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Monthly Archives: December 2017

Project Progress (2) Outline of Project

Topic chosen: Proteins that are involved in the formation of tricellular junction in developing Drosophilia melanogaster’s wing imaginal disc.
We know:
Gliotactin is positioned onto bark-beetle at the tri-cellular junction during development. However Gliotactin does not directly interact with bark-beetle. The intracellular domain of Gliotactin contains a PDZ binding motif domain which interacts PDZ-containing proteins, such as scribbled and discs-large.

SPECIFIC QUESTION:
Using blast to find drosophila proteins that are similar to those that are known to interact with human homolog of discs-large, potential drosophila proteins that interact with drosophila disc large is identified. This project attempts to identify which proteins are potentially interacting with gliotactin during the formation of the tricellular junction. A falsifiable model will be proposed.

HOW IS THIS QUESTION NOVEL AND ORIGINAL?
The question of how gliotactin is position onto barkbeetle at the tricellular cellular junction during development is a known mystery. While other researchers in Auld lab have attempted to understand which proteins may be interacting with the tricellular junction, a interspecies blast has yet been performed. This approach to fish for the proteins that are important to the formation of tri-cellular junction is novel.

POTENTIAL IMPACT OF THE PROPOSED QUESTION (WERE IT TO BE ANSWERED BY YOUR PROPOSED EXPERIMENT):

Although vertebrates do not have tri-cellular junction, they possess tight junctions which are analogous to the tri-cellular junction. In both system, fluid permeability control is affected when junctions fail to form. Some possible consequences include exposure to toxic metabolites, loss of control over ion exchange and homeostasis. These results to lethality and disease. In understanding which proteins interact with gliotactin, we can understand how the tri-cellular junction is formed during development.
Furthermore, human discs-large have been extensively studied as it is a known tumour suppressor. In Drosophila, it has been shown by Woods and colleagues (2006) that Drosophila Dlg mutations causes overgrowth in the developing larvae brain and imaginal discs. When dlg-mutant imaginal discs were transplanted into wildtype flies, cancerous outgrowths that mimic metastatic tumours were observed by Woodhouse et al. (1998).
While Dlg is not the master gene to tri-cellular junction formation, it is beneficial to understand the induction hierarchy that may control its function. The identification of proteins that may inhibit or enhance the function of discs large may be helpful in cancer research.

HYPOTHESIS:
Veli proteins, RE30311p, RE51991p, X11L proteins, Jagar, alpha-actinin and moesin proteins are necessary in the proper positioning of Gliotactin at the tri-cellular junction.

EVIDENCE ON WHICH THE HYPOTHESIS IS BASED (INCLUDE REFERENCES):
1) The alignments from blasts (refer to supplemental word document)

From this point onward, my research is based on doctoral dissertation “Tricellular Junction Regulation, Signaling and Scaffolding” by Dr. Zohreh Sharif Khodaei.
In Zohreh’s dissertation, she proposed that Dls and scribbled are involved in the tricellular junction for the bark-beetle and Gliotactin to be properly places. I will do the proper citation later.

2) Veli proteins, RE30311p, RE51991p, X11L proteins contain PDZ domains which likely interacts with the PDZ binding motif of Gli since it has also been suggested that Gliotactin interacts with Dlg and Scrib PDZ domains at the SJ strands through its C-terminal PDZ binding motif (p.15) This hypothesis is suggested because PSD-95 (a homologue of Dlg) is a binding partner of Neuroligin (Nlg; a homologue of Gliotactin) by PDZ binding interactions (p. 16)

3) when PDZ binding motif of Gliotactin is defective, extensive endocytosis occurs to form large intracellular vesicle. It has been suggested that interaction between Gliotactin and Dlg stabilizes Gliotactin (p.86)

4) there are 4 PDZ domain in Scribbled (Scrib) that mediates protein interaction and are necessary to target Scrib to septate/bi-cellular junction (p.10) perhaps PDZ domain conservation is related to targeting proteins to junctions.

