Category: Scientific Method (Page 2 of 2)

Alcohol pwns inflammation; Or, saga of alcohol dehydrogenase from Aspergillus
Aspergillus fumigatus and various other Aspergilli are ubiquitous molds. These are hardy aerobic saprotrophs, growing as easily on breads and potatoes as on plants and trees. However, many Aspergilli are capable of growing in nutrient-deficient or nutrient-absent environments, and surviving in extreme conditions, such as high temperature (up to 55oC) and pH; for example, A. niger, the Black Mold, can grow happily on damp walls. I have observed A. fumigatus grow on the surface of a highly alkaline buffer (pH9; one of the pH meter standards).

In addition, A. fumigatus has been found to be highly tolerant of a wide range of oxygen levels, from atmospheric (21%) to moderately aerobic (~14% as in the lung alveoli), low (2-4% as in tissues), or hypoxic (<1.5% as found in compost piles), even to as low as 0.1% (Source: References available with the article under review). For an obligate aerobe, A. fumigatus has evolved remarkably robust mechanisms that allow it to tolerate and thrive in extremely hypoxic conditions. Needless to say that such mechanisms are likely to come mighty handy when causing disease.

Many of the Aspergillus species are known to cause disease in human and animals; of these, A. fumigatus is the most common causal agent of invasive pulmonary aspergillosis, a frequent and life-threatening complication in several immunosuppressed patient populations. Microscopic airborne spores (‘conidia’) of the mold, produced copiously, are inhaled by the host (human and animals). Immunocompetent hosts mount an innate immune response that eliminates the conidia; however, both immunocompromised hosts (such as cancer and transplant patients), who cannot mount an efficient enough response, and patients of other chronic lung/airway diseases (such as allergic asthma), who mount an excessive and unmitigated inflammatory immune response, are both vulnerable to the disease produced by A. fumigatus conidia.

In order to cause disease, the conidia primarily colonize airways or the lungs and, if successful in breaching the innate immune defence, germinates into hyphae, long finger-like projections, that invade the tissues and blood vessels. The mechanisms by which A. fumigatus is able to survive and grow in the host environment are not all well-understood. Based on previous studies which demonstrated that inability of certain mutants of A. fumigatus and the yeast pathogen, Cryptococcus neoformans, to grow under hypoxic conditions correlated with their reduced virulence in mouse models, Grahl et al., leading a multi-institutional group of investigators, set out to discover if A. fumigatus encountered such hypoxic conditions in the host lung and how it dealt with it while infecting the host.

They found that
(a) hypoxic microenvironments do occur in three distinct immunosuppressed murine models; they discovered this by using a chemical hypoxia detection agent, pimonidazole hydrochloride, that enabled cool, visual estimation of hypoxia in tissue via immuno-fluorescence (Figure 2). From the observed extent of hypoxia, fungal growth, and host immune responses in different immunosuppressive models, the authors inferred that “the host inflammatory response plays an important, but not exclusive, role in the generation of the hypoxic microenvironment.”
(b) alcohol is involved (perhaps not surprisingly, since hypoxia must be a stressful situation!).

No, really. Using a 400 MHz 1H-NMR, Grahl et al. could detect substantial ethanol in 4 out of 10 immunosuppressed mice infected with A. fumigatus at day 3 post-infection, but none in uninfected mice.

Figure S1 Grahl et al., 2011, PLoS Pathog 7(7): e1002145

In order to ensure that the ethanol in the lung was of fungal origin (and not a result of, say, some wild Bacchanalian orgy the mice partook of in the middle of the night), the authors tested and established that A. fumigatus was indeed capable of fermenting glucose to ethanol in vitro in media containing minimal (1%) glucose under hypoxic conditions (1% oxygen, Figure 1) after 48, 72, and 96 hours of growth.

Analyzing fungal genes involved in the alcohol fermentation pathway, the authors zeroed in on the A. fumigatus gene alcC encoding an alcohol dehydrogenase whose expression is enhanced significantly in response to hypoxia. Interestingly, this gene appeared not to be contributing to A. fumigatus‘s ability to grow under hypoxic conditions, nor to the virulence of the mold – since mutants lacking this gene was as virulent as the wild type A. fumigatus in all models of immunosuppressed mice. However, in the model with cyclophosphamide induced neutropenia, as well as the one with corticosteroid induced immunosuppression, the mutant mold strain producing no alcohol had greatly reduced growth with evidence of significant inflammation when compared to the wild type (Figure 8); in the mice infected with the mutant mold strain, increased recruitment of immune effector cells, particularly neutrophils (Figure 9), and associated altered cytokine responses (Figure 10) were observed in the lung.

In other words, the alcohol of fungal origin may modulate the immune response by suppressing the inflammation, which may offer a survival advantage to the mold in the tissue. As always, alcohol makes everything better, especially when the mold brews it by itself.

