. However, in the introduction they mention the role of both gene products on ubiquitination. I don’t quite understand why they didn’t examine Prkn-/-.
Janelle: I believe that they did do the same experiment with Prkn-/-. However, it is in the supplemental data rather than the main paper. If you look at Supp. Fig. 1C, they show that Prkn-/- mice did not display increased pS65-Ub levels following EE.
- Lrrk2 is a major gene mutation correlated with Parkinson disease and has role in inflammation. What is the rational behind comparing it to Prkn and Pink1 knockouts?
- How long the different cytokines they examined in this paper stay in body usually?
- To examine the levels of cGAMP, why the authors collected serum DNA instead of blood DNA?
- They vaguely mentioned NSAIDs causing an earlier onset of Parkinson's, but that doesn't make sense to me. With their findings shouldn't it have the opposite effect? They don't even explain how this ties into their research.
- Why did levels of IL-1beta and TNFalpha not increase after EE in Prkn-/- and Pink1-/- mice? (extended data figure 1j-k)
- Only six animals were tested for the IFNAR1-blocking antibody experiment. Is this a typical sample size for immunology research in mice?
- Lauren: In my (admittedly limited) experience, n=5-8 is pretty common for the types of parameters they are looking at in mice
- Why didn't they test Pink1 mutants in the pole test experiment?
- Homma: I am confused by this as well. They mentioned that PINK1 normally is stabilized on the outer mitochondrial membrane where it phosphorylates ubiquitin. The pole test experiment was looking at neurons, which also have mitochondria? Is it because mitochondrial stress in neurons cannot be easily detected?
- They do a cytokine analysis between WT and Lrrk2 (G2019S/G2019S) mice. What is special about the Lrrk2 (G2019S/G2019S) mice and why is it relevant for this study?
- Kathrina: I had the same question! Lrrk2 (G2019S/G2019S) is a genetic mutation of Lrrk2 that is highly associated with Parkinson's disease because it interacts with Prkn. What is fuzzy to me is that I cannot tell why this gene is essentially thrown into this experiment, and they did not explain why they didn't compare Lrrk2 (G2019S/G2019S) mice with Prkn-/- or Pink1-/- mice during EE.
- In the discussion for Fig 2, they mention that some mice had body temperatures that remained elevated for 3-4 days. Do you think this could cause any unintended consequences that might affect how they are collecting and interpreting their data?
- If this study is focusing on a neuronal disease, why were they observing mitophagy in heart tissue?
- What is STING Golden ticket?
- How does these inflammation events lead to neuronal death? Why isn't this seen in other tissues?
- Did they look at any other cytokines or other parts of innate immune system that could be influencing/confounding their data? Could they even control the microenvironment to really determine whether cytokines influence neuronal loss?
- In figure 2 c and d, why did they only look at IFNB1 and IL-6 cytokines? Do these plots show significant results?
- How can mtDNA polymerase(PolG) and Parkin (which are both key in inflammatory signaling when one is not present) function similarly?
- Why doesn't parkin(-/-) show levels of PO4-Ub similar to WT? If parkin acts downstream in the pathway and PINK1 is active, shouldn't we expect similar levels to WT?
- What are the differences among some of the pro-inflammatory cytokines? Why were these the ones the paper examined?
Answer by Flic: They actually looked at 23 in total, using the "Bio-plex Pro Mouse Cytokine Standard 23-Plex" kit. My guess is that they then focussed on the cytokines which they detected at significant levels. IFNg is your typical "bread and butter" inflammatory cytokine, which activates macrophages, among other immune cells. As far as I'm aware, IL-6 and TNF (also pro-inflammatory cytokines) are involved in the acute phase response, which contributes to fever! Now, what affect they have on immune privileged sites such as the brain, I haven't the foggiest...
- How does mitophagy differ from other types of autophagy?
- What is the significance of TH+ neurons?
- The authors mention that Parkin plays a role in adaptive immunity. How does this relate to Parkinson's? Does it?
- Well, it seems that STING is part of innate immunity, whereas Parkin (and therefore PINK1, because it activates Parkin) is in adaptive immunity. Authors may be reemphasizing how damaging innate immunity (STING) is to our body because it is non-specific, whereas adaptive immunity (PINK1-Parkin combination) can deal with damage response without further disruption to other tissues/cells.
