Work goes on and on

Work has been a bit slow lately. We just got a grant sent off to the NIH and are breathing a sigh of relief. I also have 3 residents in the lab. That is taking a lot of my time. Two of them are on 10 week research rotations so I am trying to ensure that their time is well spent and that they can hit the ground running. Luckily, they are all three pretty good. I taught one of them how to do a cochlear dissection last week and he picked it up immediately. I have never had someone pick up the proceedure that quickly. I also had to re-run a Western Blot for our Post-doc. For some reason, she just can't get one to work. I am not sure why. I think it is because she is trying to take shortcuts. In any case I got it to work on the first time - thankfully. I am not sure the results are what she expected but at least she has some results.

I have a big round of mouse surgeries coming up in a couple of weeks. We had a patient come in the clinic with a huge tumor. We are going to implant pieces of that tumor into 16 mice. The tumors will be placed intracranially and monitored by MRI pre treatment and post treatment. I will treat them for 3 months with daily injections of an inhibitor that targets a signalling protein called JNK (Jun N-terminal Kinase). The surgeries will be a challenge but the daily treatments will be a pain in the rear.

What have I done lately?

Time flies when you are busy. It has been nearly a month since I last wrote. I wish I could say that we have made earth-shattering discoveries this last month but that is never the case. Science moves forward one small data point at a time.

My mutagenesis experiement worked beautifully. It is always nice when something works out the way it is supposed to. I am looking forward to getting these plasmids packaged into viruses for further experiements.

I hired a new work-study student a couple of weeks ago. I am not sure how she will work out but I have high hopes. She has been very sick the last week and a half so I haven't seen her in lab but I am hoping she gets better soon. We have a lot to do and really need her help. I have been very fortunate to this point with my undergrads. This woman is the fourth student I will have hired and I hope she works out as well as the others. The last three students I have hired have been great - particularly the last one I had over the summer. This new woman is young (she's only a sophomore) which is both good and bad and both for the same reason - no experience. It's bad because I have to teach her everything but it is good because there will be no bad habits to break.

Today I am doing more Western blots (am) and then I will be doing some animal surgeries this afternoon. On the subject of animals, we have had an outbreak of Tyzzer's disease in the rat colony in one of our animal care units. The animal care facility has not been able to eradicate it so they are requiring us to kill all of our rats and start over (sounds familiar). Although it is a big headache because I now have to start my breeding colony over, I really can't complain. I only have/had 13 rats. Other labs are out thousands of dollars and have to start many ongoing experiments over. All of us can at least be thankful that the mouse colonies haven't been affected. I have a friend whose lab has 250 different strains of mice. Some of them are worth $100,000. How can a mouse be worth that much? If you take into account the amount of work that goes into making a transgenic mouse and the complexity of the process, $100,000 is quickly reached.

My timer is going to go off in one minute. I need to get back to work.

Progress

It's been a good week in the lab. I have had a couple of successful Western blots but am not sure what the results mean in the overall picture. Our hypothesis was that there is increased signaling in our tumor cells compared to normal tissue but that doesn't appear to be the case. We are making that observation after ensuring that there are equal amounts of the non-activated protein in each experiment. That, in and of itself may be faulty. I have found that even within the tumor samples, there is a lot of variation in the amounts of overall protein expressed. Perhaps the amplitude of growth promoting signals is controlled by the amount of signaling proteins available to be activated rather than just the amount of activation of the existing pool of proteins as is the accepted dogma. That would be pretty inefficient and not very likely but it certainly can't be discounted without further examination.

I am getting ready to perform a mutagenesis experiment next week. Oddly enough, I need to mutate a mutant plasmid to a wild type plasmid. Essentially is it like picking one letter out of a page in a book, erasing it, and putting in a new letter. Next, you put the letter in the context of a few of the surrounding words on another piece of paper and then hand that piece of paper and the orginal page to someone and tell them to insert your change onto a fresh page and make millions of copies. I really hope it works. I really need this wild type plasmid to continue on with some experiments that we have going.

Can I get worker's comp for allergies???

After the last year or so of scratchy eyes, runny nose, and sneezing fits almost every time I do a rat surgery, I have come to realize that I probably have a low grade allergy to rats. That is not good. I make my living working with rodents. Yesterday, I came in (yes, I was laboring on Labor Day) to do some surgeries and sure enough my nose started running and my eyes started to itch. It wasn't too bad but enough to notice. This morning, I went into the Animal Care Unit to check on my rats and my body went crazy. I couldn't stop sneezing. I probably lost a pound of snot and the only reason I can still see is that I was smart enough to wear my glasses this morning instead of my contacts. Without my glasses, my fingers would have had free access to my eyes and they would probably both still be in the animal care facility. Later, I ran into a friend of mine and casually mentioned the incident. He just smiled and in a stuffed voice somewhat similar to mine offered me some sudafed. Apparently, the same thing happened to him this morning. Thankfully, the rate at which my trash can is filling with used tissues is slowing.