The authors discuss one important caveat of the study: the observation of ethanol production in only 4 of the 10 infected mice. They offer several possible reasons that may have contributed to this, such as the lack of a more sensitive method of detection, unsuitability of bronchoalveolar lavage fluid as the site of interest and so forth. It’d be of interest to see if better detection methods – which they say they are developing – improve upon these results.

Another important caveat that the authors didn’t discuss lies in the model, particularly the method of immunosuppression. Corticosteroid treatment impairs the antifungal action of immune effector cells; in mice treated with a single dose of the corticosteroid Triamcinolone, it is perhaps not surprising that at day 3 post infection there was a rebound increase in inflammatory cells, led by neutrophils, which are after all the principal effectors against Aspergillus. Unfortunately, the authors didn’t check cellular infiltrate status in mice immunosuppressed with cyclophosphamide which they gave at day -2 and day 3 of infection. Cyclophosphamide causes profound neutropenia – as the authors have noted – and at the given dose, the neutropenia usually lasts for 96 hours. So, by day 3, one would expect a rebound neutrophilia in these mice prior, of course, to the second dose. It would have been interesting to see the cellular composition of the infiltrates in the cyclophosphamide-treated mice. One would expect the inflammation in this case to be largely macrophage/monocyte in nature, perhaps.

Overall, a rather interesting study with some intriguing findings; a good read.

Pathogens&rft_id=info%3Adoi%2F10.1371%2Fjournal.ppat.1002145&;bpr3.tags=Biology%2CMedicine%2CResearch+%2F+Scholarship">Grahl, N., Puttikamonkul, S., Macdonald, J., Gamcsik, M., Ngo, L., Hohl, T., & Cramer, R. (2011). In vivo Hypoxia and a Fungal Alcohol Dehydrogenase Influence the Pathogenesis of Invasive Pulmonary Aspergillosis PLoS Pathogens, 7 (7) DOI: 10.1371/journal.ppat.1002145

Religious Factors & hippocampal atrophy or hypertrophy?

As a testament to humankind’s everlasting quest for knowledge and understanding of the self, a number of scientific studies in the recent times have examined the elusive relationship between the human brain and that fountainhead of human emotion and passion, namely, Religion. There have been studies on neurological correlates of religious experiences and spiritual practices, such as meditation and prayer; many studies have looked at both acute and chronic effects of such practices in relation to brain function. A recent study along the same lines, published by Owen et al. of Duke University, in PLoS One on March 30, 2011, has attempted to link religious factors with changes in a specific brain region, the hippocampus, using functional magnetic resonance imaging (fMRI) techniques.

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Confusions galore: science and superstition? Part 1 of 2

Angela Saini, a well-known London-based science journalist and author, has written a book titled Geek Nation in which she makes a case for the rise of India as a scientific superpower despite the overwhelming influence of religion in the Indian society. I’d love to read the book. It will be published tomorrow, on March 3, 2011 in the UK; I don’t know when it’d be available in the US, but soon, I hope.
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Confusion? You betcher… Part 2 of 2

In the first part of this two part post inspired by science-journalist and author Angela Saini’s write-ups on the topic of science and superstition in India, I explained my views on the real problem plaguing science education in India. In this second part, I look more closely at Angela’s writings.

What got me interested in Angela’s forthcoming book, Geek Nation (available March 3, 2011 in the UK), has been outlined in one of her blog posts, titled “The god confusion”. She indicates:

One of the more controversial themes in Geek Nation is the impact that the rise of science and technology is having on superstition and faith in India (which is, after all, said to be the most religious place in the world).

This happens to be an issue that has, frankly, troubled me for a long time. Angela offered a sorta-kinda justification in her post:

The ideas we’re instilled with as kids are far more difficult to abandon when we grow older than some rationalists would like to think they are. And of course I know lots of intelligent, rational people who cling to faith (and many more who read their horoscopes)… for many, it’s comforting and reassuring.

This immediately dredged up memories of my having grown up in India, images of people I have been around and situations I have been in: (a) the frequent practice of choosing an ‘auspicious’ time and place of a scientific convention (meeting, congress, conference) based on astrology or some other personally favorite superstition; (b) the invoking of gods and goddesses for blessings prior to the commencement of scientific symposia; © working scientists, biologists, chemists, physicists, sporting on ten fingers ten rings set with precious or semi-precious stones, all designed either to curry favor with some astrological planet or star, or to ward off the evil influences thereof; (d) biochemists and molecular biologists devoutly praying for a favorable outcome of their PCR runs; … the list can go on and on. This regrettable behavior on part of scientists is the sign of a greater malaise: Irrationality of any kind leaves our minds open for further irrationality. For that very reason, merely because a superstition appears ‘comforting and reassuring’, that cannot/shouldn’t be reason enough for embracing it wholeheartedly.

