Microscopic observation in live tissue, awesome! But don’t ignore the methods

It has been far too long since I wrote a blog post. Look out internet, I have a blogging itch that needs scratching, and it’ll probably cause a rash!

…I apologize for that mental image.

The NIH sent me an email this week (via the various government listservs I’m enrolled in) that was proudly declaring that the mysteries of the cell were being solved right now, so I took the clickbait. In it was a cool study where we were able to actively watch mitochondria oscillate inside a living animal.

Fig from the article
http://dx.doi.org/10.1016/j.celrep.2014.09.022

There are two rabbit holes to enter with this article. The first is the observation of interest, which was mitochondrial oscillation. While these slinky moves have been observed in cell cultures, the authors wanted to see if there were any differences in cells that were part of a living, breathing animal.

Movement isn’t a surprising thing. If you’ve ever drawn the ATP synthase lollipop (totally relatable experience for everyone, right?), you already know that some of the main membrane proteins in mitochondria are constantly rolling around attaching phosphates to create ATP. The cool thing though, is that since they’re synching up with each other, that means there could be cellular communication mechanisms that are helping coordinate mitochondrial efforts to produce energy as needed. Which makes sense if you’re generating ATP in response to a stress event on a cellular level.

But what if you’re an animal? A slice of muscle tissue is made up of many muscle fibers, all of which contain mitochondria. It seems like there would be a need to coordinate increased energy production if you were planning to use all those fibers in sync to move. When looking at the cellular tissue inside a living rat’s salivary gland epithelium (the covering layer of the gland), the authors observed that mitochondria oscillated in sync not only within individual cells, but in sync with other cells in the tissue. The authors describe it in their press release wonderfully:

“You look through the microscope, and it almost looks like a synchronized dance”

It’s always more fun looking at living cells and tissue, it reminds you that all of that stuff we can’t see is always buzzing around without our notice and crawling all over our skin and gut.

Hypochondriacs I apologize for that second image.

So since we like looking at living things, let’s get to the second cool part that the press release seems to gloss over, how the hell were we observing cell structures in a living animal!

The principal author, Roberto Weigert, was the first one to publish this technique, so I went to that article to better understand what’s going on. I don’t want to dig into the microscopy so much, as the technical information is a little overwhelming. I’ll just say that there are really cool microscopes that can use near-infrared light to penetrate deep into different tissues (segregated visually by the injection of fluorescent dyes). The article has some amazing images of mouse vasculature that are both easy to observe and understand. But that technology isn’t what this blog is about. What I want to know is, what did this study entail for the animals used?

For this procedure, the research rats were anesthetized and had their salivary glands “externalized”, meaning that they gained access to them presumably by opening/removing the skin, fat, and muscle layers and segregating the gland as far as they could for the procedure (you’d be surprised how far you can pull things out while they’re still attached). Then, they bathed/saturated the glands with various dyes and chemical/hormone baths depending on what they were observing in that particular instance.

Once the images were taken, presumably these brave rats were euthanized, I couldn’t find a reference in the procedure but ultimately it had no bearing on the ability to replicate the experiment and was not included.

Now imagining these experiments in vivo (in living animals) brings up nasty words like vivisection. However it’s always important that the authors of the study aren’t left to singly decide if the research is necessary or not, it’s up to the Animal Care and Use Committee to allow the use of animal subjects for research at the NIH.

In order to use and ultimately euthanize these animals, the authors had to prove that: the information learned from the study will benefit humans and/or animals, there is a rationale for using animals including why a surrogate (e.g. cell culture) would not work (the authors make a great statement in their press release by describing the observations as if you’re looking at a tree vs. the forest), and a description of how the authors have actively attempted to minimize pain and discomfort for the animals used.

Ultimately I chose to write about this article because the methods were cool, but also to acknowledge the animal use inherent, but understated, in this type of research. It’s important to remember that often new information comes at the cost of continuing to support animal research when justified, and to not hide the facts from ourselves.

In order to responsibly care for all of our domestic species, we need to remember that before they were beef, eggs, milk, nuggets, or a data point, they needed to be cared for and euthanized humanely.