- Does the parkin E3 ubiquitin ligase work through substrate adaptors (as many E3s do)? If so, what are they and what is their neuronal distribution? Could expression of these adaptors contribute to the specificity of parkin related inflammation prevention seen in dopaminergic neurons?
- What makes the concentration of creatine kinase an indicator of tissue damage?
Answer by Breanna: CK is an enzyme is responsible for the catalysis of production of phosphocreatine, a fast acting energy reservoir common in tissues that need to meet increased energy demands quickly like in muscles. When tissue is damaged the cells lyse and CK releases into the serum, causing an increase. I believe it is commonly measured in clinical applications as a quick way to measure heart attacks.
- Based on my reading, it seems this paper is suggesting that inflammation contributes to neuronal loss. A follow up research question (or something already known in the field?) then is if Parkinson's disease can be considered as anautoimmune disorder?
- They looked at the differences between of cytokine between wild type and Lrrk2 mice? why? what are these mice have that need to compare with them?
- How cGAS-STING pathway does work?
- Why use chemically mutated mice? There have to be better options for this study no?
- How does a pole test work?
- "Exhaustion was determined when the mice failed to re-engage all four paws with the treadmill belt despite negative stimulus." What was the negative stimulus?
- Does exogenous addition of pro-inflamatory molecules also cause the hesitancy do descend phenotype after 40 days?
- Do other diseases which are correlated with chronic inflammation and/or high pro inflamatory cytokine also have an increased risk for parkinson's disease?
- The paper states "When mitophagy was increased in wild-type mice, there was no change in the serum concentration of IL-6, IFNβ1, IL-12(p70), IL-13, CXCL1, CCL2 or CCL4 either immediately or 24 hours after EE." However, KO mice did show increase in these after exhaustive exercise. Can someone with an immunology background give me a short and sweet explanation of these factors so I can better appreciate the significance? These are inflammatory response elements? Or something different? What's the difference between each?
- Gabe asked about STING golden ticket mutants above. I'm curious what they are as well, and also if this "golden ticket" notation is something used widely in immunology or if it is only specific to this STING mutant
- how does L-dopa rescue dopaminergic neuron loss/movement disorder? Also, what is the mechanism of "dopaminergic neuron loss" -- is it an apoptotic loss of the cell or something like that? Or a decrease/change in activity/function?
- How does this compare to inflammation induced by viral infection, particularly with herpesviruses that infect neuronal cells?
- Is there a chance that these neurodegenerative diseases have not been selected for evolutionarily, and reflect a sloppy-but-efficient-enough mechanism for handling mitochondrial dysfuction?
- Do extreme athletes suffer from earlier onset of Parkinson’s?
- How does mitophagy actually work? The paper states that Parkin has been linked to the adaptive immune response, and the production of ANAs. Does mitophagy, therefore, work in a similar way to autophagy leading to cross-presentation, and the activation of CD4+ cells leading to the ANA response?
- I notice the used C57BL6 mice, which are Th1-biased. As the inflammasome response is typically characterised under the Th1 response, I wonder what they might see if they used a Th2-biased mouse (BALBc), or even better, a collaborative cross! Does a mouse model for Parkinson's, with a delayed age of onset, exist?
Week 3 - A liquid phase of synapsin and lipid vesicles
Layman Abstract: Synapses, which are junctions between 2 nerve cells, consists of a gap which signals pass through by diffusion of a neurotransmitter. Neurotransmitters are contained in synaptic vesicles (SVs), which are formed in clusters at synapses. These vesicles therefore can propagate the release of neurotransmitters at a synapse, and are important for constant nerve impulses and signals. SVs are released via exocytosis (i.e. transport out of a cell) during nerve impulse activity. The authors determined that many components are associated with SVs, and focused on the synapsin protein. Synapsin has been determined to be involved the regulation of neurotransmitter release at synapses. Synapsin forms a liquid phase in an aqueous environment either by itself or with binding partners or lipid vesicles. Clusters may self-organize without the need of a membrane or protein-structure to confine them.
- Could synapsin provide a good target for therapeutic treatments? why or why not? If yes, what might these look like?
