Technique: Chemotaxis concepts – Why did the chicken cross the road?

Why did the chicken cross the road? Well, frankly, who knows what induced the chicken to cross the road. Generally, we can assume that the chicken had a reason. Arguably everything we do in everyday life we do for a reason – whether voluntary or involuntary. Like most other creatures in the world, we respond to our environments.

If you show a dog a bone, and then place that on the far side of the room, the dog will run over to get it. But the stimulus doesn’t have to be food. If a person enters the room, a dog will typically rush over to greet it – though whether that greeting is friendly or threatening depends on many factors.  Lack of movement can also be due to the environment that we sense around us. For example, when I often wake up, I am tempted not to get out of bed, because it Is incredibly comfortable where I am, and, for the time being, there is no specific reason to move.  Therefore:

Movement = response to factors signalling that another place is desirable and/or lack of factors signalling that the current location is desirable

Lack of movement = response to factors signalling that the current location is desirable and/or lack of factors signalling another place is desirable

Like whole organisms, cells behave in response to the environments they sense around them. For example, consider cells in the circulation. While initially one thinks of these cells as being simply pushed around by the physical pressures exerted by fluid dynamics, these cells are actually constantly sensing their environments and can respond by clotting, leaving the leaving the blood system and moving into surrounding tissues, or continuing circulating. In contrast, think of skin cells. These cells stay in one place, because they are surrounded by similar cells and supporting layers of cells that provide the signals to stay in place. However, in response to injury, e.g. a paper-cut, the cells quickly change their entire behaviour and can both move into the wound site as well as signal to the blood and immunological cell types necessary for repair.

Outside of the body, in the laboratory setting, it’s easy to study the effect of each movement signal, also known as a chemotactic factor (chemotaxis meaning movement in response to a chemical signal) because we can add the factors in individually and in combinations to observe their exact effects. In this blog I have already discussed a way to measure random cell movement in response to growth factors (September 28: Calculating Cell Velocity). In my next post I will discuss the Boyden Chamber assay for studying directional cell movement in response to factors.

 

And now, my body is telling me I need to move to a location that provides caffeine. I’m off to get some coffee and get back to working on my thesis.

Thesis Writing: The Middle Chapters

The middle chapters of the thesis include the Methods, Results, and Discussion sections. These are, to a certain extent, the hardest chapters of the thesis to write. Unlike, for example, the introduction, it’s not simply a questions of laying out your ideas, looking up what other people have done that is relevant, and discussing their work. The Methods section, I grant you, is very straightforward as long as you have documented what you have done along the way – you, after all, should know what experiments you did. The Results section is slightly trickier, in that you need to figure out how to logically and best present your findings in a way that conclusively demonstrates your point without overwhelming the examiners with extraneous data or confusing them. The Discussion section is by far the most difficult as you must evaluate your data and explain how it fits in with the overall aims of the project.

So far, in my posts on thesis writing, I’ve discussed the general format of the thesis (August 27) and my current efforts with the results section (November 17). Particularly, in the August 27^th post, I stressed that the PhD is not simply a long paper. That said, there are countries in the world, e.g. Sweden, Finland, the Netherlands, and Australia (to name a few) that will accept several papers written by the student (and published in peer-reviewed journals) stapled together in lieu of an actual thesis.   If thesis writing were like just writing a long paper, there would be the following sections: Introduction, Materials and Methods, Results, Discussion, and Conclusions. However, many people would argue that while a thesis needs an Introduction section as well as a Conclusions and Future Work section, the middle chapters should not be so strictly organized. Rather, the belief of having several middle chapters based on various themes or questions pertaining  to the overall project which each contain subsections of Materials, Results, and Discussion, is becoming increasingly prevalent within the scientific academic community.   In fact, a friend of mine who recently defended in Oxford had organized his thesis as if a long paper and was told that for his final version the only alterations desired were that he cut and paste his sections in the latter method to tell a better story.