ResearchBlogging.org

Natalie Porat-Shliom, Yun Chen, Muhibullah Tora, Akiko Shitara, Andrius Masedunskas, & Roberto Weigertemail (2014). In Vivo Tissue-wide Synchronization of Mitochondrial Metabolic Oscillations Cell Reports : http://dx.doi.org/10.1016/j.celrep.2014.09.022

Weigert R, Porat-Shliom N, & Amornphimoltham P (2013). Imaging cell biology in live animals: ready for prime time. The Journal of cell biology, 201 (7), 969-79 PMID: 23798727

Urine protein profiles in cats with cystitis are indicative of increased epithelial damage

Cystocentesis - Image from University of Minnesota CVM

Cats and urinary problems go paw in paw, from the obstruction emergency in males, to common urolithiasis. As a cat owner, it is always important to search for a medical problem before blaming behavioral issues for “accidents” in the house. There’s a whole world of disease out there that can manifest itself as litter box trouble. This article in JAVMA discusses the protein analysis of both healthy cats and those with idiopathic cystitis, urolithiasis, or a UTI.

I’ve actually been thinking about performing a similar study now that I’m at a veterinary clinic with digital records, but do not have the control or finances to have as specific inclusion criteria as this study did (each patient that met inclusion criteria had urine cultures, bladder biopsy, and additional lab work to identify components of the urine performed). The results from this simple small study (n=60) are interpreted well and not only identify a protein present in urine correlated with idiopathic cystitis, but propose a mechanism for it’s presence. Clearly simple, specific, and thorough analysis of blood/urine chemistry have been paying off well for identifying these indicators.

The protein of interest the study found was fibronectin, a protein that plays a role in creating the extracellular matrix and adhesion, and is found in abundance around all cells. This adhesion role implies that fibronectin is crucial within epithelial tissues such as those lining the bladder and urinary tract. What’s interesting is that according to the article, while fibronectin plays large roles in wound healing, blood clot formation, and tumor invasion (Lemberger Et Al., 2011), it was not found in the same high concentrations in any of the cats in the study with conditions other than idiopathic cystitis. One would expect with any inflammation or blood present there would be the same abundance of this protein, but that was not the case. The authors proposed the mechanism that, with chronic idiopathic cystitis, there is significant fibrosis in the urinary tract which damages the epithelial walls. This damage is corrected by increased expression of the fibronectin gene so that the tight junctions between epithelial cells in the cell matrix can be repaired. Thus, with an abundance of fibronectin available to repair chronic damage in the urinary tract, some of it is released and flushed out with urine.

Further study is obviously needed with a larger sample size and more variability in cases, but if the authors’ prediction is correct, fibronectin could be an indicator of epithelial damage in other areas of the body as well. I’m not sure how exactly to go about identifying localized damage, but I am interested in the role that fibronectin could play in anticipating chronic renal failure in cats, as urine chemistry will often not yield an obvious diagnosis until loss of renal function is severe.
ResearchBlogging.orgLemberger SI, Deeg CA, Hauck SM, Amann B, Hirmer S, Hartmann K, & Dorsch R (2011). Comparison of urine protein profiles in cats without urinary tract disease and cats with idiopathic cystitis, bacterial urinary tract infection, or urolithiasis. American journal of veterinary research, 72 (10), 1407-15 PMID: 21962285

Article review: Mechanisms of Viral Emergence

It’s funny how reading these articles is incredibly relieving for me. They confirm that I actually did learn and do remember principals and details from my classes. Today’s article comes from Veterinary Research, and discusses the mechanisms and variables involved in the emergence of a virus.

A viral emergence is generally defined as the appearance of a new pathogen for a host, such as human immunodeficiency virus (HIV)-1 for humans in the twentieth century. Viral re-emergence often refers to the reappearance of a viral pathogen after a period of absence, such as the periodic human influenza epidemics or pandemics.” (Domingo, 2010)

There’s a huge amount of information on viral evolution and mutation, a concept that (like everything else you haven’t specifically studied) I had greatly oversimplified. It was incredibly fascinating to read how viruses can recombine and splice gene segments from other unrelated infections, and the various pathways new genes can be created or introduced that allow viruses to jump host species. I’m not going to try to summarize all of those, the article explains them much better than I could, but there’s still a few broad topics in the paper worth discussing.

The concept of viral quasispecies is something I’ve never heard about. I’ve discussed viral serovars on here before, but I believe that term specifically refers to antigen variation. Quasispecies refers to groups of virus that are labeled and operate as one species (for example, Influenza H1N1) but contain different genomes. For example, if I get the flu this winter, the virus I am exposed to may contain several quasispecies, that are the same virus, but contain different genes. Now any deleterious mutations or reassortments will probably be wiped out by my immune system, but the ones with a fitness advantage will reproduce and take over. Here’s the extra interesting part, even if I had been infected with just one quasispecies, I could still shed several while I’m infected. There is so much mutation and variation in viral reproduction that I would be generating new ones as a single host.

