Why do we (and why should you) care about all of this stuff??!!
OK, granted, all the stuff on the previous pages may be of limited use/interest to anyone but the few hundred of us actively engaged in this kind of research, but that doesn't mean that we all shouldn't care about this kind of thing. In my opinion, we should all become more interested in how we get to the level of knowledge we currently have since this is what drives the future of medical care throughout the World. Secondly, of course, is that if you live in the US, this is your tax money at work- understand it, don't dismiss it as unapproachable. In fact. most of us who work on the basic science side of biomedical research really LOVE to talk about what we do. Some researchers are better at it than others, but what else is new?!! So here are some facts that I hope will let you see the WHY in the research I am most interested in:
1) Hearing impairment is a significant occupational risk, even after some of the more typical venues leading to hearing loss have migrated overseas (such as steel foundries, etc.). Think about it- how many times have you seen the guy operating a jackhammer while wearing his protective ear muffs on TOP of his head, rather than on his ears? I live in a pretty urban environment, and I can say that I see this every week. The current military deployments also come with significant hearing risks.
2) Approximately 15% of the entire US population (and I'm sure this is probably the same throughout the world) suffers from some sort of hearing impairment. This number has held pretty steady over the decades. However,
3) Numbers coming into the CDC now indicate that young people are exhibiting signs of hearing loss that historically did not occur until much later in life. In fact, evidence shows that approximately 20% of kids aged 12-19 years old are now showing up with early signs of hearing loss! These people have their whole lives in front of them, and they will probably continue losing hearing function over the next 60 years of their life- what will their hearing function be like then? What will they be missing that they currently take for granted now? It's kind of paradoxical that the very thing they love is the thing that will rob them of their future enjoyment of not only that activity, but also of many other aspects of Life that are so much more meaningful with sound.
For High School, or even Middle School students: Get engaged. Ask local researchers to come to your school to talk about their Science, what kind of life a research scientist leads, etc. You'd be surprised at the reaction you will get from us. And remember, all things in moderation, including exposures to loud sound, will keep you healthy and happy!
Analytical Methods Employed
Developmental Analysis
Much of our work involves experiments designed to investigate the genes that are involved in normal developmental processes. The experimental design/model we most often exploit makes use of our ability to induce precise genetic modifications in mice. Using such molecular tools, we can then ask whether the gene being manipulated plays a role in development, and if it does, what are it's roles. This is the classic reverse genetics approach, in which we are looking for the phenotype being expressed under different genetic conditions. From these kinds of analyses, we can then build up a story pertaining to the role of any gene of interest in normal development and adult function (assuming that the genetic modification is not lethal).
Here's a cover illustration generated from some of our data describing synapse formation in the cochlea (Murthy et al., 2009).
Here is a small section of the cochlea stained for the inner hair cells (those hair cells most responsible for our sense of hearing) in red, and the nerve fibers that connect the hair cells with the brain (in green). The next 2 images come from work done by Christine Graham (see Graham and Vetter, 2011).
We ask questions concerning the role played by different genes and the proteins they encode in the normal development and function of the cochlea. Here is an example of what happens to the hair cells when the gene that makes the corticotropin releasing factor receptor 1 (CRFR1) is deleted from the developing mouse. Note the small size of the inner hair cells (red), and the abnormal manner in which the nerve fibers (green) contact the hair cells. The top panel is the normal state,and the bottom is a picture from the mutant mouse (the mouse without the CRFR1 gene).
NETWORK ANALYSIS
We do a lot of work examining the state of gene and protein expression levels and how they change under different conditions, e.g. during normal development, or under abnormal selective pressures due to genetic modifications induced in the lab (gene knock-out, over-expression, etc.) One paper we recently published (Turcan et al., 2010) demonstrates some of the kinds of analyses we pursue in order to begin assessing/generating novel gene and protein networks. For example, these two videos (.wmv format, so if they are not running on your OS, you may need a converter) show movies in which we have examined and codified self organizing maps (a type of clustering) using the GEDI (Gene Expression Dynamics Inspector, see http://www.childrenshospital.org/research/ingber/GEDI/gedihome.htm) software to relate dynamic gene expression differences during development in a wild type and a knock-out mouse line. These data relate to the set of genes expressed by the cochlea, and show how expression of gene sets change under the selective pressure of alpha9 nicotinic acetylcholine receptor gene expression. For more in depth information, see our PLoS One paper (Turcan et al., 2010).
wild type expression
knockout expression
Another way in which we have been chasing down expression changes that occur during development in the face of induced gene expression states has been via the use of quantitative proteomics. In the standard "heat map" style cluster analysis presentation below, we have included the top proteins undergoing expression level changes following the loss of the corticotropin releasing factor (hormone) receptor 2. Protein samples were processed from a cell line derived from the mouse cochlea (OC-K3 cells), and subjected to no drug (condition 114), gentamicin treatment (condition 115), urocortin 2 treatment (condition 116), or urocortin 2 pretreatment followed by challenge with gentamicin (condition 117). Note that gentamicin is an ototoxic drug, and urocortin 2 was used to selectively actibate the CRFR2 expressed by the cells. Samples were labeled with iTRAQ reagents and submitted to LC-MSMS. Expression levels were determined, and the top expression changes were then used for this clustering. You can observe that the proteins fell into 5 main categories, and relate to the fact that under urocortin treatment, cells survived the gentamicin challenge. Red is high expression level changes, while green is low expression level changes. This data also helps us begin to understand the topology of signaling cascades (i.e. the step-by-step signaling that occurs in a cell to, in this case, survive a metabolic insult).
Finally, many of these kinds of data can be put into signaling maps as demonstrated below by Dr. Johnvesly Basappa, a former postdoc of the lab. Here, we have used protein expression data obtained from the intact animal (i.e. not cell lines, but rather the cochlear tissue itself) to generate a quantitative mapping of cellular signaling changes that take place under noisy conditions in normal (wild type) and CRFR2 null mice. This first image demonstrates what happens along a variety of signaling pathways when the normal mouse is exposed to sound (approximately 50-60dB). Quiet conditions are used as baseline, and numbers reflect an up or down regulation (expressed as a ratio of quiet to noise expression levels):
In this next image, the same is done for the CRFR2 null mouse:
Of course, we can also directly compare the wild type condition to the knockout condition. Under both quiet (first image) and noisy (second image) conditions, we can begin to resolve the differences that occur between these two genetic states, and how the CRFR2 gene and protein is involved in setting baseline auditory processing signaling:
KO quiet versus WT quiet-
KO noise versus WT noise:
These kinds of data help us better understand the role of genes (in this case, the CRFR2 gene) in hearing and hearing loss.
We use many different software packages to analyze our expression data and overlay such data on protein:protein or gene:gene interaction maps. These include (but are not limited to) Ingenuity Pathway Analysis (IPA) software package, iHOP, STRING, and Cytoscape. Much of our gene expression statistical work is done in the R environment (especially using BioConductor for the Affymetrix chip data).
