Tag: scientific method

Scientific Method for the Non-Scientist? Yes, please!

NextGen Voices is a feature of the premier science magazine, Science. It is designed as a series of surveys targeted towards young scientists, asking them questions on different aspects of life as a scientist that matters to them.(For some reason, it is not very well publicized, which is a pity – because I do think that NextGen Voices is offering young scientists an important platform to voice their opinions. I got to know about it only because my colleague in the lab, a subscriber to Science, showed it to me. This is partly the reason why I wanted to blog on this today – to raise awareness).

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‘Life’ as a scientist: The Cheshire Cat effect

Another post after a brief hiatus because of work-related pressure. I’m sure nobody missed me, though. [Sniff!] Well, the pressure’s still on, but let’s say I was inspired to write this post by a chance occurrence, a question asked by a physician friend of mine. An accomplished and established surgeon in India, he is considering various possibilities and options, having recently learnt that his young son is desirous of coming to the US to pursue a career in biological research.

He asked me: how is life as a scientist in biological sciences or genetics etc? Very tough, boring life that leaves you no time? Or fulfilling and all that?

You could hear from a mile the sound of my mental machinery creaking and groaning and whirring. Naturally, I’d be delighted to welcome a budding scientist to the fold, but I also wanted to provide my friend with as true and complete a picture as I possibly could.

Shying away from the usual spiel on the quality of scientific research done at noted US universities and institutions of renown (my friend is aware of all that), I focused on the core of his question – the life as a scientist. What exactly is life as a scientist? Is it, like, life in all its glories as presented with a sonorous narration in a Discovery Channel documentary, or is it more of life, as in “Dude! Get a life!“? Does life of the latter kind come to the scientists in the manner of the proverbial Cheshire Cat of Alice in Wonderland, appearing suddenly with a mischievous grin and then vanishing slowly and unattainably until nothing but the grin is left, and then –Poof!– that is gone, too?

Pushing aside these philosophical (and ultimately useless) cogitations, I set to writing him a reply. Here’s a part of what I wrote:

There are several angles to this question, all of which – in the final synthesis – boil down to the matter of temperament.

First, as with every other profession, the rewards of a career in science are not consistent – and indeed, may even be considered insignificant under certain lights. There will be work-related irritation, frustration, aggravation and denial, some of which may even spill into one’s personal life if one cannot carefully separate the personal from the professional.

Secondly, even if one is passionate about the work to begin with, it would be difficult to sustain that same level of passion through the years. However, professional scientists can usually keep their interest aflame by diversifying into multiple research questions and/or refocusing their priorities.

Thirdly, life as a young researcher may be impecunious. One simply doesn’t become a scientist if one’s goal in life is to become a millionaire outright. I admit that in rare moments of self-doubt, I have thought about young adult basket-ball players and other athletes (especially the talented Jeremy Lin in recent times), who seem to command an exorbitant amount of money in exchange for their prowess and agility, whereas we, the science researchers in the same country, despite contributing day in and day out towards the betterment and progress of humanity, are doomed to live in relative penury.

To the discerning mind, however, the rewards are manifold, even though they may not readily translate to wads of greenbacks or pots of gold. Fulfillment is often a matter of perception, after all.

To many scientists, there is an element of thrill-seeking in what they do. Understanding a problem, analyzing it, putting forth a rational hypothesis and then performing rigorous experiments to test its validity, anticipation of the results, the joy that one feels when the observed data vindicate one’s hypothesis or the sobering effect when they don’t and push the scientist back to the drawing board – there is a lot of drama, excitement, emotional upheavals therein that can be quite enjoyable overall.

There are many scientists who find the challenge of an intractable problem very attractive and engrossing. To them, the systematic attempts at puzzle-solving, especially if the problem happens to be multi-layered, are themselves fulfilling; if they do manage to unravel the mystery, it can be very rewarding, sometimes even lucrative.

Many scientists, especially those working upon problems of immediate consequences to, say, the health and well-being of living beings, including their fellow humans, are often fortunate enough to observe the benefits of their work in relatively real-time. It can be incredibly fulfilling as well as humbling. On the other hand, even those scientists, whose professional endeavors are distally related to health, and more proximally, to basic and/or applied problems in biology, have the satisfaction of knowing that their work connects them to a larger continuum, because modern living organisms, having evolved from same or similar ancestors at different levels, often share a surprising degree of relatedness.

