Arches. Photo by Daniel Chia
Jun
26
2010

The Scientific Approach

When it comes to scientific research, the public wants results and we want them fast. This is especially true of research on chronic or fatal human diseases such as diabetes, cancer, and Parkinson’s, which affect millions of people in the United States alone. Because the public loves good news, the media is quick to report stories involving major scientific breakthroughs (or what appear to be). On June 16, 2006, for example, the Canadian press released an article entitled “Canadians cure Huntington’s disease in modified mice.”

As I learned firsthand this summer as an intern at Dr. Marcy MacDonald’s Huntington’s disease (HD) laboratory in the Center for Human Genetic Research at Massachusetts General Hospital (MGH) in Boston, the disease is far from cured, even in mice. In fact, the research community is still years, perhaps decades, away from finding drug treatments that target the genetic mutation whose deleterious effects lead to HD, a neurological disorder with symptoms that typically begin in middle age. HD is termed “neurodegenerative” because it involves a progressive loss of nerve cells in the brain. The disease affects men and women alike, occurring at a rate of about one in every 10,000 in most Western countries.

While science journalism is not, for the most part, intentionally fraudulent or misleading, it sometimes gives people the wrong impression about scientific findings by the way it interprets the data from recent articles in science journals like Cell and Human Molecular Genetics. When the Canadian scientists reported that they had inhibited an enzyme that cleaves, or cuts, the mutated HD protein (huntingtin) in mice, thus preventing the degeneration of the nerve cells in the brain, the press trumpeted it as a cure.

When the media takes such leaps or oversimplifies a complex, highly nuanced finding, it presents a skewed picture of the actual process of scientific research and discovery. In reality, the scientific method—that is, the process of empirical investigation into the validity or invalidity of a scientific claim or hypothesis—relies on replication and critical testing of each new finding, which takes a considerable amount of time. Not only does it require patience from both the scientists and the public, but it also requires a great deal of intensive effort that includes collaboration between research teams in different parts of the country and around the world.

Scientists rarely work alone or in isolation. To do so would be highly inefficient, especially since one scientist or group of scientists does not have expertise in every skill necessary to carry out an entire large experiment from start to finish. When teams of scientists work together and share ideas and materials (such as cell lines, which the MacDonald lab frequently sends to other labs), they are able to produce results in less time. However, “less time” does not mean instantaneously; collaborative work, while certainly more efficient than solitary work, still requires many years of sustained effort to find results that translate into good news for disease sufferers.

Although the scientific community values collaboration, it does not necessarily frown upon competition. Competition to test new ideas, to try and “knock them out of the ring,” is built into the scientific method (described later in The Scientific Method) and is, in a manner of speaking, one hallmark of the scientific endeavor.

Blueprints^

One of the greatest rewards of scientific research is the “Eureka!” moment—that sudden gleeful breakthrough that can occur after much effort and many months of work. When a scientist experiences this lightning flash of insight, all the smaller discoveries of years past come together in a meaningful way, like the pieces of a puzzle, forming a much larger discovery. Indeed, HD can be compared to an enormous puzzle, the outlines of which are known, and the rest of which is still a mystery.

The genetic nature of the disease provides a kind of framework for discoveries about the changes that take place in the body on the cellular and molecular levels during the course of the disease. The macro-level changes observed in people with HD also provide guidelines about the micro-level changes occurring within their brains and bodies as the disease progresses.

With a rough outline to use as a guide, scientists can begin finding new pieces of the puzzle and fitting the puzzle pieces together to form recognizable pieces of the bigger picture. By putting the newly discovered pieces in place, scientists can make strides toward finding effective treatments not just for the symptoms of HD but also for the root genetic defect, or mutation, that causes the disease.

In 1993, an international team of researchers, which included Dr. MacDonald and her colleague, Dr. James Gusella, identified the responsible mutation. They found a CAG triplet repeat expansion in a region of human chromosome 4. Found in the nucleus (the information center) of cells, chromosome 4 is, like other chromosomes (we have 23 pairs of them), comprised of the DNA and associated proteins. Lengths of DNA in a chromosome make up genes, which are the functional units of heredity in humans and other organisms.

