Deepak Chopwned

Gotta love this video. The most succinct takedown of Chopra I've ever seen.




That confused "I don't get it" look on Chopra's face makes the video all the more great.

Creationists: not so smart and honest.

Browsing Youtube today, I came across a video entitled "Why do SMART AND HONEST people laugh at Darwinsits?". With a title like that, I knew I had to watch it. Here it is, below:



The guy's argument is this: the scenarios used by evolutionary biologists to explain the giraffe's long neck are completely implausible (and smart and honest people, apparently, laugh at them). According to him, biologists claim that the giraffe's neck grew longer to reach leaves higher up on trees, and all the shorter neck giraffes died of starvation because they were "too proud" to bend their necks down and eat the grass on the ground. Why, he asks, did none of the other African animals also grow long necks? Why didn't the giraffes just eat the grass on the ground? This scenario is ridiculously laughable, he says. Silly biologists!

The problem is that he has a less than tenuous grasp on how evolution works and hasn't a clue what evolutionary biologists actually say on the issue.

The currently favoured hypothesis in evolutionary biology is that the giraffe's neck is an example of runaway sexual selection, not natural selection via resource competition. Male giraffes routinely use their neck in "necking" competitions - mating displays that males use to establish dominance and win the fancy of females. Just like the peacock's tail, the giraffe's neck grew longer and longer in response to the ladies' preferences. Shorter neck giraffes did not "die of starvation"; they had a harder time wining mates and this negatively impacted their reproductive fitness. Why don't the other plains animals have long necks?  Because this sexual selection process didn't occur in other animals.

But even in the absence of sexual selection, the giraffe's neck can be explained by ecological selection alone. Those giraffes with longer necks had access to greater resources than those with shorter necks (that is to say, not only could they eat the grass, but they could eat leaves higher up on trees, too). The long neck giraffes, then, had greater reproductive fitness than those with shorter necks. The alleles for longer necks became more and more prevalent in the population; the alleles for shorter necks became less and less frequent. The short neck giraffes did not "die from starvation", the short neck alleles were weeded out over time. The video's creator asks "why didn't the short neck giraffes just eat the grass?", and if he thought about it for a second, he would realize that they did. It was competition from the other grazing animals that prompted giraffes to find resources else where; those that could eat both the grass and the high leaves had an advantage!

So why do smart and honest people laugh at "Darwinists"? They don't. They laugh at fools trotting out tired creationist canards without doing a moment's research.

AIDS Denialism: Deadly Ignorance Part III: Fuzzy Math and Distorted Reality

In the previous two part of this series (see Part I and Part II), I tackled the forerunners of the AIDS "skeptics" community, and addressed how Koch's Postulates support HIV as the cause of AIDS. In this part, I will assess how some of the deniers distort studies and misinterpret math to support their dangerous ideas.

If you spend any time on AIDS denialist websites, there are likely a few statistics that will show up repeatedly. This is because it is a common practice for AIDS denial websites to copy and paste the same arguments verbatim from one website to the other, no matter how old the information might be, or if the arguments have been debunked already. One such statistic that the deniers constantly toss around is that half – or more - of Africans who qualify as having AIDS test as HIV-negative¹. Specifically, they routinely cite three specific studies²:

122 patients with "AIDS": 69% test HIV-negative (Brindle, 1993)

227 patients with "AIDS": 59% test HIV-negative (Hishida, 1992)

913 patients with "AIDS": 71% test HIV-negative (Songok, 1994)

But if one were to take a critical look at each of these studies, they do not support the claims of the AIDS "skeptics" in the least. Let's take a look at each of them.

The first paper by Brindle et al. is entitled "Quantative bacillary response to treatment in HIV-associated pulmonary tuberculosis"³. In this study, the authors looked at 122 patients with "culture-proven pulmonary tuberculosis". These patients were divided into two groups, and each group had their tuberculosis treated with one of two different treatments. They then looked at how the HIV-positive individuals in each group took to their treatments compared to the HIV-negative individuals. The purpose of this was to determine if patients with HIV would respond to chemotherapeutic treatments differently than those without HIV (and they found that there was very little difference).

It should be noted that the 122 patients in the study were NOT patients with AIDS. They were patients with pulmonary tuberculosis. This is a symptom of AIDS, but of course, pulmonary tuberculosis can arise in humans in a variety of ways; it is not exclusive to AIDS patients. Of the 122 patients, only some of them had AIDS. So where did the denialists get their numbers? Of the patients that were given the first treatment, 17 were HIV-positive and 57 were HIV-negative. Of those that were given then second treatment, 20 were HIV-positive and 35 were HIV-negative. In total, 37 of the 122 patients were HIV-positive, so 85 out of 122, or 69%, were HIV-negative. But this is not the number of AIDS patients who were HIV-negative; rather it is the number of tuberculosis patients who were HIV-negative. This is a different thing altogether. The AIDS "skeptics" misrepresent this number to support their case, when in fact, it does not.