I love my job

I managed to sequence through the important part of both plasmids. Sure enough, one of them showed a mutation at the 518 position just like it should have. The mutation was a serine to alanine. This means that at amino acid 518 we changed the peptide sequence from the wild type (serine - a phosphorylation site) to non-active mutant (alanine is not able to be phosphorylated). However, the other plasmid, which I thought was a wild type plasmid had a mutation at the 518 site that makes the protein always active. It is called a phospho-mimetic. That means that this mutation acts as though the protein was phosphorylated even though it is not. I need both of these constructs for the experiments that I have planned but I really wanted a wild type. Now I am going to have to go back and perform a site-directed mutagenesis reaction to change one of the plasmids back to wild type. The joys of molecular biology.

On Friday, Marlan and I did performed a couple of really challenging surgeries. One of the criticisms we recieved on our most recent grant proposal to the NIH was that the tumor placement in our mouse model (taking a brain tumor and placing it between the shoulder blades of a nude mouse) was not accurate. The reviewers wanted us to place the tumor in the mice in the same position as we find it in humans. It is hard enough to do skull base surgery in humans (from what I have observed) but to do it in mice would be quite the accomplishment. The first two mice we practiced on died. However, those failures enabled us to figure out the correct approach and to speed up the process. We managed to keep the last two mice alive throughout the surgery and through recovery. It was incredible. The surgery was performed under a microscope. By carefully exposing our site of entry, we were then able to drill out a small window in the skull, retract the cerebellum, and find the vestibular nerve which will be the area onto which we will place the tumor. This is going to be a cool experiment.

Moving on

Sequencing is back. Although I haven't managed to sequence through the whole construct, what has come through is correct. What a relief. Only one more construct to do for this project. Let's hope it doesn't take me forever like the first two did.

OH YEAH!!

I'VE GOT A POSITIVE CLONE! I still need to sequence it but the banding pattern on the gel looks correct. I did a cartwheel in the lobby outside my lab yesterday.

Happy dance!!!!

I've got colonies! So I am doing a happy dance but with limited enthusiasm. Just because bacteria grew, doesn't necessarily mean that my plasmid is there. One of the reasons I am a little leery is that nothing grew on my positive control plates. That's not a good sign. I am going to go forward anyway and see what I've got.

No wonder I am going gray

I don't have to kick myself. The one thing that I thought would fix my Merlin problem, didn't. I figured that I was heat shocking the bacteria too long and that they were all dying. It made perfect sense. Yesterday, I decreased the time I heat shocked the bacteria but when I came in this morning, there was no growth on any of the plates. This afternoon, I am going to try a different approach. Instead of using heat to open up the bacterial wall, I am going to use electricity. If this doesn't work, I am pretty much up against a wall.

Dad, you said that you didn't understand what I have been talking about. Let me attempt to explain it. What I am doing is essentially editing. I am taking a sentance from one page, cutting it out of its context, and pasting it into a new paragraph with a new context. Instead of words, however, I am doing this with DNA. I begin with a process called PCR that "photocopies" the stretch of DNA I am in which I am interested (in this case, a gene called Merlin). This strand of DNA is then pasted (called a ligation reaction) into another strand of DNA (a circular contruct called a plasmid. Plasmids contain regulatory elements that allow my gene to be expressed efficiently) that has been cut open (this process is called a restriction digest) at sites that are complimentary to the ends of my DNA strand of interest. Now I use nature to make more copies of my finished product. I do this by introducing my plasmid into bacteria (usually a strain of E. coli) in a process called transformation. Finally, I plate my transformed bacteria onto agar plates and incubate them over night. The problem I have been having is that I am not getting any colonies on my plates, not even in my positive controls. Hopefully this explaination has been helpful.

Christmas in July

This time of year is always a time of change. July 1st is the day the new residents start at the hospital (mortality rates will be up across the nation during July, I am sure) and it is also the day that a lot of the new faculty members start. We had two established members of our department leave this year. It was bad for the department but good for our lab. The reason it was good is that we got to be vultures and pick over the remains of the the labs. Any equipment that was purchased with department funds has to stay here and can be redistributed among any lab that needs/wants it. I figure we scored over $100,000 in equipment including an ultracentrifuge (~50K), two new cell culture incubators, a new biosafety hood, and a large capacity liquid nitrogen storage tank called a cryosafe. The problem with getting this new equipment is getting it all over here and installed. Most of the stuff I can move myself but several things are just too large to move by myself or need to be hooked up to central gas and vacuum lines or need to be plumbed, etc... . This means that I have to put in work orders with the Facility Management Group. Red tape, red tape, and more red tape. Whatever happened to just calling down to the university plumbing office and scheduling a plumber? Heaven forbid it could be that easy. I have three work orders in and only one of them has been responded to. It is taking over a month to get all this done.

Research is going fairly well. I think I have figured out my Merlin cloning problem. I am going to redo the experiment this week to see if my fix works. My fingers are crossed. I will kick myself on one hand for not figuring it out sooner but on the other hand will be doing cartwheels.