Each person inherits two copies (called alleles) of each gene, one from mother and one from father (the only exception being genes on sex chromosomes). Because HD is inherited as a genetically dominant character, a person needs only one mutated copy of the gene, called the expanded HD CAG allele, to inherit the disease. (For more information on genes and chromosomes, please click here

Genes are often compared to blueprints for making proteins. If the blueprint is defective, a defective protein will be made. Unlike the non-HD allele, which makes huntingtin protein with fewer than about 37 glutamines (one of the building blocks of the protein), the expanded HD allele makes an abnormal version of huntingtin, with an excess of glutamines (more than 37 or so of them in a row). Due to this mutation, the expanded glutamine version of the huntingtin protein does something—or is a byproduct of another process that does something—that contributes to the slow destruction of nerve cells in the brain. While the onset of symptoms can vary widely, the onset typically occurs between the ages of 30 and 50, after a substantial percentage of the nerve cells have died. There is also a juvenile form of the disease whose symptoms commonly appear before the age of 20. For more information on juvenile HD, please click here.

Physicians typically group the symptoms into three categories: movement, cognitive, and psychiatric. Movement symptoms include uncontrollable movements such as twisting and turning (known as “chorea”), rigidity, falling down, difficulty physically producing speech, and, in the later stages of the disease, difficulty swallowing, which can lead to significant weight loss. Cognitive symptoms include the altered organization and generally slowed processing of information in the brain. The most common psychiatric symptom of HD is depression; other symptoms include personality changes, anxiety, obsession, delirium, and mania. Denial of having HD is also a common symptom of the disease.

Presymptomatic genetic testing is available for those at risk for HD (i.e. people whose mother and/or father were diagnosed with the disease). While there is currently no cure for HD, there are drugs available to treat some of the symptoms, particularly chorea and depression. Some HD researchers, however, are beginning to develop and test drugs that target the presymptomatic effects of the genetic mutation that causes the disease.

Dr. MacDonald is one such researcher who works at the beginning of the disease pathway. She and Dr. Gusella, now director of the Center for Human Genetic Research, believe that the most effective treatments will be those that are specifically designed to reverse the first effects of the genetic mutation. These effects may impart altered physiology that is intrinsic to being born with and living with the HD mutation from birth. Scientists are still a long way from fully understanding the biology of the disease and the underlying mechanisms of nerve cell degeneration.

The Scientific Method^

HD research can also be compared to erecting a building without knowing its dimensions. As of now, researchers only have a vague idea of the shape of the “building,” as specified by the genetic information on chromosome 4. In time, as scientists learn more about the cellular and molecular basis of the disease, they will have a clearer idea of what the “building” actually looks like.

Creating a firm foundation, as the first order of business, is key. Before moving forward with his or her research, a scientist must look to the relevant data from past research and attempt to replicate the key results of other scientists. This step is to make sure that the foundation is sturdy before beginning to build the first wall, or the second. And after completing the second wall, one must make sure that it does not fall when the wind blows, so to speak, in the face of different experiments designed to knock it down.

The idea is to avoid building a flimsy house of cards, but rather to make a solid structure that can be inhabited (and tested) for many years and decades by future generations of scientists. Progress can be thought of either as building a new piece of the foundation that may not initially be connected to the rest, or it can be the addition of new pieces to the growing structure on the original foundation. Progress is accomplished by employing the scientific method as follows:

  • Repeat earlier findings in your specific area of inquiry.
  • Make one or more hypotheses—that is, succinct propositions about what you expect to find if you are right about a process or phenomenon. Be sure your propositions are suitable for empirical testing in laboratory experiments.
  • Conduct an appropriate experiment, controlling for (that is, holding constant) conditions other than those specified in your hypotheses. Carefully observe and note what happens in detail. These details, in combination with the conditions under which they were obtained, are your results.
  • Check your results against the original hypotheses: Do they support one or another of your propositions? Are they what you expected or predicted from one argument or another? Or do they require that you reject all your original hypotheses because you saw something new or unexpected?
  • Explain what you saw and what that tells us. You may need to modify the original hypotheses or, if necessary, you may need to make entirely new ones that are consistent with your findings.
  • Repeat this process until you have eliminated all but one remaining hypothesis. Note that the last hypothesis “left standing” is not what we might call “proven”; it is simply our best bet, given current knowledge. It, too, may be rejected one day when we have better information and understanding.

