Category Archives: Notebook Entry

Sheep Antibodies

Up until now, I was under the impression that the DIG-labeled probes were the colored component used to stain the slides where pathogen DNA was present, but I was mistaken. The DIG probes are actually synthesized with antigens attached to the DNA backbone. Specifically, the antibodies are from sheep. We then apply sheep serum, which will bind to the antibodies in the backbone. If the pathogen DNA is present on our slides, the DNA will stick, and if it’s not, the DNA will wash off when soaked in a buffer. The antigens in the sheep serum will bind with the antibodies remaining on the slide. Finally, we apply a molecule that will show color when it binds to the antibodies. This makes a lot more sense than what I’d thought before, since it is similar to other antibody-antigen binding assays like ELISA.

I saw some black spots settling over the RLO phage slides after applying the antibody. I’m assuming since I hadn’t added the final incubator solution needed for visualization, it was just ink from the slide labels. If that was actually the color that attaches to the antibodies, then that’s good news for the RLO slides but bad news for our sea star slides, since I didn’t see any black spots.

I’ve been searchin’ so long

To find an answer!

We’ve been continuing the search through our DEGs to fill out pathways and to investigate what the diseased stars were up to.  We’re looking at Toll and Complement pathways as components of the sea stars’ immune response.  Further, we’re trying to see what the transcriptome might be telling us about how the signs of the disease.  Echinoderms have mutable collagenous tissues, which they control with their nervous system to become softer or more rigid.  In this way, their nervous system has a very large role in their bodies’ structure and function.  The melted, twisty stars seem to imply a role of both the nervous system and connective tissue.  Disruptions in either could be influencing the changes we see in diseased stars.  It will be interesting to see what else we can see in our transcriptome, but it will likely take much more research to be sure what’s going on.

Where can a naked armadillo and a dishevelled porcupine be found?

The Wnt pathway! http://web.stanford.edu/group/nusselab/cgi-bin/wnt/reporters. Play the video to see a baby Zebrafish expressing Tcf/Lef-miniP:dGFP.

My post today will be covering the weekend as well as today, so it will be appropriately long. The last few days have been quite busy as we work through all the sea star transcriptome data and got to participate in the RCN meeting on Marine Infectious diseases. It was an honor meet so many scientists focused on disease ecology and to hear many great talks over the weekend.

I have struggling with posting every day since I like to have a complete story to tell when I write, however, looking back at my posts and those of my classmates I can see what a great tool it will be moving forward. This is something I will be working on in the future to incorporate into my daily note taking. It is different to post with an audience in mind, but good I think. My work is always better when I need to explain it to someone else and that is how this feels, just more permanent.

The last few days I have focused on the significantly enriched sea star genes falling within the “adhesion” biological function category. I took a subset of the genes, and Reyn and Ruth looked at the other. For my set, I went through each one and looked up the function of the protein it encoded and other proteins associated with it. It was difficult to find papers focusing on echinoderms for all of the genes, but for the ones that could find them for I did. I noted if the gene was higher of lower expressed in the control vs. experimental animals, what the value of the log change was, and what the p-value was.

Clear grouping of gene functions started to turn up right away. There were several genes involved in the extracellular matrix and another set in mutable collagenous tissue. Here is the list of genes I looked at with notes on the reference relating to them and a summery of their function taken from UniProt: Adhesion-ME References listed

After going through the list I grouped the genes based on function (highlighted in the doc) and started looking up papers based on the potential interacting pathways. While the Wnt pathway was not well represented in my list I had many genes that were regulated by it so started searching for the related Wnt genes in the lists of immune and cytokine genes. In the diseased animals there is a general lowering of expression relative to the controls in Wnt and Frizzled, and an increased expression of genes associated with the Adherin Junctions (cell-cell adhesion). The Wnt/beta-Catenin pathway regulates the extracellular matrix through activation of beta-Catenin, but is also involved in mutable collagenous tissue through beta-Catenin mediated integrin signaling. The disruption of the Wnt/beta-Catenin pathway is associated with disease in vertebrates. A disruption of this pathway is seen in many types of cancer. It is well characterized in echinoderms due to the sea urchin genome. I then compared the list of genes I had hand curated to the genes discussed in Croce_2006_EvolutionConservedWntPathways_SeaUrchin. Since this paper describes only the genes found and characterized in the sea star genome there is more confidence that these are coding for a protein in the Wnt/beta-Catenin pathway. My next step was to search all the significant enriched genes on the list against those mentioned in Croce et al 2006.