Merlin is still a pain in my neck

I took a couple of days last week to finish the Merlin cloning experiement but to no avail. I didn't get any colonies on my LB/amp plates. Not even in the positive controls. I decided to check my vector to make sure it was being digested properly. Yesterday, I did 8 restriction digests in an attempt to figure this out.

This project is my Moby Dick. I have been working on it for so long that I just can't conceive of not finishing it. It has been a real source of irritation. It is a very straight forward cloning experiment that should go off without a hitch - even with the variables that are inherent to biology. I think I have narrowed down the problem to the ligation reaction. I redid the experiment today so I guess I'll see if I solved the problem tomorrow morning if I have colonies on my plates.

My boss has been out of town for the last two weeks and the bulb housing for the mercury bulb in our epifluorescent microscope burned out. This means that all the immunofluorescence experiements for the last week have been put on hold. Unfortunately, nobody told the cells to stop growing and we have had to come up with experiments using assays other than immunofluorescence. Normally this wouldn't be much of a problem but I am not up to date with everybody's progress and therefore don't necessarily know what needs to be done or redone. It has been a real pain in the neck. Marlan is back tomorrow and then starting Friday, I will be out for a week and a half. The break will be nice.

Merlin is on track

Work marches on slowly. I did a PCR of wild type and a mutant (serine to alanine at position 518) Merlin. I actually got a band on the gel at the correct place but was still not sure if I successfully added the correct restriction enzyme sites to the 5' and 3' ends. So, I sent the PCR products in for sequencing and lo and behold, 8 of the 10 sequences were correct. Cause for a minor happy dance. Next, I will do a DNA clean up reaction. This will essentially filter out all the extra stuff from the PCR reaction such as residual enzyme and salts, leaving me with pure DNA. I can then proceed to do a restriction digest of both my PCR products (to "rough up" the ends) and of the viral vector that I will be dropping the PCR products into.

I just hired a student to work for me for the summer. He has no lab experience but is working out well. He is smart and is a hard worker. He has already gotten some results this week on a project that my last student (who just graduated and is going on to dental school) started. It is a pretty simple project but has been giving us headaches. One of the limitations of our work is the availablity of tumor samples. Many labs get around that problem by using cell lines. Cell lines are great in that they can be propagated for years and years (HeLa cells are cervical cancer cells that have been around since the 1950s) and are available whenever they are needed. On the flip side, immortalized cell lines are also a screwed up. You have essentially taken what in most cases are normal cells and made them into cancer cells so that they will continue to grow. This means that your results need to presented with the caveat that what you are reporting may not necessarily be exactly what happens in vivo. We all accept that and continue on with our work. We would like to be able to work with a cell line because we wouldn't have to be so dependant on someone getting cancer. However, there are no good vestibular schwannoma cell lines. As such we decided to make our own. We have two cell lines that we are working with and as part of the characterization process we are attempting to sequence the gene that encodes Merlin. We have isolated genomic DNA and are in the process of PCRing managable parts of the gene for sequencing. We have been having problems finding the right conditions but yesterday we actually got it right. With one part down, we have about 10 more to go. Let's keep our fingers crossed.

Old McDonald I am not - it just seems that way

I think Dad always wanted one of us to become a farmer and follow in his footsteps. It would appear that he has been granted his wish - kind of. Instead of raising hogs, I am going to be raising rats. We normally order in one pregnant rat every week but they are expensive. We were ordering from a company called Harlan but they were charging us just over $100/rat. I found another vendor that carried the same type of rat for about a third of the price. I was pretty happy about that until I noticed that the number of pups per litter was severely reduced. There also seems to be quite a bit of cannabalism going on. I know that happens with hogs occasionally also. I think we are being sold first time mothers who just aren't having very big littters (less than 8 pups). As such, we are going to start our own breeding colony. This will save us a lot of money and hopefully provide us with all the pups we need. Rats tend to be quite prolific. Their gestation period is only 21 days and their estrus period is only 4 days.

If it worked the first time, we would just call it search

I have been unsuccessful finding a clone that contains any one of my merlin constructs. It's a good thing that there is a plate full of bacterial colonies to pick through. I only need one colony to of each construct.

Today I am switching gears a bit and doing some cell culture. I have six plates of vestibular schwannoma cells that I am going to treat with a kinase inhibitor. I will let them incubate for a couple of hours, isolate the protein and run Western blots. Pretty straightforward and simple. I also have a couple of dissections to do this afternoon. I am going to remove the cochlea from a couple of mice. One of them is what is called a knockout mouse and the other is wild type. The knockout mouse has a gene, in this case a neurotrophin receptor, "knocked out" of it's genome. Additionally, one ear on each mouse has been deafened. I will begin by perfusion fixing the brain with paraformalydehyde. Then I will remove the temporal bone and proceed to dissect out the cochlea. After removing the stapes from the cochlea, I will make sure that both the round and oval windows are open and finally place the cochlea in fixative. Once fixed, I will de-calcify the bone with EDTA (yes, the same EDTA in the ingredient list on your box of Fruit Loops). Following de-calcification, we can section the entire cochlea into 7-10 µm thick slices, place them on a microscope slide, and label them with fluroescent tags to various proteins. By labeling the appropriate proteins, we can begin to get a sense of what role our neurotrophin receptor plays in deafness. I'll keep you updated at to our results.