In addition, the sharing and communication of one’s research outcomes within the scientific community and without is no less gratifying. Having one’s work accepted for publication in a scientific journal of repute can be quite life-affirming. Recognition and renown for one’s work, when they eventually arrive, ain’t too shabby either. Accomplished scientists often wish to spread or share their experience and life’s journey, thereby hoping to influence younger minds and instilling the spirit of enquiry.

Many of these intellectual rewards or fulfillment that I mentioned above may initially seem too esoteric and far-fetched, but they exist – they require a fair bit of hard work, but they are not unattainable goals. This is important to understand, especially for a new graduate student.

The entire period of graduate studies (leading to a PhD degree) is – as I see it – essentially a period of training. One learns not only technical skills of various sorts, one also learns how to integrate one’s knowledge in one’s work. One grasps the value of perspectives – how to view one’s own work in the context of a larger picture. One picks up valuable people skills, skills of interaction, communication, presentation and the art of networking, as well as how to work cohesively in a group setting and independently at the same time. One assimilates the ways and means of effective time management, and the benefits thereof. Most importantly, under competent mentorship, one gets a thorough grounding in the scientific method.

To me, this is the most crucial aspect of training as, and being, a scientist. A scientist is much more than what one does; it refers to what one is. It is possible to integrate in one’s life, or one’s attitude towards life, the basic tenets of the scientific method, objectivity, reliance on empirical evidence, a rational and skeptical outlook, and an ability to question, observe and analyze, to varying degrees. People who successfully do that are also able to effortlessly transition from their workbench to life outside and back.

As far as having ‘time’ to do other things is concerned, I have found that it largely depends on the individual. It is indeed possible to manage one’s time effectively, so as to be able to pursue other interests. Examples abound. Just to randomly name a few instances, Paul Z Myers is an accomplished and popular biology professor, with a tremendously celebrated blog. Stephen Curry is a noted structural biologist who still finds time to blog and write for the Guardian. Russian composer Alexander Borodin was a life-long and distinguished researcher in organic chemistry. Jennifer Rohn is a working cell biologist who is a champion for the genre of “Lab-Lit”, is an author, as well as finds time for political advocacy for science funding in the UK. Canadian physicist Diane Nalini de Kerckhove combines a career as a successful scientist with her job as a professional jazz singer. As I said right at the beginning, it is a matter of temperament. If one loves what one does, one does it well – no matter what – and garners fulfillment from it.

What do you think, gentle readers? Please throw in your comments, suggestions, bouquets and brickbats in the comment section.


“Water memory” – a myth that wouldn’t die

Holy pseudoscience, Batman!

Homeopathy websites (too many to list; I found the material for this post here) are all gleefully abuzz today** with the following factoid – New Research From Aerospace Institute of the University of Stuttgart Scientifically Proves Water Memory and Homeopathy.

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et tu…? Acupuncture and pain in Nature, part deux

Continued from Part 1

As I was saying, a study by Goldman et al. in the July 2010 issue of Nature Neuroscience, postulates that “Adenosine A1 receptors mediate local anti-nociceptive (i.e. pain reducing) effects of acupuncture.”

I stumbled a little right at the title. Anti-nociceptive effects of acupuncture? Where is the evidence that such an effect exists?

Evidence schmevidence. I needn’t have worried, for the introductory paragraph reassured me of the benefits of acupuncture in pain management, by indicating – no… not clinical evidence, but – that (a) acupuncture has become worldwide in its practice, (b) despite Western Medicine’s skepticism, a broader worldwide population has granted it acceptance, © WHO endorses acupuncture for at least two dozen conditions, (d) the US National Institutes of Health issued a consensus statement proposing acupuncture as a therapeutic intervention for complementary medicine (now, that wouldn’t be the basis of the US NCCAM, would it?), and (e) – what the article found “most telling” – the US Internal Revenue Service approved acupuncture as a deductible medical expense in 1973.