Although not all research progresses in such a linear fashion, the scientific method can nevertheless be conceptualized as a flowchart:

Fig. 1. “How scientific investigations proceed.” (from Jones et al, 1994.)

The most time-consuming aspect of research—indeed, the heart of any scientific endeavor—is the continual knocking down and building up of the various parts of the knowledge-structure. Scientists can only make progress by first attempting to disprove previous hypotheses, including their own, to ensure the strength of the structure’s foundation. Researchers must also allow time to investigate unexpected results and decide how they fit (or don’t fit) into the emergent structure.

Overall, the scientific method provides scientists with an orderly, systematic way of approaching their research that, in the end, guarantees progress. But it is a multi-step process that cannot be shortened as a result of pressures either from the scientific community or the public without weakening the entire structure. The pace of research can, however, be accelerated by adding more trained scientists, by building and using machines that can allow experiments or observations more quickly and without bias, and by increasing the rate of flow of accurate information about the research, both within the scientific community and from scientists to the public which, directly or indirectly, funds most of these efforts.

Genetic Research at Massachusetts General Hospital (MGH)^

The MacDonald lab is located in the brand-new Richard B. Simches Research Center, just up the street from the main MGH campus. Designed to facilitate communication between the various research groups, the building features wide hallways, open spaces, and meeting rooms equipped with audiovisual equipment for presentations. A spiral staircase, representing the double helix structure of the DNA molecule, connects the fifth and sixth floors, which make up the Center for Human Genetic Research (CHGR), through to the seventh floor, which houses the Molecular Biology Department.

One of five new thematic centers launched at the Hospital, the CHGR strives to facilitate the genetic research cycle, which begins with basic research, driven by scientists’ interest in questions pertaining to the biology behind a genetic disease. In basic research, biologists try to make new discoveries about the disease. For example, by studying animal models relevant to a given disease, scientists can try to observe new phenotypes in animals (that is, observable properties, particularly those associated with gene effects) that can then be looked for in human patients as well. Or researchers may try to use these new phenotypes to develop novel assays (chemical analyses) that can be used to discover drug compounds that may prevent the disease-associated phenotype.

The next stage of the cycle is the applied, or engineering-type, research, which puts the discoveries of basic research into practice. For example, researchers, usually in biotechnology or pharmaceutical companies, may use a variant of the assay discovered in the academic research lab to test a wide variety of drug compounds to see which of them effectively alter the outcome. Then, they may give the effective ones to animals and evaluate the outcomes, modifying the compounds by changing the chemical structure and retesting them, in successive rounds, to make them perform better, with fewer untoward side effects.

At this stage, researchers often look for drug targets, or molecules that can be expected to enhance or inhibit the disease. The best drug targets provide a direct route to what should be changed in a patient on the molecular level. Testing drugs in animal models helps researchers to identify targets and prioritize the best ones for further testing.

The third stage of the research cycle is clinical research, in which physicians and clinical researchers administer drugs to patients in government-approved clinical trials. Observations made at this stage often give rise to hypotheses at the basic research stage, and the cycle begins again, as illustrated in the diagram below.


Fig. 2. The genetic research cycle.
The cycle begins with basic research in academic labs, continues with applied research in biotech or pharmaceutical labs, and ends with clinical research in hospitals. Observations from the clinical phase may be used in basic research, and the cycle begins again.

Genetic research, or research of any kind, is therefore not monolithic; there are various stages of the research effort that operate in different facilities, with different kinds of people, and on different timelines for completing experiments and trials. Because each type of research has different goals, it requires funding from different sources.

The HD researchers at the CHGR are among the many people at MGH working to facilitate the genetic research cycle for HD. The MassGeneral Institute for Neurodegenerative Disease (MIND), directed by Dr. Anne Young, Chief of Neurology, makes discoveries in the basic realm and aims to translate them into prevention and treatments of neurodegenerative diseases like HD and Alzheimer’s. MIND, therefore, serves as a bridge between basic and clinical research. On the clinical side, the Department of Neurology helps HD patients to manage their symptoms through medical treatment, such as drug regimens, some of which may be experimental, in the cadre of clinical trials.