Quasispecies are important in emergence because they are a major source of viral evolution. We identify the “wild type” gene amongst quasispecies as a distribution of genes that characterize that viral species (consensus gene); even though amongst different quasispecies they may be found in different places in the genome. This is where the existence of quasispecies propels viral evolution forward. When you mix up the genome so much, you can create mutations that change nothing in the functional portions of the genome, but create subpopulations that are fine tuned to conditions that may not yet be occurring. As an example, it’s been assumed that SARS was introduced to humans through Civet cats, when it was originally a virus transmitted by bats. Contact with civets is common in areas surrounding SARS outbreaks (where they are raised for food), where contact with bats is much less common. It is possible that there was a quasispecies subpopulation of SARS carrying a mutation allowing it to jump species to humans long before it was even introduced to civets. When the disease was contained to bats, this mutation has no fitness advantage, but it can sit idle until a change in the environment (higher infection rates in civets, civets brought from more rural areas, there are unlimited possibilities) gives it an advantage. This essentially allows virus to be proactive in its evolution. Instead of waiting for selective pressure, new genes are created spontaneously in the event that they may become useful.

Civet, image from healthjockey.com

The article continued to discuss different factors in emergence, primarily focusing on new host emergence, and brought it all together as an example of biological complexity. My personal favorite example for biological complexity is the realization that we’ll probably never fully understand the complete pharmacology associated with feelings of hunger and satiety. Alternatively you can illustrate this principal by asking someone to model the weather. The idea is that there are so many variables, all interdependent on each other, that it is pretty much impossible to trace all of the factors that led to an event such as the host change SARS made from civets to humans. They illustrate this by showing that even if viral variation is sufficient to promote a host change, there are numerous other roadblocks that have to be surmounted to allow it to take place (interaction with the new host, sufficient amounts of virus introduced to the system of the new host, fitness of the mutated virus, exposure to multiple quasispecies or serovars, future shedding of virus adapted to that host species, etc.). It’s a paradoxal idea, because we have been able to successfully model systems like these through the CDC and WHO, and we have been able to successfully model complex systems like the weather, even when there are countless interactions that we can’t even begin to measure.

Read this article if you have any interest in virology at all. I learned a lot, and it’s interesting and fluid reading if you have a basic understanding of cell biology.

ResearchBlogging.orgDomingo, E. (2010). Mechanisms of viral emergence Veterinary Research, 41 (6) DOI: 10.1051/vetres/2010010

Article Review: Leptospira and Leptospirosis

In my latest ScienceDirect purge, I came across this article covering Leptospirosis. I had no idea it would be such a dense read, figuring it would be a simple review of the disease with emphasis on new discoveries. I ended up using a lot of immunology references and Google searches. This article isn’t just an entry from the Merck manual.

The article does a very good job of covering Lepto microbiology, but I was especially impressed with the point they made to identify everything we don’t know. Indeed that was the emphasis of the article, that lepto contains so many pathways unique to it as a bacterium that we don’t know nearly as much about it as we do something like E. Coli. Additionally, Lepto is extremely hard to culture, as you end up with non-virulent colonies. They identify and isolate the virulent daughters by inoculating lab animals.

You might assume that immunity to Lepto is a simple thing, given how prevalent Lepto vaccination is due to the zoonotic risk. However the article makes the point multiple times that immunity to one Lepto serovar does not grant immunity to others, though occasionally it can help grant passive immunity or resistance across different species. While the exchange of genetic material between parent and daughter lepto colonies is not well understood, it appears to be slow mutating, which is interesting given how unique the antigens between serovars seem to be.

There’s a lot of complicated immunology discussed in the article that I don’t feel qualified to comment on, but it’s very interesting, and I recommend glancing through. The more microbiology I learn the more I understand that 99% of the workings of the cell happen on membranes (a statement that probably produces a loud “duh” from any student, biologist, or doctor). For a more clinical discussion of Lepto, a simpler reference like Merck or Blackwells will help, as well as several peer-reviewed sources that the article itself recommends for information on clinical presentations.

Adler, B., & de la Peña Moctezuma, A. (2010). Leptospira and leptospirosis Veterinary Microbiology, 140 (3-4), 287-296 DOI: 10.1016/j.vetmic.2009.03.012