PREDICTION(S):
Because veli proteins, RE30311p, RE51991p have highly conserved domains, they likely interact with Gliotactin’s PDZ binding motif. Jagar contains SH3 domain which is likely to interact with proline rich motif. Alpha-actinin and moesin had high percent identity and alignment score conservation and interacts with the cytoskeleton. In binding the model, these probably indirectly interact with Gliotactin but help pull it into place by interacting with microtubules or actin filaments.

EXPERIMENTAL APPROACH TO TEST PREDICTION (INCLUDE ANY DETAILS THAT YOU HAVE WORKED OUT SO FAR):
First, create a stock fly line that has engrailed-GAL4/balancer. The stock genotype can be used to cross with UAS-geneRNAi. This knocks out the candidate gene’s mRNA at the posterior compartment. The anterior compartment acts as an internal control. The wing imaginal discs can be dissected from 3rd instar larvae and immunostained with antibodies specific to Gliotactin and Disc-large. The knockdown phenotype can be observed. After identifying which proteins delocalizes Gliotactin and Discs-large at the wing imaginal discs. To identify if each protein directly interacts with Gliotactin and/or disclarge, PLA can be performed with antibodies specific to proteins that has passed the screen and antibodies targeting Gliotactin and Discs-large.

 Need a technique to track microtubules and gliotactin in vivo

LIST OF RELEVANT PRIMARY AND REVIEW ARTICLES READ, AND SUMMARY OF RELEVANT INFORMATION FROM EACH (this is the start of the annotated bibliography that you will need to include in your portfolio):

I’m organizing this part.

POTENTIAL WAYS TO MAKE YOUR QUESTION KNOWN TO THE PUBLIC AT LARGE (e.g. TO YOUR NON-BIOLOGIST FAMILY AND FRIENDS):
I will start a research blog to conclude my findings
I can also write a review article and post it on my facebook

ANY OTHER PARTS OF THE PROJECT COMPLETED SO FAR:
Supplemental documents, and all the blasts and alignments

ANYTHING YOU WOULD LIKE SPECIFIC FEEDBACK ON:
Experimental design

Project Progress (1) Draft of Research Question

> Given Disc Large 1 interacts with Gliotactin , protein candidates that may
> be involved in positioning Gliotactin on anakonda at the tricellular
> junction were identified. I would like to propose an experiment which
> tests whether CASK proteins, veli, uncharacterized Dmel amd PTEN2 proteins
> are necessarily in Gliotactin positioning.
>
>
> The rough experimental design part 1:
>
> Tag Gliotactin and Disc large with GFP and RFP. It is expected that the
> wild type control would exhibit co-localized signals at tricellular
> junctions in the wing imaginal disc. Using engrailed to drive GAL4 ,
> UAS-RNAis specific to candidate proteins would be expressed in the wing
> imaginal disc. Upon comparing the dorsum of the wing imaginal disc, where
> engrailed is known to express, to the rest of the wing imaginal disc
> which lacks engrailed expression, one can determine the necessity of
> candidate proteins in positioning. If the knock down of a protein causes
> delocalization of the disc large or gliotactin, it would suggest the
> protein to be necessary in Gliotactin positioning in the wing imaginal
> disc.

Our infographic on a nanochip that can “reassign” roles for cells

The infographic assignment was really fun.
What we did:
we choose a technique
Made a poster
had in-class poster session

One of my group mate is an engineer so he advocated for our infographic to be more application-based rather than an experimental technique.

Everyone else did the drawing (mostly the engineer) because I’m not a really good artist.