- What other protein groups aggregate in a similar liquid phase manner? How does this help cell functions or specific protein functions? Are there other cell-type specific examples of this phenomenon?
- What might be giving synapsin/the synapsin liquid phase SV specificity?
- What is IDR? How is it made and what are its function in general?
- The authors confirmed that the IDR region is lipid forming region rather than the ATP site, my question is that why didn’t they make mutations in IDR and see if the droplet forming capability of synapsin is affected or not?
- What role synapsin has in other tissues than nervous system? When authors made synapsin knockdown mice, was it whole body knockdown or conditional?
- How does the phosphorylation of synapsin lead to a conformational change?
- In figure 1a, why aren't there any droplets formed at 50 minutes?
- What size are the droplets?
- What are SH3 domains – how have these domains been implicated in neuroscience?
- What is the phenotypic importance of the TKO?
- All of the experiments were done at RT, why didn’t they use physiological temperature?
- How come they chose to do the reaction at room temperature and not physiological?
- If you didn't want to change the dynamics by adding the large GFP, what is the best way to measure without fluorescence?
- this is a test answer
- Does the order of phosphorylation in different sites of synapsin 1 matter?
- What does "biphasic effect" mean?
Breanna: Feel free to correct me if I'm wrong. Biphasic literally translates to "two phases". In the context of this paper it mentions "biphasic" when talking about the SH3 interaction which earlier in the paper was discussed as being able to generate "two liquid phases". This is what I assumed it was referring to. I would like to know more what these different phases are.
Julia: I don't believe that's quite what they're referring to in this instance. The biphasic effect refers to a phenomenon when varying concentrations of a substance have different effects. The most common example is alcohol, which acts as a stimulant up to a certain BAC %, and then acts as a depressant. Here, they are specifically talking about the ability of intersectin to promote vesicle formation at low concentrations, and inhibit vesicle formation at higher concentrations.
- As the authors describe what synapsin consists, they hypothesize that it would be a "bimolecular condensate that includes SVs". Which of the compartments ("ATP-binding module... N-terminal short region that partially penetrates membranes... and a C-terminal IDR (15) with multiple... [SH3] domain–binding motifs" is indicative of a protein creating condensate for SVs?
- Why was photobleaching and measuring the recovery of fluorescence used? I can't figure out what this tells us about the order and organization of these droplets.
Thanks to Colin I now understand that it is representative of the ability of the nonbleached lipids to disperse the photobleached ones indicating a liquid phase.
- Is there a way to measure this phenomenon in vivo?
- how does FRAP work? how analysis data based on this way?
Janelle: FRAP means Fluorescence Recovery After Photobleaching. Basically you fluorescently label proteins or lipids on the membrane and zap it with a laser. This leaves a photobleached spot and you can see how fast or slow the label moves based on the recovery of fluorescence (if the bleached area becomes colored again).
- Is the SV have different size? and if yes how the size control?
- They only talked about synapsin relative to synaptic vesicles. Would synapsin had other effects on other types of vesicles used in different body systems?
- They determined that synapsin's binding partner SH3 influenced the formation of the liquid condensate at low ratios. Why did they observe inhibition at high ratios? What kind of conditions cause the synapsin IDR to want to interact with each other at high ratios? Is it energetically favorable for these IDR's to interact with one another?
- Why were intersectin and GRP2 selected for co-incubation with synapsin? Were they selected solely based on the presence of proline-rich SH3 binding domains? Or would there be a possibility of these actually interacting in synapses?
- Is synapsin expressed in other areas of the brain/nervous system? What about in other parts of the body? Could there be other synaptin-like proteins modulating similar "trafficking" (not sure if that's the right term) between cells? The paper mentioned other liquid-liquid phase proteins had been discovered recently, I'd be curious to know the implications of those findings.
- This paper talks more about the structure of synaptic vesicles: it looks like they are quite complex structures. Yes, they contain synapsin, but they also contain at ton of other proteins. I'm wondering how the synapsin droplets studied here relate to SV struture/function? In this study, phosphorylation of the synapsin droplets caused them to disperse, but since SVs are much more complex, would this be a suitable model for what is actually happening in SVs? Is there a way to study this?