Back to Merlin

I am finally getting back to some molecular biology. Although I don't particularly enjoy working with DNA, it is vital to what we do. Last night I inoculated some media with a colony of bacteria that I am hoping contains my plasmid. When I came in this morning, I noticed that somebody had turned the incubator down to 30 degree C. I should have checked it when I put my cultures in but in reality, the incubator has never been at any other temperature but the 37 degrees C necessary for optimal bacterial growth (at least for the vast majority of bacteria used in molecular biology). My cultures still grew but not as well as they should have. I turned the incubator back up to 37 and am letting the cultures grow for a couple hours more. After they are done, I will isolate the plasmid DNA and do a restriction digest to see if my desired DNA sequence is actually there.

Hah!

Well, I redid the experiment and got cleaner results. At first I was going to mess around with the amount of protein I loaded but as I thought about it, I got the distinct impression that the only thing I should change was the concentration of blocking buffer - which is something I never do. Blocking buffer is usually a milk or albumin solution that helps to reduce non-specific antibody binding on the Western blot. I have always used a 5% solution. Yesterday, as I was planning the experiment, I got the distinct impression to use a 10% solution. I did and my results today are easily understandable and interesting. I found that pERK signalling is increased in vestibular schwannomas while not in control tissue. Although we expected this, the results are gratifying none the less. However, I didn't see any differences between the two tissues with regard to pAKT signalling. We didn't expect this. So what does this mean? At first glance, it simply means that growth of schwannomas is regulated through the ras-raf-MEK-ERK signalling pathway and not so much through PI3 Kinase. So why is this important? Well, the more specific a response is within a cell, the easier it is to control abnormal behavior. Perhaps someday this knowledge will lead to more effective treatment options for patients with this type of tumor.

ARRGGG!!!

I am a little perplexed right now. I have been doing protein work for 10 years now and usually when something is what I expect I can at least come up with a reasonable explaination and then a follow-up experiment to test my hypothesis. My results from last week are so contradictory that I am just running the experiment over again - same conditions. If I get the same results I will at least know that the odd results are not due to my technique but are due to tissue variability. I just want to get this set of experiments done and move on to the next thing. I think all researchers are a little ADD. Routine doesn't sit well with most of us. That is the primary reason I left my last job. I was doing the same thing day in day out. After a year and a half of that, I was going nuts. Here's hoping for good results.

Fluorometer angst

As I predicted, I haven't gotten as much done this week as I would have liked but overall I have made progress. I successfully taught Cho how to electroporate bacteria and plate them on Amp-LB plates. He did a good job. Today I will teach him how to inoculate growth media so that tomorrow we can isolate his DNA.

A couple of months ago I bought an instument called a fluorometer. It uses fluorescent tags to measure protein, RNA, and DNA. Most fluorometers are very expensive ($10,000-$20,000) but this one is a simple unit that measures one tube at a time. It seemed to meet our needs perfectly so I spent the $700 our sales rep asked for. Sure enough, the Qubit (that's the model name) is easy to use but I have been having second thoughts about its accuracy. After measuring protein levels, I adjust each individual sample so that I am loading the same amount of protein in each lane on my gel. However, when I go back to the gel and stain it with silver or with coomassie blue, I am finding that there are different protein levels. As a notoriously poor mathmatician, I went back over my number to make sure I calculated everthing correctly - I did. This means that my raw data is incorrect. I wonder if there is anything in the cell lysates that autofluoresces? You would think not. If so, that is a problem that should have been addressed long ago by the many instrument manufacturers. I ran a protein assay again yesterday and am going to run four gels today. The first two I will stain with coomassie blue to double check my protein loading. If my results mirror my calculated data, I will rerun the gels this afternoon and prepare them for Western blotting. If they don't agree with my calculated data, I will have to make the necessary adjustments by visually gauging how much to load. Then I can rerun them. Enough writing. I have a lot to do.

I get paid for this? How cool is that?

Another Monday. I have a lot to do this week but realistically, I will probably only get to about 50% of it. Part of this afternoon is already shot because Michelle and I are refinancing our house so I have to go to the bank to sign papers. Here's hoping that I get more done this week than last. I got sick last week and ended up taking about 2 days off. Luckily, the university gives us great benefits so I have a lot of sick time (about 30 days accrued) and a lot of vacation time. Even with my recent trip to London, I will have maxed out my accrued vacation time. The university gives me 16 hours of vacation a month. Additionally, I elect to trade 12 hours of monthly sick leave for an additional 4 hours of vacation. The University of Iowa allows us to accrue up to 480 hours of vacation time. I have hit that mark and will not be able to recieve any additional time. I am going to have to take some more vacation. Darn. Now that it is warming up, I will probably take a couple of days off to get my flower beds ready and planted. I find that to be very therapeutic.