The second paper is "Clinically diagnosed AIDS cases without evident association with HIV-1 and 2 infections in Ghana" but Hishida et al⁴.The authors' aim in this paper was simple: to test patients suspected of having AIDS for HIV. It is important to remember that the patients they looked at were suspected cases of AIDS – the authors state "CD4 cells were not counted [in these patients] because of insufficient facilities". They looked at 227 cases and found the following results : 48 were positive for HIV-1, 17 were positive for HIV-2, 11 were positive for both stains, and 16 were of indeterminate HIV status. The remaining 135 patients were determined to be HIV seronegative. Those 135 of 227 patients amounts to the 59% the AIDS denialists toss about. So at a superficial glance at the numbers might be seen as giving support to the denialist arguments.

But the denialists apparently didn't read any further than the raw numbers, because the authors continue: the 135 seronegative samples were from patients that had "weight loss, prolonged diarrhea, chronic fever" – so is this AIDS? The authors didn't think so. They state "We believe that many patients of this group were perhaps improperly diagnosed or had other unidentified diseases…[t]he existence of other agents causing AIDS-like syndromes might be possible for these so-called HIV-negative cases." In other words, it's likely the HIV-negative cases were from patients who did not have AIDS to start with – remember that these were samples from suspected AIDS cases, and not confirmed cases. The denialists take a quick look at the numbers, point out the 59% HIV-negative cases and ask "How could this be?" without bothering to read further and realize that the authors have answered that very question!

I also feel that I should point out that the authors used serological techniques to detect the presence of HIV antibodies in the samples, rather than using more sensitive PCR techniques. I am confident that if this study was repeated today, using PCR and improved diagnostic techniques, the suspected AIDS cases that test HIV-negative would be quite fewer.

The third study the denialists refer to is Songok et al's paper "Low Prevalence of Human T-Lymphotrophic Virus Type 1 (HTLV-1) in HIV patients in Kenya"⁵. They claim that this study showed a huge 71% of AIDS patients were HIV-negative; such a bold claim better have a wealth of evidence supporting it. The first thing one might notice when reading it, however, is that it isn't a peer reviewed research study; it's just a letter to the editor. Nevertheless, the authors report on some original research they were doing: they wanted to check to see the prevalence of HTLV-1 infections in HIV infected individuals. They did this by collecting 913 samples from suspected AIDS cases in Kenya. Again – remember that these are not confirmed cases of AIDS. Using these samples, they tested for HIV-1 using both ELISA and western blots. They reported that 265 (or 29%) of these tested positive for HIV-1 on both tests. This is where the denialists get their statistic. However, this number represents the samples that were positive on both the ELISA and the western blot – it excludes those samples that tested positive on one or the other tests. They also tested for HIV-1 alone; they did not test for HIV-2, and this undoubtedly reduced the number of positive results. And again, the researchers did not use more sensitive PCR techniques, so it is entirely plausible that many positive cases were missed.

What all three papers have in common is that the estimates of HIV-positive individuals are underestimated. All three papers were written before sensitive PCR-based methods for detecting HIV were developed and put into common use, and before doctors were able to accurately diagnose AIDS and differentiate it from other immunodeficiency syndromes. And what deniers need to realize that it is impossible to obtain a 100% detection rate. Multiple factors exist that prevent the presence of HIV from being detected 100% of the time. If the AIDS "skeptics" really wish to show that HIV does not cause AIDS, then perhaps they should find a study to cite that isn't two decades old.

So as usual, critical examination of denialist claims shows that they don't have a leg to stand on. Better luck next time, guys. Come back when you have recent research that you can cite without twisting the statistics into misleading conclusions.

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1. See http://www.healtoronto.com/nih/ for an example.
   
2. These three statistics were also stated by the Youtube user who prompted this series on debunking AIDS denial nonsense.
   