- Why might synapsin-1 form different size droplets?
- What other neuron-expressed proteins might be relevant to synapsin-1? Does the liquid droplet structure change which proteins synapsin interacts with?
- Does the binding mode of synapsin-1 affect its ability to be phosphorylated? (i.e., T vs R?)
- How does fusion with pre-synaptic membrane for neurotransmitter release occur if phosphorylation of synapsin causes SVs to fall apart?
- Do synapsins at different types of synapses interact with different SH3 binding partners?
- Is it a specific domain of synapsins responsible (or partly responsible) for the packing/clustering of SVs in the WT mice?
- The synapsin has an ATP-binding module of unclear function, do you think introducing CTP or GTP would affect this protein?
- Why are negatively charged lipids necessary for synapsin binding to vesicles?
- Homma: They mentioned that when they increased the salt concentration, it impaired droplet formation. This implicated that there were charge dependent interactions with their formation. I assume from this, that negatively charged lipids has electrostatic interactions with synapsin when binding to vesicles. Synapsin probably has some positively charged character that interacts well with a negative charge.
- Much of the imaging was done with synapsin 1 bound to Enhanced Green Fluorescent Protein (EGFP). Synapsin 1 is a ~74 kDa molecule. EGFP is a 32.7 kDa molecule. How could the addition of a molecule half its weight not change its binding properties and the way it would cluster? It'd be be like adding a nitrogen to ethanol then seeing how it behaves. The nitrogen should change that behavior right?
- How physiologically different are the two phases – e.g. pH. Do you think there could be a pathogen that take advantage of potential physiological differences?
- Does this liquid phase phenomenon occur in other cells, in additional to neuronal cells?
- How closely does PEG mimic the density of the presynaptic bouton? Why did they use 3% PEG?
- What other domains does the synapsin IDR bind?
- They mention that packing of SVs was lower in the TKO mice. It looks like there may be some clustering, could this be due to another factor that also has a role in SV clustering?
Week 2 - High speed of fork progression induces DNA replication stress and genomic instability
Layman abstract: DNA replication is a complex, finely-tuned process that is regulated by a wide range of proteins. One such group of proteins are poly(ADP) ribose polymerases, also known as PARP proteins. PARP proteins have been shown to be an important part of recognizing and repairing DNA damage that occurs during replication. Inhibition of these proteins has proven to be an effective therapy in certain cancers, including subtypes of breast and ovarian cancers. In this paper, the authors show that the therapeutic effectiveness of PARP inhibition might be due to how nullifying these proteins causes an acceleration in DNA replication. At high enough speeds, some DNA repair pathways cease to function properly. This dysfunction is overcome by backup repair pathways in healthy cells but leads to cell death in tumors that lack those same repair pathways. These results highlight the relevance of replication speed in the scope of DNA replication damage control and the authors propose how PARP proteins may have some regulatory control over such speed.
- The authors mention that their findings apply to both cancer and aging. They make the case for cancer, but how do their findings relate to aging?
- Breanna: I would assume it has to do with cancer and aging are very linked through the use of DNA damage repair pathways. If PARP inhibition causes BRCA (part of dna damage repair pathway) deficient cells to fail, it would also make sense that it could work similarly in aging. I also read outside of this paper that PARP is part of the BER pathway (specifically detection of breaks and signaling for repair) which again is important in both ageing and cancer *I'm not an expert tho so I could be totally off*
- All of the experiments were done in a cell line would this data be supported in primary cells?
- TUNEL positivity in cells is usually used as an indicator for apoptosis. Do these cells undergo apoptosis after PARPi?
- They briefly mentioned that PARP1 physically binds the P21 protein, but what does it do? They mention that PARPi inhibits nuclear localization of P21, so does it PARylate P21 to localize it to the nucleus?
- What is the mechanism for PARP regulation of fork speed progression?
- Did the authors discuss what breaks down when the fork speed becomes too fast? If not, what makes determines the threshold for "too fast"
- Is helicase or polymerase the rate limiting step for replication?
- Does temperature affect the rate of replication?
- Do slower polymerases generally have higher fidelity?