Comforted thusly, I now proceeded to the premises of the study. Accepting a priori the analgesic effect of acupuncture (which is ‘well documented’ according to the article), the study sought to find a biological basis for that effect.

Let us examine one of the articles used by Goldman’s group to formulate their hypothesis, namely, a review article written by ZQ Zhao, titled “Neural mechanism underlying acupuncture analgesia” (Prog. Neurobiol. 85, 355-375, 2008). Zhao notes in his review that

Traditional acupuncturists remarkably emphasize ‘’needling feeling’’ in clinical practice. It seems that acupuncture analgesia is manifest only when an intricate feeling occurs in patients following manipulation of acupuncture… described as soreness, numbness, heaviness and distension in the deep tissue beneath the acupuncture point. In parallel, there is a local feeling in the acupuncturist’s fingers, the so-called ‘’De-Qi.’’ The acupuncturist feels pulling and increased resistance to further movement of the inserted needle…

In other words, dermal and subdermal tissue reacts to the presence of a foreign body, so much so that even the patient is able to feel the sensation; in fact, in a recent clinical trial studying acupuncture as adjunct therapy to proton pump inhibitors in refractory heartburn, patients were told to expect “de Qi”, described as a heavy aching sensation. Quoting other studies, Zhao goes on to indicate that since the deep tissue beneath the acupuncture points (or ‘acupoints’), including epidermis, dermis, subcutaneous tissue, muscle and tendons, were found to be richly supplied by peripheral nerves, the process of acupuncture might involve the manipulation of pain carrying Aδ and C nerve fibers. Although Zhao dismisses the effect of C fibers in the putative acupuncture analgesia, the authors of the heartburn study used the effect of tactile sensation carried through C fibers to argue against the inclusion of sham acupuncture controls in their study!

Zhao also takes note of the clinical observation that acupuncture needles inserted into the lower limbs fail to produce the ‘’de Qi’’ feeling or have any analgesic effect on the upper part of the body in paraplegic patients, and goes on to conclude that mere insertion of acupuncture needles don’t relieve pain, and deeper manipulation of the needles (rotation, electrical stimulated or heating) that results in tissue soreness in the patient is essential to produce the desired analgesia. Goldman et al. used this hypothesis to design their protocol.

Of course, Zhao also concludes from some other studies that the effect of acupuncture analgesia is highly subject to individual differences; in one study he quotes, only 5 of 11 healthy volunteers reported reduction in pain. In addition, it has been shown in patients of osteoarthritis (Pariente et al., 2005, quoted in Zhao’s review), as well as patients following dental surgery (Bausell et al., 2005), that even sham acupuncture, or for that matter, the mere expectation of receipt of acupuncture by patients or the belief that it would work produced the same level of pain reduction as that by acupuncture. So much for various neurally-mediated mechanisms of acupuncture analgesia!

Although Zhao has presented what he considers compelling evidence on a role of centrally-released endogenous opioids, such as β-endorphins and enkephalins, in the alleged analgesic effects of acupuncture, Goldman et al. in their paper disregard that possibility, noting that acupuncture has to be applied locally to the pain, or even on the same side as the pain focus.

The comparison with Tooth Fairy Science is getting stronger, then.

In the Goldman et al. study, induction of pain in a mouse model was achieved in two ways:

  • Neuropathic pain: Induced by ligation of the right leg sciatic nerve in anesthetized mice.
  • Inflammatory pain: Induced by injection of Complete Freund’s Adjuvent (which would cause painful peripheral inflammation) in the plantar surface of the right hind paw of mice. As a control, the study used injection of an equal amount of physiological saline (which should not cause any inflammation) in the left hind paw.

Effect of the inflammatory pain was assessed by two techniques. Once the paw was inflamed, the mouse became more sensitive to –

  • Mechanical allodynia (pain induced by agressive use of a normally-painful stimulus): Evaluated using repeated stimulations with a Von Frey filament exerting 0.02 g of force onto the plantar surface of the paw, and observring the withdrawal of the paw when the pressure becomes uncomfortable to the mouse. (Find here a description of the process.)
  • Thermal hyperalgesia (pain from heat): Assessed using a mobile radiant heat source focused on the hind paw (for a maximum of 20 seconds to avoid tissue damage), and observing the time taken for the paw withdrawal.