Part of the reason why this approach of having self-contained chapters that each contains several parts, is that it lends a better flow to the thesis. Because theses tend to be so long, it becomes difficult for an examiner (one dare not hope that anyone other than the examiner will actually attempt to read the full document) to read each section in its entirety and keep it fresh in their mind as they go on to the next section.  Indeed, if organized thusly, the method, results, and discussion of a particular method could be spaced out with 20 or more pages in between them – hardly allowing for the formation of a sense of direction. However, by splitting the middle section of the thesis into topical chapters, this allows for the examiner to focus on a smaller topic at each time, with a sense of how each experiment was performed, what the results are, and how to interpret these results in relation to other experiments which also answered the same question. Then, with an idea of how each topic was addressed, the parts can be brought together in the final Conclusion section to show the larger picture.

The simplest way, perhaps, of explaining this concept is to think of the difference between a nursery, and a garden.  If the Methods, Results, and Discussion sections were three types of flowers, then the more traditional method would be akin to a nursery; each section would be taken care of, but separately and in neat little rows. However, the newer method would see that although a garden contains these three types of flowers, in contains them in several beds which contain clusters of each type, so that the flowers can be seen in relation to each other, followed by the design and layout of the beds.

Fatigue related to outdoor darkness

Living in England in the heart of winter can make for quite a dark time, literally. For a number of months the sun does not rise until after 8 AM and sets before 4PM.  Even  people who have lived in the country all of their lives will tell you that this has the effect of making it seem much later than it is in the evenings. There is no denying that the environmental darkness that we experience  an effect on our psychological state, and possibly our physical state as well. I recently experienced this first-hand after spending almost 2 weeks in South Africa on holiday, where the sun rose before 6 and set after 8pm. Upon my return to Oxford I immediately lost over 6 hours of sunlight with no jetlag (time difference is only 2 hours). I find myself asking for dinner around 4:30 in the afternoon because it looks like dinner-time outside, and simply getting more tired in the evenings.

But what does science have to say about all this? The science of how our bodies regulate themselves on a daily cycle is known as the study of circadian rhythms. And indeed, the concept of circadian rhythms is not just psychological, with changes in body temperature, gastrointestinal, endocrine, and respiratory functions affected. Metabolism is also affected (though whether that or my brain is more at work with my 4:30 desire for dinner is debatable). Popular topics of study with circadian rhythm scientists are jet lag, shift work (i.e. if you have to work nightly shifts), and seasonal affective disorder (SAD). All of these are instances where our bodies experience changes in environment from what is considered the norm.

Jet lag is easily explainable through the study of circadian rhythms. By shifting time zones, your body immediately senses the new environmental conditions, and gradually your rhythms shift to become in line with this. However, as the cycles take time to align, this is what results in the feelings of jet lag – with changes in behaviour and performance observed. Working the night-time shift is more difficult than switching time zones because it involves using the body against some of the environmental cues (sunlight) that normally govern its functions. In these instances other environmental cues, such as social interactions, become more important to try and shift the cycle. However, studies have shown that individuals who work these shifts do exhibit behavioural problems and social isolation, compared to those working normal shifts.

So how does this all play out with respect to the English winter? Well, in winter we get a conflicting set of cues. True, the time in which we experience sunlight diminishes, but our work schedules and social interactions with others stay on the same time scale, in effect, helping out where the environmental cues are now absent. The hypothesis is that our circadian rhythms are phase-delayed in winter as contrasted to summer, but this has not been conclusively demonstrated. Interestingly, much research has been done with special bright light therapies, in order to reduce the symptoms of SAD. While these therapies do generally improve symptoms, they somehow do not appear to be effected by when they are administered. This confuses scientists, who suspect that it might be having a “sophisticated placebo or expectation response” effect.

The general outlook of the English is to simply accept the situation as it is, and take the occasional winter holiday to Spain or Mallorca. But perhaps the finding that night-shift workers deal with the situation in better in older age, due to advanced coping mechanisms, is the most illuminating.

Disorders of the sleep-wake cycle in adults

On New Year’s Resolutions

Do New Year’s resolutions work? Do they set us up for disappointment? Are they a marketing ploy to increase sales, particularly of fitness equipment and gym memberships, at the beginning of every year? Or can they actually be helpful, an honest effort and impetus, however, brief, to genuinely improve our lives?

Different studies over the years have shown high levels of initial success, e.g. 1 week, diminishing more rapidly by a month, and with limited success after one and two years. In general, it appears, individuals fail more frequently when they make weight resolutions versus non-weight related resolutions, e.g. smoking or drinking.