- The authors are studying PARP and fork progression, is it safe to assume that all cells they obtained data from are in S phase? I didn’t see any cell cycle analysis or control.
- How did they measure fork speed and symmetry?
- Is there any disease related to mutations in PARP?
- Can DNA pol moderate the speed it moves along the DNA?
- Possible answer (by Kathrina): It probably can; DNA pol has exonucleolytic activity and it could perhaps "feel" if incorrect bases have been added.
- What factors change DNA pol's speed? Is the replication fork speed limited by DNA helicase, etc.? What is the limiting factor?
- What role do the other PARPs play? Could non-catalytic PARPs be involved?
- At times it was very confusing to follow the experiments and inhibition pathways they used. I feel like maybe the authors could have put in similar figures to Figure 4k to help visualize how they are modifying the PARP pathway.
- I'm a bit confused about one of the methods. How does the TUNEL assay work and what are they looking for by using this method?
- Julia: TUNEL assays screen for breaks in DNA. I believe the assay works by adding fluorescently labeled dUTP and the enzyme terminal deoxynucleotidyl transferase (TDT) which catalyzes the addition of the nucleotide to any free 3'-OH. Since the dUTP is then added to any free 3'-OH groups, it is effectively labeling any fragemented DNA which would have free 3'-OH groups. It is usually used to detect apoptotic cells (I think Haley touched on this a bit in her comment above) so I'm also wondering about Haley's question regarding the fate of the PARPi treated cells...
- Why did the authors not really look at other cell cycle stages in their analysis other than the S phase?
- The authors do mention that in one of their experiments, they looked at the PARP1 knockdown affecting fork rate and looked at the "cell cycle towards G1 enrichment." Why would they be looking at the G1 phase instead of the S phase in this case?
- They mentioned that PARP1 inhibitors would enhance deleterious effects? Do they mention the deleterious effects like increased cell death, mutation, and increasing the progression of cancer and aging of cells?
- How come fork acceleration is independent of cell type?
- Possible answer: all cell types, regardless of what type, have the same replication and DNA damage repair response proteins/pathways.
- Have there been protein-protein interaction assays to check to see which PARP proteins may be redundant to each other?
- What does PARP2 do in the elongation process? Is it a redundant protein of PARP1? And isn't it a given that a disabled p53-p21 interaction promote fast elongation if those two proteins inhibit DNA synthesis? (Referring to line: "We predicted that in cells that lack PARP2 and with disabled p53–p21, forks should elongate fast even after PARP1 knockdown, because PARylation might not be maintained and p21 may remain low.")
- How measure the distance between origins of replication?
- why use U2-OS as sample?
- At the end of the paper it conclusion present concept is also relevant for ageing? How it would be for ageing?
- Cancer cells are more difficult to treat because they often leave room for adaptation, especially under a selective pressure like the PARP inhibitors. I wonder if long term, adaptation studies would show cancer cells activating other repair mechanisms to decrease the effect of these PARPS.
- They also mentioned the use of PARPS in conjunction with chemotherapy. I wonder if there is a transcriptional mechanism that a small molecule can perturb to add another instance of mutagenesis within the cancer cells.
- How exactly does the PARPS not effect normal cells as much?
- Taking these findings into account, could olaparib or other PARP inhibitors find justifiable use against other forms of cancer, other than BRCA-dysfunction derived cancers?
- Do we see analogous fork-speed regulation mechanisms such as PARP in prokaryotes? How does their average replication fork speed compare to eukaryotic cells?
- Are there any observed cases of fork-speed that is too low? Would that phenomenon similarly trigger DDR pathways, or simply result in slower-replicating cells?
- If PARPs are important parts of DNA damage repair pathways, wouldn't employing PARP inhibitors increase the risk of an offsite secondary cancer?
Answer from discussion: Yes, but chemotherapy also has this issue so our tolerance for that risk is high.
- Can someone explain a Comet Assay?
- Considering PARPs are part of an important DNA repair pathway, have the PARP genes been investigated as potential oncogenes? Are there diseases known to be a result of a PARP mutation? If you can inhibit them without catastrophic results, I would think a mutation wouldn't outright be embryonic lethal.
- Is replication speed ever increased to induce mutagenesis for a process like V(d)j recombination?