Anyway, I have a couple of tissues to process for protein today. I will probably run the gels tomorrow. Also, tomorrow I will be teaching one of our research fellows how to extract DNA from a filter, get the DNA into bacteria, and grow the bacteria on a selective plate. If we are successful, then I will teach him how to amplify the colonies, extract the inserted DNA, and sequence his insert. Hopefully this will go smoothly. I just hope this guy doesn't expect me to do it for him. My job is to teach him how to be independant. Unfortunately, for many of these surgeons, it is easier for them to rely on someone else since they have a relatively limited amount of time in the lab (usually 1-2 years). However, if they want to be successful researchers, they at least need to know the principles behind the science and have a working knowledge of the techniques.

What I am perhaps most interested in this week is doing the phospho-merlin Western blots of the tissue I collected before a couple of weeks ago. The last Western gave me inconclusive results. Hopefully this one will be a little more convincing.

Oh, we also just got a paper accepted for publication. It is a project that I worked long and hard on. As a preliminary study, it is pretty good but there is much more that can be done with it. The only drawback is that it is labor intensive - the mice require daily treatments. Oh well, that is what I am here for.

I like to be busy

This afternoon just flew by. I spent this morning getting caught up on email and other things that come with being gone for a week. This afternoon was spent isolating protein from tissue samples. I had seventeen samples and after about 3.5 hours I finally finished. Tomorrow I will start running gels again.

More surgery

The sutures I made last week on my rats that I thought were dehiscing actually didn't. Well, one of them did partially but the others actually looked pretty good. No infection either. Today I performed the second part of the experiment. It only took me two hours to implant the pumps and catheters in the four rats. Not bad. Most of the rats went under very quickly but one rat just wouldn't give up. I had to inject her three times with ketamine. While they were under, I also injected them with an analog of uridine (a nucleic acid) called BrdU. When cells divide, this compound is taken up during the DNA replication phase (the S-phase). We have antibodies that recognize BrdU and will therefore allow us to get an idea of which cells are dividing. Since BrdU has a short half-life, I will do two more injections tomorrow before I sacrifice the animals and harvest the nerve samples. I am anxious to see the results of this experiment. Our hypothesis is that we will see an increase in phosphorylated Merlin in the nerve section distal to the cut. We also believe that we will see an increase in cell proliferation distal to the cut.

My morning is blown.

I really wish something would just work. My job is a lot like my golf game. I am a lousy golfer but every now and again, I pull off a tremendous shot. That one shot out of 100 (my brothers may remember that 321 yard drive straight down the fairway on the first hole we played right before Adam's wedding) is all it takes to bring me back. Research is a lot like that. Most of the time, we fail but it only takes one good result to keep us going. I've been swinging a lot lately but just haven't connected.

Last night I innoculated three flasks of growth media with bacteria and not one of them grew. This would either indicate that my bacterial stocks have gone bad (unlikely) or that I am using the wrong antibiotic (also unlikely, but I went by memory so I could be wrong). This process is about as basic as washing dishes so not a lot should go wrong. I am going to check my notes to ensure that I used the correct antibiotic.

This particular project has been a real pain in the neck. We got these three DNA constructs (A DNA construct is generally in the form of plasmid or circular DNA. Your gene of interest is inserted into a larger chunk of DNA that contains sequences to enhance the production of your gene of interest such as a promoter and a poly A tail. The plasmid backbone also contains a gene that for antibiotic resistance to assist in the selection of bacterial clones that contain your gene of interest) from another researcher but didn't get much information about them. Generally, when you send someone a plasmid, you also send them a plasmid map and a sequence. We got nothing. I didn't even know what plasmid backbone my gene was in. I had to design some DNA primer so that I could sequence my gene from the inside out so that I could see what the DNA sequence upstream and downstream of my gene was. I then took this data and ran it through a data base at the NIH (a process called BLAST). The results indicated that my gene was in a plasmid called pCDNA 3.1-. pCDNA 3.1- and its family members contain an ampicillin resistance gene. The typical way to store and mass produce this plasmid DNA is to insert it into E. coli bacteria. It is stored at -80 degrees C and when needed, a small stab of frozen bacteria is placed into growth media and incubated overnight with the appropriate antibiotics. Unfortunately, my bacteria didn't grow last night. Back to square one.

At least they are not chewing on each other

I left my rats in the lab overnight so that I could keep tabs on them to make sure that they recovered properly. They are all alive (but not kicking - they don't have a sciatic nerve anymore) but the wounds on three of the rats have dehisced. That means that I am going to have to re-anesthetize them and resuture the wounds. I purposely used a non-absorbable suture material this time but the rats still managed to chew through the stitches. I am going to have to contact the university vet to see what he suggests.