In addition, behavioral correlates of pain were evaluated in the certain mice – before intraplantar injection of CFA or nerve ligation, and a few days to a week after the process.

Building on the local effect hypothesis, Goldman et al. wanted to test if Adenosine – a by-product of the breakdown of the cellular energy currency, ATP, that is released during mechanical or electrical or thermal stimulation – could produce analgesia by binding to a receptor called the A1-Adenosine receptor. Indeed, acupuncture applied with deep manipulation sharply increased the extracellular concentrations of all purines, including Adenosine. The group also demonstrated the requirement of the A1-Adenosine receptor by showing that 2-chloro-N(6)-cyclopentyladenosine (CCPA), a substance that binds to that receptor, reduces the sensation of pain in the both above-mentioned mouse models when applied locally. The authors went on to postulate that the effect of CCPA was possibly mediated by C-fibers as well as Aδ fibers.

Acupuncture with deep manipulation achieved the same effect as CCPA in reducing pain. However, the local effect was evident, and – as authors note in supplementary data – acupuncture without deep manipulation did not achieve the same effect.

Substances (such as Deoxycoformycin, a nucleoside analog drug approved for Leukemia) which cause an accumulation of Adenosine were able to potentiate the analgesic effect of acupuncture in inflammatory and neuropathic pain. Strangely enough, Deoxycoformycin appeared to be subject to the same local effect phenomenon, and had no effect unless it was combined with acupuncture in the two models of chronic pain.

The authors admitted in the discussion that mechanical stimulation of the skin, including non-penetrating needles as placebo, can activate epidermal A1-receptors, as well as release adenosine, thereby decreasing pain, but they claimed that this is different than the deep penetration of the acupuncture needles reaching muscle and connective tissue. Is the adenosine release at the deeper level more difficult, since it requires the vigorous manipulation of the needles? Combine this with the fact that adenosine is rapidly cleared from the extra-cellular fluid. Is the length of the time for which adenosine is active and binds to A1-receptor sufficient to give rise to the putative analgesia through acupuncture?

Of course, the authors’ hypothesis does not explain the equally well-observed analgesic effect by sham acupuncture, or the expectancy of acupuncture, in human patients. The interventional mouse study, testing very specific types of experimentally induced pain, with a small sample-size (n=5-8) and without proper placebo controls could hardly be an adequate study to establish a causal relationship between acupuncture and analgesia.

Tooth Fairy science: despite low prior probability or weak premises, there is an over-dependence on deductive reasoning to arrive at a conclusion, and not enough application of inductive reasoning to check the falsifiability of the said conclusion.

And yet…

Nature Neuroscience!!!

Main articles cited:

1. Goldman, N., Chen, M., Fujita, T., Xu, Q., Peng, W., Liu, W., Jensen, T., Pei, Y., Wang, F., Han, X., Chen, J., Schnermann, J., Takano, T., Bekar, L., Tieu, K., & Nedergaard, M. (2010). Adenosine A1 receptors mediate local anti-nociceptive effects of acupuncture Nature Neuroscience, 13 (7), 883-888 DOI: 10.1038/nn.2562

2. Zhao, Z. (2008). Neural mechanism underlying acupuncture analgesia Progress in Neurobiology, 85 (4), 355-375 DOI: 10.1016/j.pneurobio.2008.05.004

et tu…? Acupuncture and pain in Nature, part one

Physician and blogger Harriet Hall, MD, once coined an exceptionally apt phrase to describe research in many alternative medicine modalities – “Tooth Fairy Science”; it refers to research undertakings into a phenomenon whose existence is yet to be established. In a post in her blog Science-based Medicine, she explained:

You could measure how much money the Tooth Fairy leaves under the pillow, whether she leaves more cash for the first or last tooth, whether the payoff is greater if you leave the tooth in a plastic baggie versus wrapped in Kleenex. You can get all kinds of good data that is reproducible and statistically significant. Yes, you have learned something. But you haven’t learned what you think you’ve learned, because you haven’t bothered to establish whether the Tooth Fairy really exists.

Priceless. And of all the modalities championed by modern peddlers of pseudoscience, acupuncture most certainly qualifies as a prime example of Tooth Fairy Science.