3. Brindle et al. Quantative bacillary response to treatment in HIV-associated pulmonary tuberculosis. 1993. American Review of Respiratory Disease
   
4. Hishida et al. Clinically diagnosed AIDS cases without evident association with HIV-1 and 2 infections in Ghana. 1992. The Lancet
   
5. Songok et al. Low Prevalence of Human T-Lymphotrophic Virus Type 1 (HTLV-1) in HIV patients in Kenya. 1994. Journal of Acquired Immune Deficiency Syndromes

AIDS Denialism: Deadly Ignorance Part II: Koch’s Postulates, AIDS and more Wackaloonery

(Click for Part I and Part III of this series)
In my previous post, I wrote about some of the faces of AIDS denialism, and the arguments (and I use that word loosely) that these "skeptics" present. What their arguments revolve around - and indeed the arguments put forth by virtually all members of the AIDS denialism community - is that there is no evidence that AIDS is caused by HIV. Certainly, by all accounts, the medical and scientific communities have demonstrated otherwise. But how do we know that HIV is the causal agent behind AIDS? How do we know what the causal agent is behind any disease? Do we have a standard method of determining causality, and is the HIV/AIDS connection supported by this method? The answer is yes.

In 1884, a Prussian physician by the name of Robert Koch, along with is colleague, the bacteriologist Friedrich Loeffler formulated a set of criteria that they used to establish a causal relationship between an illness and a causative agent. These criteria were published in 1890 by Koch and have since been known as Koch's Postulates. Koch used this set of postulates to determine the etiology of both tuberculosis and anthrax¹, and in the 120 years since their first publication, the causes of many other diseases have been determined by successful application of Koch's Postulates. The four tenants of Koch's Postulates are as follows:

1. The causative agent must be found in all organisms exhibiting the disease, and not in healthy individuals.
   
2. The causative agent must be isolated from a diseased individual and grown in culture.
   
3. The causative agent, when introduced to a healthy individual, should cause the original disease.
   
4. The causative agent must be reisolated from the inoculated, diseased host and shown to be identical to the original agent.
   

Note that the first postulate is not universally required, as many diseases exhibit asymptomatic carriers (as is the case for HIV). The third postulate is unique in that it states "should" rather than "must" – this is because infection rates are never 100%. The fourth postulate was originally required by Koch, but fulfilling the first three postulates is generally considered to be sufficient to indicate causation. If all four postulates are fulfilled by a particular causative agent, then it can be said that the agent is the likely cause of the disease.


So, when these four postulates are applied to HIV/AIDS, does the result support the claim that HIV causes AIDS? The answer is a resounding "yes". Let's take the postulates one at a time.


The causative agent must be found in all organisms exhibiting the disease, and not in healthy individuals.

As noted above, this first postulate is not universally required, as asymptomatic carriers of HIV appear as healthy individuals. Nevertheless, those with HIV-1 and no symptoms at time of diagnosis, eventually progress to AIDS. And in 95% of cases, individuals suspected to have AIDS test positive for HIV virions, HIV antibodies or positive for HIV viral loads². This association between HIV and AIDS is supported by a wealth of evidence from countless studies, and cannot be dismissed.

The AIDS denialists, however, will insist that this first postulate has not been fulfilled. They will point out that there are cases of individuals exhibiting AIDS-defining characteristics who have not tested positive for HIV antibodies. This, they are quick to claim, is in violation of the first postulate, since these cases show the disease without the putative causative agent. There is a very straightforward answer to this, though: immune deficiencies can develop in more ways than just being induced by HIV infection. There is a virtual plethora of ways immune deficiencies can arise, and these will exhibit symptoms similar to AIDS – bacteria, genetic conditions, environmental factors, and immune suppressing drugs can all cause symptoms associated with AIDS. In such cases, it is not surprising that patients test HIV negative! But in cases where individuals present all or most of the AIDS-defining characteristics – particularly a depletion of CD4+ lymphocytes – the HIV virus is found³. Koch's first postulate remains fulfilled.


The causative agent must be isolated from a diseased individual and grown in culture.
Culturing the proposed pathogen is not only the second postulate but also, perhaps, the most important step in identifying the suspected pathogen. Viruses can be more difficult to grow in culture than bacterial strains, but this has not prevented HIV from being cultured from samples taken from AIDS patients. Over the years, it has been cultured in T-cells, macrophages as well as in HeLa cells. Furthermore, isolated and cultured strains of HIV have had their genomes sequenced and cloned, with literally thousands of unique sequences added to sequence data bases. Isolation of HIV from samples is not an issue, and there have been multiple^4,5 protocols established and published on how to accomplish this. There are even commercially available kits which allow HIV to be isolated from infected samples. Culturing HIV has become a routine practice in HIV research to the point where it is more or less a triviality. Postulate 2 has been fulfilled and continues to be filled on a daily basis.