Back to Merlin

Today I am starting another experiment designed to look at Merlin phosphorylation in deenervated schwann cells. It is a relatively easy experiment. I have four rats - two controls and two that will be treated with a PKA (protein kinase A) inhibitor. I will begin by making an incision in the right rear leg and cutting the sciatic nerve. I will then suture the wound up and let the rats recover for one week. Next Thursday, I will go back in, insert an osmotic pump that will release my PKA inhibitor over the cut nerve for 24 hours, and resuture the wound. On Friday, I will remove the pump, and take samples of the nerve proximal (closer to the midline of the body) to the cut and distal to the cut as well as taking the uncut contralateral nerve. Samples will be taken both for microscopy and protein analysis. The purpose to this experiment is to see if Merlin phosphorylation is controlled by PKA when a nerve is cut. We know that when a nerve is cut, the axon distal to the cut dies. This stimulates schwann cell proliferation. Our hypothesis is that PKA, at least in part, is responsible for this stimulation. Previous data have shown that PKA causes Merlin (a tumor supressor gene) to be phosphorylated. When Merlin is phosphorylated, it becomes inactive thereby allowing a cell to re-enter the cell cycle. We want to see we can prevent Merlin phosphorylation in the distal nerve section by treating with a PKA inhibitor. The first time we did the experiment there wasn't any difference between treated and control nerves. I think that was more a protein concentration problem when we loaded the gels but I could be wrong. The second time I did this experiment, the results were inconclusive. Hopefully the third time will be the charm.

I just finished the first round of surgeries and all of the rats are still alive. Hopefully they will be tomorrow also.

Slow week

This has not been the most productive week. I ended up going home early on Monday because my lower back hurt too bad to sit down. Yesterday I set up a maxi prep (a method of havesting DNA by inoculating 100 mL of media with bacteria that contain your gene of interest) but when I came in this morning, the culture media was as pristine as it was yesterday - no growth. I am not sure why the cultures didn't grow except maybe I over treated with ampicillin. I doubled the AMP concentration in an effort to decrease the number of false positives. A friend of mine told me he regularly does that so I thought I would give it a try. Whether that is the problem or not, I don't know.

When I came in yesterday morning, the lab was a mess. Our post-doc had done a protein isolation from a plate of primary schwannoma cells and had left everything out. Apparently she did the experiment on my bench because that was where the majority of the mess was. I am getting sick of having to clean up her messes. I have spoken gently to her about the necessity of cleanliness in the lab but apparently she is not listening. I have worked with quite a few foreign scientists over the past 10 years and most of them have been very good. Several of them, mostly Chinese, have been quite sloppy though. I am not sure if they leave messes because they are used to people like me cleaning up after them or if that is the way they are. This is not just a Chinese thing though. I have found that a good number of physician/scientists are the same way. Maybe they are just used to having nurses around to clean up their messes. I guess I shouldn't pick on my Chinese collegues too much, though. I have known a lot of American scientists who are so messy that I can't stand to even go near their benches. Several weeks ago in lab meeting I brought this problem up by telling everybody that I really didn't care what their own work space looks like but I do care what our shared work spaces look like. It is simply common courtesy to clean up after yourself - didn't their mothers (and fathers) teach them that? How can you do good science in a pig sty?

Grants

We had a bit of a set back yesterday. We turned in a grant application to the National Institutes of Health in Feb. The NIH is made up of many smaller sections and when you turn in your grant, it is assigned to one of the sections - usually the one deemed most appropriate based on your grant. Our grant kind of straddled three of the sections - cancer, audiology, and neuroscience. We asked that it be assigned to Audiology but someone got the great idea to assign it to cancer. That means that we are competing for a fist full of dollars with people studying breast cancer, lung cancer, prostate cancer, and the like. Our cancer doesn't affect or kill anywhere close to the number of people as the ones I just mentioned. As such, the chances of our grant getting funded with Pres. Bush's extremely tight grip on the NIH are non-existant. Truthfully speaking, those cancers do deserve the bulk of the funding from the Cancer section of the NIH. That is why we applied to a different section. In the last 15 years, the Cancer section has not funded one grant on Vestibular Schwannomas. After hearing that we were being assigned to the Cancer section, we asked that the grant be reassigned but to no avail. We are considering reapplying to the Veteran's Administration for funding. I guess we'll have to see what happens. NIH grants have been very hard to get the last few years. Even well established researchers are getting their grants tossed.

Eventhough we have two currently funded grants, we are always writting the next one. This is a never ending process. I am grateful the responsibility for funding a lab does not fall on my shoulders. Research is very expensive. Even basic consumables are expensive. The only thing that comes cheap is the labor. Although I like to complain about my pay, I really am lucky to have this job. I really do love what I do. I guess there really isn't much to complain about.

Well, my timer goes off in 12 minutes so I need to wrap things up and get back to the next step of my Western blot.

Slower than a snail

Research goes at a snail's pace. For every "discovery", there seems to be at least 10 setbacks. I guess if it worked every time we could just call it search instead of research. I have more Western blots to do this week. Hopefully I can get these last few blots wrapped up so that we can finally get this paper submitted. We are going to try to get it published in the Journal of Biological Chemistry (JBC). I have my doubts that we will be successful getting it into JBC but it is good research so hopefully we will have a chance. Journals are rated first tier, second tier, or third tier (not offically, though) depending on the influence of the journal. Journals like Science and Nature are considered tier 1+, JBC, the American Journal of Physiology, Cell, and the like are considered tier 1 journals and on down from there. I don't like a lot of what comes out of Science and Nature because these are the breaking reseach stories and due to space limitations, I can never get all the information I want. Also, in the excitement to publish ground breaking research before someone else does, the experiment(s) is not repeated as often as it should be. This leads to retractions. There are probably more retractions in Science than any other journal. Having said that, retractions are still few and far in between.