I hope everyone who came to check out our infographic had as much fun as we did. I really enjoyed discussing the potential of this technique with my classmates.infographic poster

The following is our notes. Feel free to read through them and comment to generate discussion.

goal: In vivo cell programming
nano-transfection by applying electric field that nanoporates cell membranes and moves DNA of interest (Tissue Nano-Transfection, TNT)
goal was reprogramming skin cells into induced endothelial cells (iECs)
reprogramming factors (all genes): Etv2, Foxc2 and Fli1 (referred to usually as EFF)
“Co-transfection of these three genes into the dorsal skin of C57BL/6 mice resulted in marked stroma reprogramming within a week”
“successful anastomosis [interconnected blood vessels] of the newly formed blood vessels with local functional cutaneous arteries.” happens due to new iCEs
the TNT technique is then used to recover mice limbs suffering tissue necrosis due to low access to blood

[Work to be done (and by who):
Talk about transformation technique (TNT) -> giulio
Talking about iECs made by reprogramming -> Ida
Talking about new blood vessels and how that stops necrosis (the main application of this tech) -> Katrine
Talking about applications, the future of the tech, potential harms/sideffects and more things to test out -> Rosalie
Put things together -> Giulio]

Summary of chip: (by giulio)
This chip has nanoscopic (500nm) holes etched onto its surface with lasers, just like most commercial electronic chips.
These holes are the only exit of a reservoir filled with an electrolyte solution full of the target plasmid (that has EFF genes).
When pulses of voltage are applied between this chip (negative pole) and the inserted needle (positive pole), the electrolyte and target DNA are shot out of the chip and into the skin.
Because the holes are so small, the electric fields (the path of electric charges) are very concentrated on the top of the membrane of the topical cells, so they are extremely likely to get transfected, but also not have too many holes in their membranes.
Because the current going through the tissue is smaller than in BEP (bulk electrophoresis), it is much less likely to cause inflamation.
Bullet points:
nano holes
focused electric fields
more transfection
less unnecessary membrane damage and inflamation

In vivo cell reprogramming and induced endothelial cells
In vivo cell reprogramming circumvents the need for isolation and induced pluripotency by using already available cell sources. To try out the transfection method, the researchers first used the previously established ABM (Asl1/Brn2/Myt1) reprogramming factors. ABM is known to reprogram fibroblasts into neurons in vitro. Reprogramming into neurons was confirmed by measuring Tuj1 expression and electrophysical activity. To confirm the origin of the reprogrammed cells, K14-Cre reporter system was used, as well as eGFP tagging of Col1A1 and the results suggested fibroblastic origin.

They also wanted to reprogram skin cells into induced endothelial cells, and therefore identified a set of reprogramming factors: Etv2, Foxc2 and Fli1 (EFF). Co-transfection of EFF was sufficient to reprogram fibroblasts into iECs in less than a week. This was confirmed by Pecam-1 and vWF expression and enhanced proliferative activity. K14-Cre and Col1A1-eGFP reporter systems showed that the iECs had dermal origin.

Restoration of blood flow (full text):
In relation to promoting reprogramming of somatic cells to iECs (induced endothelial cells) with EFF (a set of reprogramming factors to promote somatic cells –> iECs) TNT-mediated treatment, the research group study whether it also can lead to functional reperfusion (restoration of blood flow) of ischaemic tissue and whole body limb rescue. In both experiments laser speckle monitoring showed higher blood perfusion in EFF treated compared to control (limiting the tissue damage), whereas the control showed more pronounced signs of tissue necrosis compared to EFF treated.

Test of whole limb rescue: Transection of the femoral artery in mouse model, laser speckle monitoring recorded a significant reduction in blood flow to the limb immediately after surgery. EFF TNT showed improved perfusion 7 days after TNT-treatment (conducted on the inner thigh skin, 3 days post-surgery)

In the whole limb rescue an immunofluorescence analysis revealed revascularization far beyond the treatment area and inducing angiogenesis (formation of new blood vessels) in distal location within the limb (gastrocnemius muscle/calf muscle). By injecting EVs (extracellular vesicles), isolated from EFF TNT treated skin, directly into gastrocnemius muscle in a hindlimb ischaemia mouse model it also had the potential to induce blood vessel formation. In the article they propose/suggest that EV’s derived from EFF TNT treated skin might also play a role in spreading pro-angiogenic signals (preloaded with pro-angiogenic vascular endothelial growth factor) within the first hours after treatment besides its expected mechanism for propagating of EFF signals the target tissue.