An interesting study, published in 2002, aimed to not just measure the rates of resolution success, but also the driving forces behind it, and to directly answer whether resolution actually effected change as contrasted to individuals contemplating change. The study indicates that its success rates may be slightly inflated due to the nature of self-reporting, with less successful applicants declining follow-up interviews or making up more successful results, and with some of the participants being motivated to stick to their resolutions because they know they will have follow-up interviews. However, it is important to note several things from the study. First, that 40% of American adults make New Year’s Resolutions every year. Second, that non-successful resolvers are likely to make the same resolution the following years until some degree of success has been achieved. And third, that resolving is not in vain and that most participants do achieve initial success, even if this is not particularly long lasting. As the authors point out, the rate of success should be compared to non-resolvers, i.e. people who don’t make a conscious effort to improve at all.

Most importantly, and surprisingly, once the initial resolution had been made, desire to change had no correlation with resolution success. Instead, success was found by participants who used techniques of “self-liberation, stimulus control reinforcement management, positive thinking, and avoidance strategies”, whereas those who were unsuccessful were characterized by “self-reevaluation, wishful thnking, self-blame, and minimized threat”. That is, those who were more successful better managed and kept on top of the situation and goals, whereas those who were unsuccessful were more likely to get frustrated, blame themselves, and/or talk themselves out of the need to change.

Thus, it’s better to try out a New Year’s resolution than not at all. And above all, stay positive. What’s your New Year’s resolution?

Auld Lang Syne: Success Predictors, Change Processes, and Self-Reported Outcomes of New Year’s Resolvers and Nonresolvers

Technique: The Simplest Way to Make a Collagen Scaffold

Collagen scaffolds are very common within the field of tissue engineering. They are varied in composition as different types of collagen can be used, and virtually anything added s a supplement. In this post I’m going to discuss the simplest method of creating a collagen scaffold, a method that I employed for two out of the three materials I made for my PhD project. The first material was a plain fibril bovine collagen type I scaffold, and the second was the same except with an added hydroxyapatite component. I actually made the hydroxyapatite myself, through the standard precipitation method (topic for a future technique post) in order to eliminate any byproducts present in commercially available product.

Basically, all materials in the final scaffold are mixed together to form a homogenous aqueous solution, which is then centrifuged and vacuumed in order to remove any air bubbles. The solution is then placed in a mold (we used PVC) and frozen at the desired temperature (in my experiments I used -20 degrees Celsius, though in past experiments I have also used -80 degrees Celsius freezers). The samples are then lyophilized (a.k.a. freeze-dried) for a few hours, pushed out of their molds, and then lyophilized some more. Note: beware of removing the samples too early; they dry from the outside, so if moisture is still present in the center they can collapse later. Finally, before use, samples are trimmed and cut as desired. I found that some trimming was necessary as sem micrographs showed walls of collagen on the outer edges, hindering cell and nutrient penetration.

Like any other polymer scaffold, the size and shape of the pores can be easily modified for different desired pore sizes etc. Given a set amount of collagen in an area, pore size is directly controllable by modifying the freezing temperature. At lower temperatures, ice crystals nucleate faster, and therefore the crystals formed will be more numerous and smaller. Since final pore size is directly related to aqueous crystal size, this means that the lower the freezing temperature, the smaller the pores. If the pores are more numerous, this means that the number of walls between pores also rises; but, since the total amount of collagen remains the same, this means each individual wall is thinner. One of the advantages to collagen and this fabrication method is the variation of the scaffold in its micro-porosity under 100 micrometers. It has actually been shown that variation on this scale is more conducive to biological activity, a long as the larger pore structures average the same as an otherwise similar scaffold.

Growing more enamel in our sleep?

The following is a draft of a short piece written for the National Institute of Dental and Craniofacial Research.

The concept of circadian rhythms, that is, the regulation and fluctuation of biological processes over a 24-hour period, has always been a popular idea. Are blind animals still attuned to what time of day it is? Can humans kept in special chambers on 28 hour days function normally? How do long-distance airline pilots cope with multiple time zone, and the accelerated/decelerated daylight exposure?