The causative agent, when introduced to a healthy individual, should cause the original disease.
The third postulate is perhaps the most controversial of the postulates. For one, it cannot be fulfilled solely on the basis of epidemiological data but must rather rely on experimental evidence. When looking at diseases in animals or when using animal models, the third postulate is easy to demonstrate experimentally. When it comes to the transmission of diseases in human subjects, however, the third postulate is tougher to demonstrate. No ethics board would ever allow researchers to experimentally infect healthy people with cultured HIV to see if they progress to AIDS. Nevertheless, this postulate has been fulfilled due to the unfortunate accidental transmission to humans who were doing research on HIV⁶.

In this case, three lab workers were working with HIV-1, and all became accidentally infected with the virus. None of these workers belonged to any of the high-risk groups; they were not drug users, had not had blood transfusions, and did not engage in homosexual behaviour. All three of the workers exhibited depleted CD4+ cell counts (two of which were low enough to warrant an AIDS diagnosis) and one progressing to pneumonia⁷. AIDS "skeptics" who insist that HIV does not cause AIDS and that AIDS is caused by drug use are at a loss when it comes to explaining this case study. How else could the infected workers, who were not part of any risk groups, have developed AIDS unless it was due to HIV infection? How do they explain the onset of symptoms in these workers if HIV is, as they claim, pathologically asymptomatic?

Evidence from animal studies has also given support to the third postulate. Studies in which baboons have been infected with HIV-2 have shown these animals to develop AIDS-like symptoms including depletion of CD4+ lymphocytes⁸ (and I highly doubt these baboons were drug users!). Mouse strains used to model SCID which have been given human peripheral blood lymphocytes (hPBLs)⁹ also develop severe immunodeficiencies when infected with HIV¹⁰. These mice also show depleted CD4+ cell counts.

Studies like this show experimentally that cultured HIV, introduced into healthy individuals, result in the hosts progressing to AIDS. This is sufficient to fulfill the third postulate.

The causative agent must be reisolated from the inoculated, diseased host and shown to be identical to the original agent.
This fourth postulate, as noted above, is now often considered extraneous to suggesting causation by a suspected pathogen. Nevertheless, the example of the infected lab workers cited above is a good case of this postulate also being demonstrated. The virus was reisolated from the infected lab workers, and sequenced¹¹. The divergence between the sequences from the strains isolated from the workers was very small – about the same as the divergence in sequence between HIV-infected infants and their mothers. In other words, it was the same strain that they had been working with. Consider Postulate 4 demonstrated.
 
So it is plainly obvious that all of Koch's postulates are fulfilled, and HIV can be said to be the likely causative agent behind AIDS. Koch's postulates demonstrate reasonable criteria for determining causation, and provide reasonable evidence that the AIDS "skeptics" are wrong.

I will continue in Part III, where I examine the fuzzy math used by AIDS "skeptics" to distort reality and push their dangerous nonsense.

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1. The infectious agents behind these diseases, Mycobacterium tuberculosis and Bacillus anthracis, respectively, were discovered and first isolated by Koch himself.
   
2. See the references in Part I for more on this.
   
3. There is an exception to this. A rare condition exists called hypogammaglobulinemia where individuals are unable to mount an immune response to invading pathogens. People with this disorder who are infected with HIV will not show HIV antibodies present when tested.
   
4. See http://www.miltenyibiotec.com/download/protocols_macsmolecular_en/516/SP_Iso_HIV1.pdf
   
5. See http://www.currentprotocols.com/protocol/im1202
   
6. Weiss SH, Goedert J J, Gartner S, Popovic M, Waters D, Markham P, Veronese FD, Gall MH, Barkley WE, Gibbons Jet et al: Risk of human immunodeflciency virus (HIV-1) infection among laboratory workers. Science 1988, 239:68-71.
   
7. I have been unable to find any information on these three workers more recent than the above referenced article. If anyone has updated information, I would be interested in hearing it!
   
8. Barnett SW, Murthy KK, Herndier BG, Levy JA: An AIDS-like condition induced in baboons by HIV-2. Science 1994,266:642-646.
   
9. SCID-model mice are unable to produce B and T lymphocytes. Transplanting hPBLs into these mice reconstitutes a functioning immune system.
   
10. Mosier DE, Bulizia R J, Baird SM, Wilson DB, Specter DH, Spector SA: Human immunodeficiency virus infection of human PBLSCID mice. Science 1991,251:791-794.
    