The study of HD at Massachusetts General Hospital via the scientific method can be compared to what scientists call a “fractal,” a geometric pattern that is repeated at ever-smaller scales, as in the diagram below. Whatever the size or scale of the problem—whether a researcher is looking at a molecule, an organism, or an entire population—the process has a regular structure (derived from the scientific method) and resembles the greater whole.


Fig. 3. “Construction of a Fractal Snowflake.” (from MSN Encarta.)

The basic triangle shape is reflected at every stage in the process of forming the larger design, just as the scientific method is reflected at every level of research from the smallest to the biggest detail.

As part of the CHGR, the MacDonald lab performs basic research and takes a molecular genetic approach to understanding HD. The researchers examine the DNA sequences of genes—the HD gene, in particular—to understand how changes in gene expression and protein structure are affected by the HD mutation. Gene expression is the process by which a gene’s DNA sequence is converted into proteins that are involved in cellular processes both structurally and functionally.

Studying the genetic expression of the HD gene (both the HD and non-HD causing alleles) can provide scientists with clues about how the nerve cells stay healthy or get sick. Determining the temporal order of the early steps in the disease pathway will eventually lead to the development of drug compounds that prevent these steps from occurring. As biological models for the disease, the MacDonald lab uses genetically altered mice and cells derived from them. Because the mice have high numbers of glutamine repeats in the huntingtin protein, as a result of the same HD CAG mutations that cause HD, they are likely to reveal the earliest presymptomatic changes to manifest with HD in humans.

From Journalism to Science^

When I arrived at the lab at the beginning of July, I was eager to make a discovery of my own that would, in some small way, help scientists to find a cure for HD. I imagined myself working feverishly under the fume hood, swirling neon-colored chemicals in Erlenmeyer flasks. I envisioned myself peering into a high-powered microscope to observe the elusive structure of the huntingtin protein. As ridiculous as it sounds, I even imagined jumping up from my chair and crying, “Eureka!” as I bounded down the hallway in triumph.

As I settled into the daily routines of the lab, however, I saw the fantasy evaporate before my eyes. My biggest discovery this summer was that, while some discoveries may come in the form of intense flashes of insight, this is a rare event—except in the movies, of course. Getting to this point is much more prosaic.

Working in a lab was a brand-new experience for me. I am, however, familiar with the biology behind HD. For the past three years I have worked for Huntington’s Outreach Project for Education, at Stanford (HOPES), a student-led educational service project working to build a global Web resource on HD. Our site is a “layperson’s guide” to the scientific intricacies of HD and HD-related research. Akin to science journalism, my work has consisted of writing news briefs on the latest research, drugs, and other treatments, as well as interviewing eminent scientists and writing articles about their work on HD. My first interview was with Drs. MacDonald and Gusella during the summer of 2004, published on the website as the first chapter in a section called Research Frontiers. The two researchers discussed at length their approaches to HD and touched upon some of the myths of scientific research, including the ever-popular notion of a sudden cure or “magic bullet.”

Several months ago, just prior to my graduation from Stanford, Dr. MacDonald invited me to do an eight-week internship at her lab in this summer. After earning my BA in English with a minor in Human Biology, I headed east to Boston to begin my work. I was going from writing about science to actually doing science—a big leap.

Dr. MacDonald introduced me to Drs. Gill Gregory and Surya Reis, the two postdoctoral fellows who would be supervising my independent project. In preparation for conducting future research in their area of specialty, postdocs are in the last phase of their training, preparing them to start their own research laboratories, each working on a piece of the research puzzle. Research technologists, on the other hand, are responsible for performing one or more experiments that may either be varied or more routine, requiring long-term concerted expertise. For instance, Lakshmi Mysore, who has been working on HD for twenty years, specializes in genotyping, or determining the genetic makeup of an organism. The lab also depends upon the work of animal technicians, such as Edith Toral Lopez, who oversees the breeding of the animals and provides the genetically altered mice used in experiments.

During my first week I was outfitted with a white lab coat and notebook, given a tour of the lab, briefed on safety procedures and experimental protocols, and taught basic lab skills such as pipetting (using a syringe-like instrument to measure and transfer liquids from one container to another), taking care of tissue cultures, and transferring cells onto cover slips to be mounted on slides for viewing under the microscope.