I have listed my views on the topic. I am, unfortunately, not aware if Angela is a scientist by training or not (Her Blogger profile mentions her industry as ‘Communications or Media’). If she is not, it’d certainly be refreshing to get a perspective from a non-scientist on the strange, and strangely easy, coexistence of science and superstitions in India – all the more reason to wait eagerly for the book.

Pending the arrival of the book, I turned to the column that Angela has written for the New Humanist, titled the same as her blog post, “The god confusion”, in order to get a feel of what was to come in the book. It is well-written and insightful. Angela’s conversational style is a pleasure to read. She has explored the situation from a personal as well as historical perspective, noting past efforts at injecting rationality into the Indian societal mores. She has examined a couple of the reasons why the juxtaposition of science and superstition seems to have endured in the Indian psyche, such as high levels of adult illiteracy and the apparent fluidity of Hinduism (which is the predominant religion in India). She has questioned the foundations of a so-called spiritual resurgence among India’s urban or semi-urban, educated youth.

But there is also something oddly wrong and out of sync in her piece. It is almost as if the confusion she underscores in her article is not the confusion that the Indian people appear to face in having to choose between science and irrationality; it is rather a confusion that is her own, as if reflecting her own ambivalence about the relative place of science and religion in her life – perhaps borne out of the confusion of ideas from her childhood, the invisible-yet-present struggle between her unashamedly geeky, rational and skeptical father and horoscope-wielding mother (judging from her own words).

In odd places in Angela’s otherwise interesting account, a strange credulity, a desire to look at the Indian science situation through rose-tinted glasses, has shone through – evident in the facile ease with which she refuses to acknowledge what her inner rationalist says. When she passed by the Swaminarayan Akshardham temple in New Delhi, a sprawling religious edifice purportedly for showcasing “the essence of India’s ancient architecture, traditions and timeless spiritual messages”, the rationalist in Angela did note that “In a poor country, it’s a sumptuous and expensive testament to faith”, and yet she is “impressed” by the motivations of the people who built the place. Is it really that hard to imagine how many poor, hungry people could have been fed and clothed, how many little girls given the light of education, how many endeavors – towards empowerment of women, the disadvantaged, and the marginalized – financed through the amount of money and/or effort spent on building an edifice that does nothing but look pretty and rehash some perennially ineffectual words?

Towards the end of the article, Angela also whimpers about how difficult it is to let go of religion in India. Don’t get me wrong. Her observation is astute when she notes:

In India that struggle is multiplied because the culture is so dominated by it. Beliefs are burned into the minds of children – Hindus often keep shrines at home, pray daily and have their fates decided by their horoscopes at birth. It’s common to appeal to the gods to guide you in your choices and to give you luck. Not only this, superstition and religion are big business: astrologers have their own television programmes (sic); homeopathic drugs and traditional medicines are sold in the millions; and fashionable gurus attract stadia full of fat-walleted worshippers. Ditching god isn’t easy when you’re surrounded by an infrastructure built on belief.

(It may be difficult, Angela, but it is not impossible. I have done it and freed myself from the shackles of religion. It is an unbelievably liberating feeling.)

However, it is important to understand that unless Indians grow up as a nation, unless the unholy reliance on faith and superstitions is burnt at the altar of reason and sanity (the religious imagery of this allegory seems oddly appropriate here!!), unless rationality and skepticism is made the mainstay of the basic education, including science education, the nation can never grow, prosper and thrive intellectually – no matter how ‘desperately religious’ modern, so-called educated Indians try to rationalize their transcendental bond with faith and superstitions, no matter how much they attempt to reconcile scientific facts with fundamentally incompatible religious stories.

But perhaps Angela’s problem – despite her obvious understanding of the crux of the situation – has a different root. She epitomizes it when she engages in a rather disappointing, spacious, strawman-beating statement in her blog post, where she says:

“Unlike some scientists and radical atheists like Richard Dawkins, I’m actually quite sympathetic to the emotional reasons behind religious belief.”

Yes, the sympathy. Angela’s article’s title The god confusion may possibly be a play on Dawkins’ The god delusion, but the former ain’t nothing like the latter. Nowhere has Richard indicated that he is not sympathetic to the emotions associated with religious belief in people; in fact, in The God Delusion, he has dedicated chapter after chapter towards understanding the basis of religious belief, of faith.

But Angela’s ‘sympathy’ towards the faithful – likely stemming from her own internal confusion – seems to have effectively blinded her towards the fact that rational atheists object to the religious beliefs, myths and superstitions per se, and not to the individuals holding those beliefs – until and unless those beliefs lead to harm and injury to others (and they do; there is enough evidence of that in the real world – but that’s a topic for another day).

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.

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