Although long based on empirical observation, modern science has allowed researchers to find the biochemical evidence of a circadian clock, and to study this phenomenon all the way down to the genetic level, and in organisms as far back as a simple fungus. A group of scientists funded by the NIDCR set out to answer the question:

“Do we grow more enamel in our sleep?”

In order to answer this question we have to discuss previous work that has been done in the field of enamel biochemistry. Previous scientists had done staining experiments that showed bursts of enamel being deposited on a daily basis, as contrasted to a slow, steady, build-up. Because this was on a 24-hour rhythm, speculation arose that the process might be circadian.

Enamel growth has been shown to be two-step process: the production of a protein matrix made of amelogenin and other enamel-specific proteins followed by the deposition and growth of bicarbonate crystals as directed by this matrix and concomitant removal of matrix. Snead et al. set out to show that these two steps were directly related to the circadian clock, with protein matrix being grown during light hours and crystal growth occurring during dark hours.

The group of researchers attacked the problem through three different analysis methods. Gene sequence analysis was carried out to see if promoter sequences known to be circadian-clock-regulated (E-box sequences) were associated with enamel-specific protein and crystal production genes. Through the culturing of circadian-synchronized cells and the dissection of molars from pups kept in a strictly circadian environment, RNA was extracted and analysed at 4 hour intervals to see if enamel genes showed fluctuation in a circadian-specific manner. Additionally, to demonstrate the presence of circadian-regulating proteins in the mice, molars were extracted and stained for these proteins. Thus, through DNA analysis, gene and protein expression analysis, the scientists were able to conclude that the two steps of enamel production did indeed take place sequentially and on a 24-hour cycle.

The circadian clock modulates enamel development.

The NIH-OxCam Program

I thought I would give a brief shout-out for the NIH-OxCam program that I’m on, for any people considering graduate programs who are reading this blog. Applications are now open until January 2nd, and the official website and application information can be found here:

NIH OxCam Webpage

Just over a decade ago, as graduate students were first starting to make their way onto the National Institutes of Health (NIH) campus, it was decided that the NIH would form what is now known as the Graduate Partnerships Program (GPP). It was decided that the NIH was not going to be degree-granting institution. However, they wished to provide funding for students to do research on campus and to work towards degrees at partnering Universities.  One of the first partnerships was with Oxford and Cambridge.

Each graduate partnership at the NIH works differently, but the OxCam program, as it is known, gives American citizens the opportunity to study a biomedical –related field at either Oxford or Cambridge as well as at the NIH. (There is a similar program called the NIH-Wellcome program for non-US citizens.) While the NIH funds the degree, the university in England is in charge of the academic side of affairs, including examination and degree conferral. The goal is for students to evenly split their time between the two locations, working in collaborating labs and pulling together a cohesive project.

As with any course of graduate study the success of the project is dependent on many variables, the three main ones being mentorship from supervisors, tenacity of the student, and topic choice. The great thing about the program is that it does in many respects offer a middle-ground between British and American attitudes towards PhD projects. One of the main aspects of this is the fact that funding is given for five years, which is longer than the British timeframe, but shorter than the American time-frame. This seems to work out just right for the students in the program who generally seem to graduate after about 4.5 years.

The program is certainly not for everyone in that in addition to the normal criteria for a PhD candidate, you also need to add a good set of communications skills, self-motivation, and extra tenacity. But it can, and does, work out, and I would definitely encourage anyone interested to apply. And please do contact me if you have any questions.

Holidays Can Be Dangerous

In searching for relevant Hannukkah-related articles on Pubmed, an interesting article jumped out at me.

Child Injury in Israel

In this article, the authors studied trends in injuries admitted to the emergency room at the Petach Tikvah Children’s Medical Center in Israel. Interestingly, they found that injury rates in children rose in specific ways related to certain Jewish holidays. In particular, they found that there was a significant rise in burn injuries surrounding the holidays of Hannukkah, Lag BaOmer, and Passover. Hannukkah involves the nightly lighting of candles, and Lag BaOmer traditionally involves bonfires. While Passover does not explicitly involve any fire-related activity, the authors speculate that this is due to an overall increase in cooking in the house around this festive period. What is clear, however, is that there is a sudden jump in accidental poisonings around Passover, due to cleaning products used during holiday-mandated spring cleaning. Around the days surrounding, and including Passover, there is a spate of bicycle and skateboard related injuries. Cars are not driven during Passover, so this means that children have free-rein of the roads, and use the opportunity to promptly injure themselves.