- Is it possible that PARPs do not lower the general speed at which the fork moves, but instead act as a breaking mechanism which slows/stops the fork when damage is detected?
- Possible answer: I think this would be possible, and the results seen would be similar. I don't know a lot about DNA repair and the methods discussed in the paper, so I could just be missing something simple.
- What is being PARylated in the replisome?
- Does PARPi literally speed up elongation or is it simply removing the breaks?
- In what ways does their new model vary from the currently accepted model? And were there reproductions of the current model or is that model just based on a single groups work?
- PARPi inhibits the SSB repair pathway so it makes sense that this would result in cell death in cells with non-functional DSB repair, but does this lead to mutagenesis in healthy cells? Does the DSB repair pathway in healthy cells completely compensate for defective SSB repair?
- How did the researchers here draw the conclusions they did from the comet and TUNNEL assays – how did this help them determine that fork speed was accelerated rather than stalled? Neutral vs basic comet assays – what information do the different pHs give about the DNA damage present?
- What is treslin?
- ssDNA breaks activate PARP1 - how?
- When they talk about “analysing symmetry between first and second pulse in double-labelled DNA fibres”, are the referring to a pulse-chase experiment? How does this work?
- Would an overactive PARP result in increased DNA damage and therefore increased risk of cancer?
- The paper touches on the fact that PARP1 inhibition speeds up replication without stalling the replication fork and comes back to this point a few times. Is there a known limit to replication speed? Would these ~60% speed increases typically result in a stalled replication fork? In other words, is this something to pay attention to, or are speed increases of this magnitude well tolerated at the replication fork (aside from introducing DNA damage.)
- The PARP family consists of 17 different enzymes. Was the PARP inhibitor used in this study specific for PARP1? If not, or if this is unknown, is it possible that there are multiple functions/pathways being affected by PARP inhibitor? How would this affect the interpretation of the results?
Week 1 - The Neuronal Gene Arc Encodes a Repurposed Retrotransposon Gag Protein that Mediates Intercellular RNA Transfer
Layman Abstract: There is little known about the molecular function of Arc or how it was evolved. Currently, Arc is shown to be present in neurons and has been implicated as to play a role in neurodevelopmental disorders. This paper shows that Arc is capable of self assembling into a protein capsid or shell, similar to those assembled to viruses. These Arc based protein capsids encapsulate mRNA in one neuron and then export it to neighboring neurons. This article further suggests that, during evolution, the machinery necessary for this function was possibly repurposed from retroviruses or their ancestors.
- Based on the paper, Arc appears to nonspecifically bind RNA. Why is RNA specificity not important (or is it)? Are there ways to still release a specific signal without RNA specificity?
- Although RNA binding is not specific, RNA is encapsulated in a concentration dependent manner. Arc mRNA is specifically trafficked to the dendrites where translation then occurs. This partially rectifies the non-specificity problem as mRNA becomes highly localized and thus more likely to be transferred.
- The Authors note that in Schizophoran's Arc homologs have undergone multiple rounds of duplication vs. tetrapods that only contain a single copy. Does this confer some kind of benefit? How do expression levels compare between the two clusters?
- How might glia-neuron interactions mediate Arc dependent/EV dependent communication?
- Do these extracellular vesicles fuse with other cell types?
- Do other non-neuronal cells secrete these capsids?
- Julia: This Nature review from earlier this year talks about other forms of extracellular vesicles: https://www.nature.com/articles/nrm.2017.125 It seems like there are some intracellular signaling pathways that make use of mechanisms which are similar to the Arc gene function described in this week's paper.
- How does the expression and secretion change during development?
- Why would the body use these protein heavy capsids to transport RNA, do lipid vesicles not offer the same level of protection to the RNA?
- Wouldn't it make more sense for us to have a transport mechanism that wasn't directly introduced by viruses?
- They mention this being a gene linked to Alzheimers, but how could this mechanistically lead into the formation of B-amyloid plaques?
- Are these capsids found in other locations of the body that act similarly?
- Do these capsids specifically form around mRNA? If so, how is this selected for?
- Were these systems acquired from an outside system like a repurposing of viral machinery?
- Does this system use a receptor-mediated endocytosis or a different mechanism?