I won't get much done today. I attended a funeral this morning for a friend that died last week. I am glad I got to go but I just don't have the time I need to run another gel. Hopefully I will get the rest of the blots run this week so that I can move on. I have some cloning that I need to do. It has been hanging over my head for months. When it works, its great but when it doesn't it is very frustrating. One simple base pair misalignment and the whole thing goes down the drain.

More fun with Westerns

I have been doing a lot of Western blots lately and thought that I would show a picture of one so that you at least have an idea of what I am talking about. The blot I am showing you is actually three blots - one looking at phopho-AKT, another at phospho-JNK, and the last (far right) at phospho-ERK. There are two lanes in each blot, one is of tumor sample, and the other of control tissue. This has been my life the last couple of weeks. The tumor samples are showing darker bands but that is probably because there is more protein in the tumor lanes than in the control lanes rather than an actual upregulation of enzyme activities.

I can't help but laugh sometimes

More protein problems. Actually the proteins are doing what they are supposed to, we are the ones with the problem. Our post-doc did a Western blot of her protein purification process and found a protein that was enriched in a couple of the elution fractions just like she saw on her silver stained gel. That is good except the protein band appeared to be about twice as large as it should have been. We think that that is probably because this protein likes to self-dimerize. However, we don't want the protein to be dimerized. I had her do a couple of things that would solve the problem and after running the blot again, she excitedly showed me the film (Western blot results are recorded either digitally or by film) that showed protein bands much lower on the blot. After taking one look, I saw from the shape of the bands that she had the film upside down. She assured me that she didn't, showing me where she had creased the film to ensure proper orientation just like I had taught her. Sure enough, her mark was in the correct place but she had placed the blot upside down in the film cassette. I managed to only grin a little bit while explaining the problem. I need to sit down with her and help her design the next couple of experiments to improve our yield and purity as well as our dimerization problem. These next few experiments are going to be a bit more involved but should yield good results. Of course now that I have said that, I have cursed the experiment. We'll see.

Results are always good

I finished another Western blot, well, actually 6 of them. The results were encouraging I think. I am not sure what I expected. I ran two gels with control tissue (2 different Greater Auricular nerves - they are sensory nerves that transmit touch from the ear). I transfered the proteins to nitrocellulose membranes and incubated them with antibodies raised against Erk, AKT, and JNK, as well as the phophorylated, or activated, versions of those three signalling proteins. Both Erk (pronounced irk) and AKT are generally considered to be enzymes involved in proliferative signalling pathways while JNK (pronounced junk) is considered to be a player in cell death pathways. The non-activated versions showed equal amounts of protein between both samples - thankfully - but only AKT was activated in both samples. Now I need to go back a do the same experiment with my vestibular schwannoma samples so that I can compare the two. This will give us the beginning of an idea as to what signalling pathways are activated in this tumor. We have so much work to do and of necessity it crawls at a snail's pace.

Let's not lose focus

We had lab meeting yesterday. We usually hold it in one of the conference rooms over here near our lab (we have two of them on our floor) but since our boss was in between surgeries, we went over to the departmental offices to have our meeting. Now, keep in mind that our lab is as far away from our department as possible without having to go outside. I actually counted the steps one day - it's 1008. That is over 0.5 miles. On days that we have to make several trips to the department, we semi-jokingly tell our boss that he needs to spring for a Segway (sp?). Anyway, I was the only native English speaker in the group and somehow we got on the subject of farms. I spent half of our trip to the department explaining the difference between a ranch and a farm as well as the types of animals on ranches and farms. It was somewhat comical. The two Chinese women giggled when I told them a male pig is called a boar. I am not sure what was so funny about that.

When my friends and I get together, we sometimes discuss the merits of having a PhD for a boss vs. an MD for a boss. I personally prefer an MD. Usually they are in the lab less than a PhD and the good ones know that they aren't science's gift to research. That is sometimes detrimental also. I certainly don't know everything and it is nice to have someone to turn to with questions. I have a very intelligent boss but he doesn't have the bench experience that a PhD would have. What he does have, however, is the ability to remind us what we are doing. So many researchers get so tied up in their one protein or their one gene that they fail to see the patient. Yesterday, during lab meeting, Marlan was presenting a journal article about menigiomas. Meninges are the membranes that line our brain and spinal cord. A menigioma is a cancer that can be quite aggressive although not metastatic. By aggressive I mean that a menigioma will eat its way through bone and tissue. As we were talking about Merlin (a tumor suppressor protein that is missing in menigiomas and schwannomas) Marlan pulled up some of MRIs of a couple of his patients that have menigiomas. One of them was clearly inoperable due to its placement and widespread location. This patient will die. It really helped me to re-focus my efforts and remind me what I am doing. I know that finding a "cure" for this type of cancer (or any type of cancer)will not be a Eureka moment but rather a series of discoveries that will eventually help us to tailor treatments to individuals. So, it is time to get back to teasing apart the molecular pathways these tumor cells use to survive and senesce.