So…what is going on in our challenge experiment Pycnopodias? The table below contains the list that I have evaluated and placed as a likely part of the wnt pathway or linked to through the literature. I have started to map them onto a pathway (human) of the wnt/beta-catenin pathway. I will wait until that is more complete before uploading it. There are several more wnt genes found in the immune category that I have yet to look at in relation to the ones I have here to see where they fit. A few of the ones here (the 2 spondins in particulare) have literature on them but I have not determined if it is relevant to echinoderms. This list does not reflect the interactions but the one I will post tomorrow will.

log2FoldChange pvalue ID Protein names Broad Group Found in Croce et al 2006?
6.69463 8.14E-21 Q8IUX8 Epidermal growth factor-like protein 6 Adhesion no
5.569046 3.87E-17 Q9BUD6 Spondin-2 Adhesion no
4.483540943 1.79E-06 Q9QYP1 Low-density lipoprotein receptor-related protein 4 Immune no
3.07154 6.60E-09 Q00651 Integrin alpha-4 Adhesion no
2.939162 4.26E-06 Q9Y2D8 Afadin- and alpha-actinin-binding protein (ADIP) Adhesion no
2.638696126 2.61E-10 Q99N43 Kremen protein 1 Immune yes
2.612389 0.00782 Q90W79 Contactin-5 Adhesion no
2.178693 7.64E-04 Q9C0A0 Contactin-associated protein-like 4 Adhesion no
2.147782 4.10E-03 Q5RD64 Contactin-associated protein-like 2 Adhesion no
1.801941 1.07E-03 Q4JIM4 Presenilin-1 Adhesion no
1.539407 0.001994 O14522 Receptor-type tyrosine-protein phosphatase T Adhesion no
1.467166 0.000402 P35223 Catenin beta Adhesion yes
1.226512 4.60E-03 O94985 Calsyntenin-1 Adhesion no
-1.41984295 0.006131503 Q8NCW0 Kremen protein 2 Immune Yes
-1.930118114 0.003009238 Q6FHJ7 Secreted frizzled-related protein 4 Immune yes
-2.262594994 0.001609021 Q5BL72 Frizzled-7 Immune yes
-2.41329 6.24E-07 Q9HCB6 Spondin-1 Adhesion no
-3.66822 5.43E-07 Q9VBW3 Tyrosine kinase receptor Cad96Ca Adhesion no

Clevers_2006_WntBetaCateninSigDisease covers wnt/beta-catenin signaling involvement in human disease. Deregulation of the system leads to cancers in many cases. Another paper on inhibitors and activators is particularly interesting since it several of the genes I have identified are inhibitors (Cruciat_2013_WntInhibitors&Activators).

After completing the analysis of the wnt gene I will look at the adhesion set of genes. The two are linked so there will be overlap in the interacting proteins. The first step in looking at this (as with the last set) will be to find as many of my significantly enriched genes as I can that are associated with echinoderms. The echinoderm adhesome is described here: Whittaker_2006_TheEchinodermAdhesome.

The last step is to merge to the two sets of interacting proteins that I am most confident in (from the echinoderm literature) so the connections between them become clearer. After that I will add the genes annotated to other organisms to fill in where they fit.

 

 

Group Projects

On Sunday we were all able to present to the RCN our group projects! It was a great opportunity to share with everyone our projects and to illustrate what has been keeping us busy these past few weeks.

I thoroughly enjoyed listened to the SeaStar Transcriptome Project. It was great to see how far everyone has come in developing results that are quite substantial and SO AWESOME! Go Team SeaStar! I look forward to keeping posted on their project! They have all been working so hard and appear to continue to work extremely hard to produce more great results!