- Where do arc capsid monomers bind the mRNA?
- How does this binding promote capsid formation between monomers?
- Is there any immune response from the recipient neuron?
- Are ACBARs endocytosed by glia? Could this mediate synaptic plasticity?
- What is the mechanism of RNA binding induced capsid formation?
- Ashley et al. (2018) found that the darc1-mRNA 3' UTR mediates loading into the capsids.
- Are there regional variations in Arc expression?
- Did the fact that Arc transposons seem to have independently originated in fly and tetrapod lineages speak more to the propensity of Ty3/gypsy retrotransposons to integrate and perpetuate themselves or to the evolutionary advantage conferred to organisms having incorporated and repurposed this signaling mechanism?
- How do the capsids disassemble and release the mRNA once inside the target cell?
- What is the specific response in cells receiving Arc capsids carrying Arc mRNA?
- Did the phylogenetic comparison conducted include non-coding regions of the sequence?
- Is there enough structural similarity between Arc and Gag, such that an infection with HIV might induce antibodies against Gag which are cross-reactive to Arc? ?ould this result in an autoimmune-type disease?
- If there have been multiple round of duplication, are there other genes that have a similar function?
- Within one species like mice (since we know many organisms have independently evolved a protein to have the same effect), are there other genes/retrotransposons in neurons that react like Arc and provide similar phenotypes to Arc? Different phenotypes?
- Does the Arc mRNA get sent out to specific neurons, depending on what the organism is learning?
- Is Arc constitutively expressed?
- Is there a mechanism or reason to why prArc capsids show little specificity for particular mRNA?
- How does the capsid molecules actually bind to the mRNA and how does RNA binding facilitate capsid formation? Does the region where RNA binds to important for transport between cells or is it non-specific?
- The authors predicted that they could use genetically engineer Arc and its ability for the capsid to harness RNA, so in what situations would it be beneficial to deliver RNA into cells without an immune activation response? Would this be important for gene manipulation and drug delivery to immunocompromised areas?
- What role do zinc fingers play and how can a cell compensate for a lack of zinc fingers?
- For the knock out mouse, they mention that they use both male and female mice and noticed no differences between sexes. Why might one expect differences between sexes?
- In figure S2, they used 10% SDS-PAGE Gel to analyze their results. How would the resolution of the gel change if you increased or decrease the percentage of SDS?
- Did Arc arise endogenously or from viral infections?
- It sounds like the hypothesis is that arc genes may have existed before defined viruses even existed (retroviruses, at least). There might have been an ancient divergence where some unicellular organisms favored arc-induced transmission as their primary form of propogation, leading to retroviruses, while others simply treated arc as a utility for their more complex replication pathways, leading to other early eukaryotes. -Colin
- Assuming arc has a role in memory development, is it possible/feasible to raise an arc-KO mice to maturity, then compare its learning ability to wt mice? What tests would you use?
- Julia: Yes, in fact this has already been done! The Arc knockouts were unable to form long lasting memories. This is studied using behavioral "learning" tests -- in this case, a Morris water maze which tests how the mouse is able to use spatial cues to navigate a round pool. WT mice were able to develop spatial "strategies" for navigating the maze, Arc KOs weren't. Check out this paper: https://www.sciencedirect.com/science/article/pii/S0896627306006465?via%3Dihub
- Do patients with neurodegenerative diseases have altered levels of arc protein in their neurons? If so, is the alteration global or localized, or is it different across individuals?
- Cont. from above, how would you be able to measure functional arc levels in patients in vivo? Some form of specific protein labeling
- What is the MVB pathway?and what is the end results of it?
- What are the differences between Cry-EM and Negative Stain EM?
- What is the rational behind using HEK293 cells? why not HEK392-FT cells?
- They mentioned that the knockouts were a "gift" from a collaborator, however, I was unable to find their method of knockout.
- How specific are these capsids for certain cell types, and are they specific to the synapse for neurons?
- If this is not a synapse specific transportation, do these capsids enter systemic circulation?
- What is the diffusion distance, are these impacting adjacent neurons or is it farther reaching?
- Does disrupting other parts of the TET2 pathway (maybe before TET2) also develop hematopoietic malignancies?