Tissue cultures are a means of keeping populations of cells alive outside the body in a nutrient-rich liquid called a medium. I was responsible for monitoring the cells’ growth rate from day to day and splitting up the cells onto new dishes with fresh medium when the old dishes became too full because the cells had multiplied. The cells came from the brains—specifically, the striatum, the part of the brain that is first affected in HD—of mutant mouse embryos (those with 109 glutamine repeats in the huntingtin protein) and their normal (“wild-type”) counterparts.

My Summer Project^

My task as an intern would be to complete a small project within the context of Surya’s and Gill’s research. Each of the postdocs takes a slightly different approach to detecting the subtle differences between mutant and wild-type nerve cells. Surya uses immunocytochemistry (IC), a method of staining cells with antibodies so that she can pinpoint the location of the huntingtin protein, for example, in the nuclei. Meanwhile, Gill uses immunohistochemistry (IH), a method of staining tissue slices (from the striatum, in this case) with the same antibodies, also to locate huntingtin in the nerve cells.

Antibodies are proteins made by the body’s immune system as a defense against foreign material, such as bacteria or viruses, which enters the body. These Y-shaped proteins attack and neutralize the substances, called antigens, that triggered the immune response. Each antibody has a specific antigen to which it binds. The IC and IH methods make use of an antibody’s ability to recognize a particular antigen, rather than its ability to attack and neutralize it. Please see below for a diagram of an antibody.


Fig. 4. The structure of an antibody. (from Wikipedia.)

To visualize the location of the huntingtin protein in the nucleus of a mouse nerve cell, researchers use a technique called immunostaining as part of the IC and IH methods. After fixating, or preserving, a cell sample or tissue slice on a cover slip or slide, they add a small amount of a primary antibody. The primary antibody recognizes and binds to a specific place on the huntingtin protein’s surface, called an epitope. Then, a secondary antibody that comes from another animal is used to detect the first. The secondary antibody contains a fluorescent molecule, which allows the researchers to see the position of the huntingtin in the cell under the powerful confocal microscope. Multiple secondary antibodies bind to the primary, thereby amplifying the fluorescent signal. Please see below for a schematic diagram of immunostaining.


Fig. 5. Immunostaining.
The primary antibody recognizes the polyglutamine tract of the huntingtin protein, and the secondary antibody recognizes the primary. The secondary antibody contains a fluorescent molecule to help scientists visualize the huntingtin under a microscope. Multiple secondary antibodies bind to the primary for fluorescent signal amplification.

Using confocal microscopy, scientists can visualize the huntingtin protein molecules to which the primary antibody binds. They can therefore show the localization of huntingtin and make conclusions as to the site-specific function of the protein (both mutant and wild-type) in the cells.

My project fit neatly into this experimental framework and had two objectives. The wet lab component of the project, performed at the lab bench, involved immunostaining with a primary antibody made at two different commercial laboratories. My goal was to determine which of the two versions was better, and under what conditions, for seeing differences between the wild-type and mutant cells with regard to the staining pattern. See below for a picture of me performing an immunostaining procedure.


Fig. 6. Taylor Altman performs an immunostaining procedure.

She applies a primary antibody stain to mutant and wild-type cells on cover slips.

The dry lab component, performed at my desk, entailed building an electronic database of information about huntingtin antibodies. In a Microsoft Excel spreadsheet, I organized the information by such categories as antibody name, epitope, and animal host (the animal from which the antibody is taken). The database will eventually be turned into a website for use by HD researchers all over the world.

Accomplishments^

Before long, I realized that my goals were unrealistic for the two months I’d be spending at the lab. I saw that I couldn’t build an entire database on huntingtin antibodies in one summer because there are hundreds that have not yet been properly described, and information is often scarce. I did manage to gather sufficient data for eight antibodies, which is a good start.

As for the wet lab project, I completed three modest experiments, each with its own purpose and goal, on the path to determining which of the batches of primary antibodies was better suited to seeing differences between the mutant and wild-type striatal cells. Although I was a bit disappointed that I couldn’t see my project out to its end, I did get a good feel for bench work and for the extensive planning that goes into each experiment.