Acupuncture is a procedure under the system of Traditional Chinese Medicine (TCM), which involves shallow insertion of needles into the skin at specific points. This system relies on quaint notions that mix pre-scientific ideas about physiology and disease with Eastern mystical philosophy. In TCM, diseases are considered to stem from a disharmony between yin and yang, two abstract ideas that are supposedly complementary in nature; this disharmony may result in blocking of the flow of a vital (life) energy, known as Qi (pronounced ‘chi’), along mystical pathways called meridians. Acupuncture supposedly unblocks Qi flow through meridians, resulting in the balancing of yin and yang, and consequently, cure. Never mind that no one has measured Qi ever, or that there are no anatomical structures that correspond to the prescribed locations of the meridians.

Acupuncture is currently the darling of the media as well as many Complementary and Alternative Medicine (CAM) professionals. Its popularity is attested to by the fact that many of the well-known medical centers and hospitals in the US are increasingly offering acupuncture as a therapeutic option. This situation per se is utterly amazing, considering that all the actual research involving acupuncture done till date seem to corroborate the hypothesis that it is nothing more than an elaborate placebo.

There is no dearth of studies on Acupuncture; a casual PubMed search of the term “Acupuncture” yielded more than 8000 English-language primary research articles (including clinical trials) and close to 2000 reviews (including meta analyses). Many of these studies have made extraordinary claims about the efficacy of acupuncture and related procedures in a variety of diseases and disorders. However, careful and meticulous scrutiny of these studies, as well as those claims, have often demonstrated that many of these studies are poorly designed or carried out, and that the claimed outcomes are based on wishful thinking on part of patients (a.k.a. conditioning) and investigators, as well as ignorance about the action of placebos and how inaccurate placebos can confound the interpretation of data. These have often necessitated prodigious hand-waving and post hoc rationalizations in the discussions.

One of the common themes that most studies in alternative medicine adhere to is that they are often NOT published in high-quality, prestigious, peer-reviewed journals. This observation, while attributable to the lack of rigorous science in those papers, have nevertheless engendered within the alternative medicine community the suspicion of a conspiracy. Rational researchers, dealing with empirically sound science-based medicine, are by and large content solely with the critical deconstruction of the absurd claims by CAM proponents, bolstering their arguments with published and verifiable experimental evidence and analysis.

Imagine my surprise, then, when I became aware of a relatively recent study claiming efficacy of acupuncture and offering a mechanism of action in… [Wait for it…]

Nature Neuroscience!!!

The study was republished as a part of a recent Nature Supplement on Traditional Asian Medicine. Curiously, the issue appeared to be sponsored by two entities which have a significant financial interest in the field of alternative medicine. There is, of course, no law that prohibits such an entity from advertising or popularizing its products, but that a premier scientific journal like Nature should be an instrument to such promotion of pseudoscientific modalities raised quite a few eyebrows. Physician and Scienceblogger Orac has eloquently expressed his outrage, which has found echoes in the skeptical blogosphere and Twitterverse. In this post, I, however, would restrict myself to discussing the study by Goldman et al., that appeared in the July 2010 issue of Nature Neuroscience. The study postulates that “Adenosine A1 receptors mediate local anti-nociceptive (i.e. pain reducing) effects of acupuncture.”

To be continued… in Part Deux! [Suspenseful Music]

Of Correlations, Causations and the Divide Therein – Part Deux

In the first part of this post, I mentioned the important maxim in science, Correlation does not imply causation, providing a glimpse of its logical framework, and discussing how the scientific method is utilized to establish causality in observed relationships between/amongst variables.

And what happens when scientists, study authors, investigators ignore this prime maxim?

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Of Correlations, Causations and the Divide Therein – part Un

One quick disclaimer before I proceed. When I have quoted one or more Wikipedia articles in the text, it is because I have found them well-written, informative, and adequately illustrative; however, I shall make no claim as to their veracity and/or authenticity because I have not been able to access and verify all the background references therein. If you find an error, please feel free to chide me in the comments.