So be careful when lighting those Hannukkah candles.

Oh, and if you’re Christian, this is advance warning to beware pokey objects near eyeballs and other Christmas-related injuries:

Christmas-related eye injuries: a prospective study.

Propeller and jet-ski injuries during Christmas and New Year in Western Australia

Oxford – Bring on the Thesis

I’ve been back in Oxford for two weeks now.  Finally got over my jet lag. Maybe I shouldn’t have slept during the daytime the entire flight over from the States. But it took me longer than usual to get over the jetlag – in part due to a newfound love of afternoon coffee and partly due to what is possibly the best duvet in the world.

 

So how’s the thesis writing going, you ask? Well, I must say that the Radcliffe Camera in Oxford is an amazing place to work. Opened in 1749, it’s a place full of history, and the sound of students working. There are also many coffee places I go to when in need for more ambient noise, and caffeine.

 

But the writing, I hear you ask, how is the writing going? Well, the first thing to do is to figure out the data that you have. To this end I need to finish analyzing some data, which is what I’m currently focusing on. Also, for the writing part, I’m working on the words that go in the results section. The Results section is always trickier than it might appear because all the figures and relevant graphs need to appear there in order to present the data, but in terms of prose it’s important to provide context for the figures, while at the same time not including that which should instead belong in either the Methods or Discussion sections.  Often, so far, I’ve found myself writing and then thinking that a paragraph really belongs in another location in the thesis.

 

So here are the steps now:

1)     Finish analyzing data

2)     Write Results section

3)     Make figures and insert into Results section

4)     Reformat Methods and Intro to fit around the Results section

Stuck in a Hurricane – Don’t Know When I’ll Be Back Again!

Hurricane Sandy will go down in history for what it did to New York and New Jersey. But it will also be a story I will tell my grandchildren.

 

It’s stressful enough to move out of an apartment.  You have to sell or give away all of your furniture. You have to throw out all the detritus of a private life that you would keep using if you were still there – but which everyone else already seems to possess and doesn’t want more of: stainless steel pots anyone?  You have to clean the apartment thoroughly if you expect any of your deposit back. And you have to pack your entire life into 2 check-in bags (I paid extra and brought 3), one computer bag, and one “personal item”.

 

It’s also stressful to finish your PhD experiments (see my previous post: “Experiments – Done!!”). To simply circle a date on a calendar and say, “I’m going to finish this day and what I’ve got is what I’ve got. It is, what it is, and I’m going to write up and be done.” Particularly stressful when it gets to 3 days before you leave and you’re starting an 18 hour experiment and still have immunohistochemistry to work out, and to make sure you have all your files off all the various computers in the department.

 

And then: Hurricane Sandy. Oh, how I love you Hurricane Sandy. I’ve seen stronger wind and rain growing up in Albuquerque; and yet, for some reason, Washington D.C. completely shut down. I had just gotten back from my birthday/going away party on Sunday night, when they announced that the metro was going to be shut all day Monday. I therefore had a choice: I could either be stuck in the apartment with no way to get into work, or I could be stuck at work, with no way to get back to my apartment.  The choice was simple, I had an experiment that had to run overnight Monday to Tuesday. Either that experiment was going to be in my thesis or it was not. Also, my immunohistochemistry wasn’t working yet. I had to go into lab. So, I grabbed a set of pajamas, and hoofed it into lab.

 

I spent two nights in the lab.

The experiments got done.

The files were obtained.

The immunohistochemistry finally worked.

I ate the cheese, salami, and cracker platter leftover from the Friday birthday party from the lab fridge. I ate leftover pie.

Did I mention I spent my birthday in the lab with a hurricane outside?

 

Then I went home.

I packed everything I could.

I threw out everything I couldn’t.

I scrubbed every appliance and floor in the apartment.

I slept one night on an air mattress (a step up from the chairs in the lab).

Then I went to the airport and flew to England.