The first of my small experiments was a primary antibody dilutions test. Dilution is the process of making something weaker or less concentrated. To get different dilutions of the antibody, I added the same amount of antibody to increasingly large amounts of the diluting solution. Then, I applied the dilutions to the mutant and wild-type cells to determine the optimal dilution for seeing the greatest differences between the two types of cells under the confocal microscope. As the dilutions increased, I expected the strength of staining to decrease more rapidly in the wild-type than in the mutant. However, I saw the exact opposite. See below for pictures of the wild-type and mutant cells at the highest and lowest dilutions. Notice the difference in strength of staining between the two.

Fig. 7. Wild-type and mutant cells at lowest and highest dilutions.
Pictures taken at 20x magnification on the confocal microscope. As the dilutions increased, the strength of staining decreased more rapidly for the mutant than the wild-type.

Part of science is dealing with unexpected results. When a scientist initially gets results that seem to go against the hypothesis, he or she must repeat the experiment in order to rule out chance or human error. My second experiment, therefore, was a repeat of the first. Again, I got the same results. Clearly, this was no coincidence.

To look for clues that might explain my results, Surya directed my attention to the method I used to fixate the cells. Using a detergent, I had made tiny holes in the cells’ membranes that allowed the primary and secondary antibodies to flow in. Perhaps I’d used too much detergent and had made the membranes too porous, thereby letting in too much antibody or letting out too many important cellular components. Or there may have been other explanations for the results—maybe the cell types had been mixed up, or maybe the antibody no longer worked. The latter seemed most probable.

My third and final experiment, then, was a test of the amount of detergent, in which I kept the antibody dilutions the same while I varied the concentrations of detergent. On the whole, the results were inconclusive, but Surya plans to run another test in the near future.

Conclusions^

Reflecting on my summer at the lab, I am grateful to Gill and Surya, and to their supervisors Marcy McDonald and James Gusella, for the rewarding experience I had at MGH. Not only was I able to learn about the process of scientific discovery firsthand, but I was also able to become a better science writer by enhancing my knowledge of scientific materials, methods, and terminology. By conducting my own experiments, I began to think like a researcher and to understand the scientific method from the point of view of one who puts it into practice on a daily basis.

I saw a side of science that the public rarely, if ever, sees. The actual process of scientific discovery is much lengthier, more complex, and more nuanced than the media’s portrayal of it. Science is about careful observation and planning, good record keeping, and building a strong foundation for future experiments by continually attempting to disprove previous hypotheses.

Above all, science requires great patience and perseverance. Scientists do not leap from their lab benches crying, “Eureka!” every day, nor do they produce a continual stream of results. Moreover, results do not come cheaply. To my surprise, I learned that it cost about $5,000 for the supplies and small equipment, and about $400,000 for the confocal microscope, to perform one of my “simple” immunostaining experiments (not to mention the still-higher cost of paying everyone’s salaries and keeping the lab running on a daily basis).

Although my future plans don’t include working as a researcher, science will always be a part of my life, whether I choose to become a science journalist or simply a science enthusiast. I now have a much greater understanding and appreciation of the arduous work that scientists do, which gives me hope that someday they will solve the mystery of HD and other neurodegenerative disorders.

For further reading^

  • “Construction of a Fractal Snowflake.” MSN Encarta. 28 Aug. 2006
    <http://images.encarta.msn.com/xrefmedia/aencmed/targets/illus/ilt/T046583A.gif>.
  • Jones, Allan, Rob Reed, and Jonathan Weyers. “How scientific investigations proceed.”
    Practical Skills in Biology. Essex: Addison Wesley Longman Limited, 1994.49.
  • “Schematic of antibody binding to antigen.” Wikipedia. 21 Aug. 2006
    <http://en.wikipedia.org/wiki/Antibody>.
  • Ubelacker, Sheryl. “Canadians cure Huntington’s disease modified mice.” The Globeand Mail. 16 June 2006. 15 Aug. 2006
    <http://www.theglobeandmail.com/servlet/story/RTGAM.20060616.whuntingtonS016/BNStory/Science/home>.

T. Altman, 2006