An important maxim used in science, or more precisely, in the scientific study of relationships between/amongst variables, is that ‘Correlation does not imply Causation’. Indeed, until and unless such causality has been verifiably established through independent means, any attempt to indicate that it does falls under the logical fallacy of questionable cause, cum hoc, ergo propter hoc (Latin for “with this, therefore because of this”).

It is important for all to understand this concept – those who are engaged in scientific studies, as well as those who read about and interpret such studies.

Correlation is a statistical relationship between two or more random variables; for simplicity’s sake, let’s consider two, say, A and B, such that if changes in the values of variable A statistically correspond to changes in the values of variable B, a correlation is said to exist between A and B. This reflects a statistical dependence of A on B, and vice versa, and therefore, statistically-computed correlations can be used in a predictive manner. To pick a completely random example, the epidermal growth factor receptor (EGFR) is expressed on neoplastic cells in colorectal carcinoma. Number of cells expressing EGFR was found to be correlated with the size of the tumor (adenoma), i.e., cells from a larger tumor express more EGFR. Therefore, EGFR expression may be useful as a prognostic biomarker for adenoma progression.

Those who have already identified the problem in this assertion, congratulations! As the paper cautions, although EGFR pathway is important to colorectal carcinogenesis, it is unknown at this point whether the observed increase in EGFR expression is because neoplastic cells make more EGFR per se for some reason, or because a larger tumor would house numerically more of the cells that are capable of making EGFR. This, as you can understand, is an important distinction, and therefore, the authors conclude correctly that “Further larger studies are needed to explore EGFR expression as a biomarker for adenoma progression.”

Such examples abound, all illustrating how correlations can be useful in suggesting possible causal or mechanistic relationships between variables, but more importantly, such statistical interdependence between the said variables is not sufficient for logical implication of a causal relationship. In other words, while empirically A may be observed to vary in conjunction with B, that observation is not enough to assume A causes B.

But what happens when one makes such an erroneous assumption? For starters, one is then disregarding four other possibilities, any or each of which may be true and account for the correlation.

  1. A may cause B.
  2. B may cause A.
  3. An unknown or uncharacterized third variable C may cause both A and B.
  4. A and B may influence each other in presence or absence of C in a feed-back loop, self-reinforcing type of system.
  5. The two variables, A and B, changing at the same time in absence of any direct logical or actual relationship to each other, besides the fact that the changes are occurring at the same time – a situation also known as coincidence. A coincidence may allude to multiple, complex or indirect factors that are unknown or too nebulous to ascribe causality to, or may reflect pure, random chance.

Each of these five hypotheses is testable and there are statistical methods available to reduce the occurrence of coincidences. Therefore, the mere observation that A and B are statistically correlated doesn’t lend itself to any definitive conclusion as to the existence and/or directionality of a causal relationship between them.

Determination of causality is an entirely different ball of wax, and that discussion is beyond the scope of this post. Suffice it to say that in the sciences, causality is not assumed or given. The scientific method requires that the scientists set up empirical experiments to determine causality in a relationship under investigation.

The scientific method works in logical progression.

  1. Initial observations (of a putative relationship between variables) are made.
  2. an explanation is proposed in form of one-or-several hypotheses about possible causal relationships, including one of no relationship (the Null hypothesis).
  3. Certain predictions or models may be generated on the basis of each of the hypotheses, which in turn guide the experimental design.
  4. Experiments are designed to demonstrate the falsifiability of the hypotheses, i.e., to test the logical possibility that the hypotheses could be proven false by a particular empirical observation. Indeed, testing for falsifiability or refutability is a key part of the scientific process.
  5. Once designed, the experiments are used to test the hypotheses rigorously, and the data, analyzed critically to reach a conclusion, accepting or rejecting the hypotheses.
  6. But the method doesn’t cease there. All empirical observations are potentially under continued scrutiny, which involves reconsideration of the derived results, as well as and re-examination of the methodology, especially in the light of newer techniques that are capable of taking deeper and more accurate measurements. Such is the dynamic nature of the scientific method.

Establishment of causality, therefore, has to pass through the same rigorous filters before it can be accepted. But if it does, the conclusions may be considered unimpeachably valid, within the given set of circumstances.

So… Correlation doesn’t inherently imply causation.

Some modern examples are in Part Deux. Please don’t hesitate to comment.