Necrosis vs. apoptosis (simple explanation):
Apoptosis: programmed cellular death (tightly controlled and regulated)
Necrosis: (unprogrammed) cell death that is triggered by external factors/diseases e.g. infection. In this case low access to oxygen.
Keypoints:
EFF TNT-mediated treatment –> restoration of blood flow in treated tissue/whole limb rescue and induced angiogenesis in distal location within the limb in mouse model.
Pronounced signs of tissue necrosis in control tissue/mouse model compared to EFF TNT-treated.
Proposed model of EV’s spreading pro-angiogenic signals after treatment.

Application( by Rosalie [author of this blog])

In this paper, two different cell re-programmed demonstrated
From epithelial cells to neurons and from epithelial cells to endothelial tissue (blood vessel)

-Cell- reprogramming has been used before, this chip is unique because
Its application is non-viral
When delivery system is viral, there is a chance of introducing mutations into the genome
b) this approach is non-invasive and it is topical while rapid
c) unlike other transplant surgeries where tissue is taken from donor, this tissue is endogenous and there is no risk of tissue/organ rejection( because surface proteins of an individual)

Some potentials:
In-vivo correction treatment for degenerative disease
-when neurons are degenerative (as in alzheimer’s), the induction of epithelial cells into neurons to replace dying neurons help control disease

In-vivo regeneration of nerves damaged
-regrow short nerves to continue neural transmission when injury damaged PNS

Rapid treatment for Burn victim
-the reprogramming back to fibroblast to encourage tissue growth, direct cell growth to repair epithelia and increase perfusion by reprogramming cells into endothelial

Combination of Crispr/Cas9 or genetic engineering to treat genetic defects(cystic fibrosis )
-reprogram tissues (lung epithelia ) to fibroblast
-deliver gene editing technique ( mindful of off-target activity that may make new mutations → cancer) fix CFTR mutation
-re-differentiate cell that can expression functional copy of CFTR

R&R 5

This reflection was submitted at the end of the term. As you can see I have come a long way

I think imprinting the one thing I learnt that stands out to me. Before this course, my understanding of imprinting was limited to imprinting via DNA methylation. In learning imprinting in X-inactivation, I discovered imprinting to be a more complex and interesting topic. It can invovle complex interactions between methylated sequences, histone modification and lncRNAs. As mentioned in one of my R&R previously, I came into this course with the impression that a lot of biological mechanisms may be rather rigid, which is why this stood out to me. In a sense, biological processes are tightly regulated and are rigid in some developmental processes. However, most mechanisms are dynamic as they can involve many factors and components. I have realized most of what I learnt as classical examples of regulation are really oversimplification of complex systems. Therefore, I was under the impression that most biological mechanisms are unchanging, and rigid processes. This course has made me realized, contrary to my previous impression, most biological processes are very dynamic, involves many components and can be flexible to accommodate mistakes. While mutations, misregulation could result in observable phenotypes as we have learnt throughout our undergraduate courses, it is possible that mistakes in development were compensated or does not cause any phenotypes.
I was not very confident in my paper reading skills in the beginning of the term. I have become more confident in reading papers because I have learnt to read figures critically with all the practise we have in class and in assignment.
I have learnt that I am rather impatient as a learner. This is important because it affects my reading and learning efficacy. While a lot of things may capture my interest, I get easily sidetracked/distracted, so reading papers become difficult when the papers are more than 20 pages long and I’m too impatient to carefully read it in one sitting. This is something I think I should work on to help myself learn better.

R&R 4

This is a reflection on my exam question response strategies.

1. On an exam I try to word my answers as clear as possible. When I am answering a question in an inconsequential situation, I may get lazy and may not be as specific.

On an exam, I already attempt to think about the full answer before I start to write to minimize the errors and the need to cross out words. When answering questions in inconsequential situations, I tend to write or say the words as they come to me. Sometimes this results in grammatical error and poor sentence structures.