This is my favorite time of day at work. I try to get in right around 8 am because nobody else is here. It is quiet and I can get my work started. We just had another snow storm last night and I have a great view to the north. Everything is frosted white and with the sun out this morning, it is beautiful.

This morning I am finishing a Western blot I started yesterday. I wish I could say the data will be earth shattering but it is just an attempt to find a good loading volume. My protein assay isn't working reliably so I am doing it empirically. When this is done, I am going to teach a person in the surgery department how to perform a rat surgery - kind of ironic, isn't it? It is actually a very basic procedure. We're just making a pouch under the skin between to shoulder blades, placing an estrogen pellet in the pouch and suturing the wound back up. The only tricky part is getting the anesthesia right. I really enjoy doing animal surgeries. They are challenging and satisfying when you get them right. Also, the amount of data that you can get is well worth the work. The only problem I am experiencing is that I am becoming allergic to either the rats or their bedding.

You have got to be kidding me.

After receiving my MS in Biology at Idaho State University, I came to Iowa to complete a PhD. After two years in a great program, I left. Partly because I just didn't have the time to devote to my studies. As the father of 6 children, I felt I needed to spend more time focusing on my young family rather than ignoring them in order to further my own desires. I probably could have stayed in and finished with a mediocre thesis but I have too much respect for the degree to have done that. I write this because I have seen so many people come out of graduate programs with doctoral degrees that really didn't deserve them. Case in point. We have a post-doctorate fellow in our lab who has been with us now for 7 months. I still have to remind her to write in her lab notebook. My boss asked me to teach her how to column purify protein. I first had her transfect a flask of HEK 293 cells (a well established kidney cell line) with a DNA construct containing our gene of interest. The cells would then use their machinary to produce our protein. The protein was engineered to have what is called a histidine tag expressed with our protein. This enables us to purify our protein by running the cell lysate over sepharose beads that have Cobalt attached to them. Cobalt and histidine form a chemical bond which allows you to keep your protein of interst bound to the beads while everything else just passes on by. After washing the beads a number times to remove anything that is bound in a non-specific manner, you can then elute your protein of interest by washing the beads in a solution containing Imidizole and if the Voodoo king is smiling on you, you will have purified protein. Anyway, this is a fairly long and drawn out proceedure. After guiding our Post-doc through the proceedure, having her run the samples on a polyacrylimide gel, and then staining the gel with silver to see the protein, I was pleased to see the elution profile I had predicted for her. I then told her that since the silver stain was non-specific, I wanted her to run the gel again, transfer the proteins to a nitrocellulose membrane and probe the membrane with an antibody specific for our protein. I told her that if we saw the same profile, we could be assured that we had pulled off the purification. She looked at me blankly and said, "How can we do that? There is no his-tagged protein in that solution." I stared at her for a moment, not really sure what she meant, but assured her that this is what we needed to do. She again told me that wouldn't work. I was still scratching my head. She informed me that she had done the entire experiment with a solution from a non-transfected flask of cells. She tried to tell me that this was a good negative control. I continued to stare at her not believing what I was hearing. Why would you even try to purify a protein that you knew wasn't there in the first place? That is not a good negative control - especially since we got a result.

I find it hard to believe that someone with a PhD in the biomedical sciences never once thought to question me or the procedure. The difference between a PhD and a lab tech is that a PhD is supposed to be able to think critically. This Post-doc has yet to rise above the level of a newly minted lab tech. I am sure she will eventually but it is getting tiresome waiting.

Hey

First to explain the title of my blog. I work in the Otolaryngolgy department (the cochlea part) at the University of Iowa (the corn part) where I study the cell physiology of hearing and hearing nerve-related tumors. Most Americans seem to confuse Iowa with Idaho or Ohio. Why? I am not sure. Maybe they are stupid or too self absorbed to realize that there are states in between New York and California. Those who know something about Iowa think of corn (which comes in ears) and hogs. And rightly so. Iowa is a huge agricultural state. What people don't realize is that Iowa is also home to one of the best public university's in the nation - at least from a biomedical standpoint. My department is actually ranked the second best in the nation behind Johns Hopkins University. In additon to having top notch clinical care, we also have great research efforts going on. In my job as a lab manager, I have what in my mind is the best of all worlds. I get to be involved in the day to day administration of the lab (which does have its drawbacks) as well as being involved in training new lab members and doing my own research at the bench. I am going to use this blog as a way to catalog some of the stuff that goes on around here - both the good and the frustrating. Anybody in research will tell you that being frustrated is one of the side effects of the job. Having said that, I really do love my job. I have an incredible boss and work with some very bright people.