I was also proud of Team Laby! We presented what results we had to far! And while some results may not be statistically significant, we are seeing trends with both our field and laboratory experiments. So hopefully in the future (once are replicated experiments are finished) we can confirm (with statistics) some of the trends we are seeing :).

Research Coordination Network (RCN)

This weekend we were lucky enough to be involved in the RCN!

Saturday we were able to listen in to some of the most brilliant scientists of today! A wide range topics were  covered and they were all very informative and interesting! Some to note include…

Kevin Lafferty: The Oceans are downstream, economic impacts

Jeff Maynard: Marine Disease Projections

Paul Hershberger: Pathogen persistence and perpetuation strategies in marine fishes: Perspectives from Pacific herring

And these are only a few! Many more speakers spoke that day and they were all AWESOME! It was very interesting to hear about all the research that is currently being conducted ..locally and internationally!

 

 

 

Prehybridization and finally hybridization

We needed to make up some probes for the RLO-infecting phage, so it made sense to hold off on running ISH on our sea star probes until we could run the RLO batches with them. The control tube for the probe boiled off again in the thermocycler last night. The lid was closed but there was a tiny crack in the upper part of the tube. Since this is the second time it’s happened, we think that we’ve bought a low-quality batch of tubes that don’t hold up well to being pulled off the 8-tube line into smaller groups. After using some probe for the gel, there is under 1uL of control probe left. We’re hoping that this is enough, since we dilute it at a factor of 1:1000 to bathe the tissues on our slides.

To prep our slides, we washed off all the paraffin with SafeClear, a less toxic alternative to xylene, and then slowly rehydrated the tissues with different proportions of ethanol and water. While not taxing, it took a lot of waiting time. When the tissues were finally hydrated, we had to applied a prehybridization solution to ready the samples for binding with the probe DNA. To save on reagents, we needed to bathe only the parts of the slide containing tissue. We got to use a pap pen to create a hydrophobic boundary around the tissues. Watching the fluid not escape the ovals we’d drawn was very cool, even though it’s not a complicated phenomenon. Finally, we applied our diluted probes directly to the thin slices of tissue and left them to incubate overnight. Fingers crossed the probes bind and we get some visible staining that indicate where they’ve attached.

2014-08-18 12.23.01

Awesome-ish

Fishing for complements (no, not that kind!)

This just in! Based off a preliminary look at the genes in the sick stars, the complement cascade and coagulation pathways are both undergoing changes in expression levels. The complement cascade, an integral part of the innate immune response with downstream effects including opsonization and phagocytosis, also has changes in gene expression of key complement factors.

vWF is an important clotting protein, both there are also several ADAMS (vWF-cleaving) proteins that are changing expression levels. Interestingly, I’m having a hard time finding any serine proteases (clotting factor activators) with differential expression levels.

What a weekend! The RCN talks were extremely interesting and I could go on about the exciting strides these scientists are making in marine disease diagnostics, outbreak forecasting, and marine disease communication.

complement and coagulation cascades

RCN and more

I am so grateful to be able to attend the EIMD RCN Meeting. It was really nice to see familiar faces (Bette Willis, Katie Sutherland, Melissa Garren) and of course it was good to meet new people and put a face to a familiar name.

Of the full 15 mins talks from yesterday, I think that I liked Katie Sutherland’s the best. Shifting targets of pathogens is something that our lab has struggled with during the process of discovering tho three bacteria that can cause tissue loss in the Hawaiian rice coral. Also, could Acropora palmata be developing mechanisms to control Serratia marcescens infections?!?! I had not heard of this phenomenon. Makes me wonder if the Coral Probiotic Hypothesis is at work.

Updates from the Oyster Filtration Experiment Part 2. Yesterday was 48hrs of oysters in the beakers, so they were removed and replace with the eelgrass. We dissected and saved tissue from 3 out of the 10 oysters from each beaker. Casey, Cody and Rachel noticed that many of the oysters didn’t look healthy. Instead of the digestive glands looking orangeish-pink, they were often brown to black looking. We did save i tissue slice for histology. I hope that the oysters were healthy enough to feed normally.

Today when we photographed the eelgrass, there were very small dark spots only in the SL treatment. I am really excited to see if these  dark spots really turn into progressing lesions.