Now that I am writing this reflection, perhaps my way to answer inconsequential questions is the best, However, if the question is asked verbally in conversation, it would be difficult to pause mid-conversation for an extended period of time to carefully phrase it then verbalize my response. I find myself often give a response verbally the first time but my audience would not understand it so I would then rephrase it better since I have had thought about the entire response during my first attempt at explaining myself

2. I ensure I think carefully and plan my sentences before I write, I often use the margin of the exam paper to jot down some key elements and words that I would like to include in my response. Sometimes I would make myself a short outline or one worded outline so my response flows logically.

In situations where my response is inconsequential, i tend to look at body language and facial expression of my audience to see if my response made sense. If they look rather confused, I would immediately rephrase my response. In settings where my response is written, sometimes I would send my response to a friend to read it over to see if it made sense to him/her.

3. The biggest challenge for me is to really understand the paper’s techniques and the logic behind doing all the experiments in the preparation phase. When I encounter difficulties in understanding a paper, I often google for simpler explanations for a new technique or a review for that paper. I expect myself to have difficulty with time and writing a good response in the exam because it is something I have always struggled with. To overcome that struggle, I find it best for myself to be as prepared as possible so I can spend less time thinking about the response and more time organizing how to phrase my response.

R&R 3

This is my R&R in the middle of the term.
Here you will read about my reflection on my progress.
If you are curious about in my progress at the end of the term, please check out R&R 5!

My view on gene regulation

1. How would you describe your progress so far?

I have progressed much more in understanding various mechanism and critically thinking about different gene regulation system than I expected from the beginning of the course. I think this is due to having discussions in class and reading a lot of papers to experience the advances in our understanding of gene regulation directly from recent research.

2. Are you satisfied with your progress so far?

I am satisfied with my progress.

3. What evidence/pieces of evidence did you use to determine whether you have made progress?

In the beginning of the course, I thought everything in gene regulation is rather rigid and absolute ,which is only accurate in some cases. For instance, conservation of Hox gene expression is important for body plan development, however not all Hox genes expression is necessary for survival. I have never thought such variation in the effect of gene expression in this conserved pathway is possible. Moreover, I have come to realize that as in everything in biology, gene expression is very dynamic and can be rather plastic. In some regulatory pathway, many components are necessary for proper gene expression. In other pathways, some components come to interact with gene of interest and is sufficient in producing a different morph( as in caste differentiation in queen bee) but they are not necessary for proper development into adults.

I have also come to read papers with ease and critically think about what the take home message is from primary literature. I have also noticed as I understand the material more, I generate better questions to challenge my knowledge in gene expression.

(Thanks, Pam! It has been amazing to be part of your class because it really pushes my brain to think more)

You may either record your answers in your portfolio and provide the url link here, or type tour answers here and then copy-paste them in your portfolio.

R&R 2– Developing my Research Question

In Biol 463, we do not have finals, instead we have a final project. Our final project is writing a research proposal on a research question we want to answer.
In my directed studies at the Auld lab, I study what proteins affect the localization of Gliotactin at the epithelial tricellular junction. I have stumbled upon literary on an interesting protein which is associated indirectly with Gliotactin–Disc large.
Here is my R&R as I was developing my research question on disc large.
FYI: Disc large mutants are found to have enlarged wing imaginal disc, that is why the protein is named disc large.

1. In developing the draft research question, I have learnt to utilize Blast and NCBI extensively. I was expecting to learn to use these databases extensively because I was aware how much information was available through NCBI. I have also learnt how to prioritize which research articles and journals to read while consulting peer reviews. I was expecting to learn how to properly read articles and journals as I was coming up with a research question for the first time.

2. I expected I would have trouble finding the proper candidate proteins to test to answer my research question. This expectation was contrary to the outcome. I ended up with close to 100 candidate proteins to choose from after blasting for related proteins from a known system. Systematically organizing information helped prioritize which proteins to test as some homologs of candidate proteins have been tested in other systems.

3. The best strategy I have developed to prioritize the testing of proteins by evaluating how many of the following criteria they fulfil.

-Proteins with high percent identity with human proteins known to be involved with DiscLarge1

-Proteins with highly conserved domains which are known to interact with DiscLarge

-Proteins with multiple isoforms can be tested with one RNAi which carries consensus sequences among all the isoforms.

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