11. Reitz MS Jr, Hall L, Robert-Gurofl M, Lautenberger .I, Hahn BH,Shaw GM, Kong LI, Weiss SH, Waters D, Gallo RC,Blattner W: Viral variability and serum antibody response in a laboratory worker infected with HIV-1 (HTLV-IIIB). AIDS Res Hum Retroviruses 1994, 10:1143-1155.

AIDS Denialism: Deadly Ignorance Part I

(Click for Part II and Part III of this series)
Those of us in the skeptical community are no strangers to whacko medical theories. It seems every week there is another quack promoting a new "naturalistic" diet or treatment. By far, though, the brunt of a skeptic's time is dealing with denialism – groups of individuals denying that contemporary medical practices don't work or are even dangerous. In the last few years, the anti-vax denialists have been in the media spotlight due to their elaborate campaigns and the outspoken celebrities like Jenny McCarthy who have taken up the anti-vax fight. But there is another camp of deniers festering away in the underbelly of alternative medicine. These people are the AIDS deniers; people who claim that AIDS is not caused by the HIV virus, but rather that it is actually caused by drug use. Some even go so far as to claim that HIV does not exist! Given the horrifying prevalence of AIDS in third world nations and even its alarming proliferation in developed nations, increasing support for AIDS denialism is not simply worrying but outright dangerous.

AIDS denialism has been known to me for some time, having learned about it from reading about Nobel Prize winner Kary Mullis, who is one of AIDS denialism's best known proponents. Though, given Mullis' colorful personality I figured he was just a lone crank. It was not until this past week when a good friend of mine, SofiaRune, began to get comments on a Youtube video she produced about debunking the link between HIV infection rates and male circumcision. A user by the name of "mykoolaidtastesfunny" made a variety of claims about how AIDS and HIV are not linked, and how HIV has not been shown to exist. He also posted a video by a variety of AIDS "skeptics" repeating his points. Immediately we began to investigate these claims. In the following series of posts, I'll address the claims of the AIDS "skeptics". In Part I, I will focus on the video that started it all…

Mullis and More: A Menagerie of Morons

The video that was posted is as follows:



Note that I will only critique the claims made by the scientists in this video. It's not worth my time to debunk the two journalists in the video since they are not the ones making any scientific claims (especially the first one, Neville Hodgkinson, who's entire argument boils down to "I spent a week in the lab of an unnamed German scientist and he showed my unexplained data which I now believe".)



Kary Mullis: Mullis is a man known to virtually everyone in biology. His claim to fame is winning the Nobel Prize in Medicine and Physiology for the development of PCR. However, as a perfect example that even eminent scientists can hold completely insane beliefs, Mullis also believes in astrology, denies anthropogenic global warming, and believes he has conversed with an alien in the form of a glowing raccoon. He is also completely convinced that he will die in the presence of redwood trees, and is thus completely reckless when redwoods are not around, going so far as to ski down the centre of a highway far from the sight of a redwood. In the video, Mullis recalls the tale of applying to a grant from the NIH and needing to find a source for the claim that AIDS is caused by HIV. He claims that he did a search through the literature but look as he may, he could not find one. Furthermore, he claims to have asked Luc Montagier (one of the co-discoverers of HIV, along with Robert Gallo) and Montagier was unable to provide him with any sources. Shocked that there was no source for the HIV/AIDS link, Mullis became a skeptic and has denied a relationship between the two ever since. Whether or not this story is true, I have no way of telling. But it is possible to do a search of the literature to see if there are any papers showing the link between HIV and AIDS. And does Mullis' claim stand up to scrutiny?

Hell no.

A very preliminary search on PubMed allowed us to find 6 different papers¹ which show a link between infection with HIV and AIDS, more than enough necessary to support the claim that AIDS is caused by HIV. All of these papers were published between the years of 1985 and 1993, so many – if not all of them – were available to Mullis at the time he was writing his grant application². Either Mullis is incapable of doing a cursory search of scientific literature, or he isn't being entirely honest with his story. I'm inclined to believe the latter.

But I'm feeling generous. Let's give him the benefit of the doubt; maybe some of these papers had yet to be published, and perhaps he didn't have access to the journals the other papers had been published in. Perhaps Mullis could be excused for being skeptical at the time. But what about now? Those papers, and many others, are now readily available. Arguing that there was no evidence available two decades ago doesn't mean much today. Saying "Oh HIV doesn't cause AIDS because I did a search of the literature twenty years ago and found no proof" is akin to arguing that evolutionary theory is flawed because Darwin's original work from 150 years ago wasn't entirely accurate. Such an argument completely ignores scientific findings and advances made since then. This argument fails because there ARE papers that show AIDS is caused by HIV, those papers have been published as far back as 1983, and such papers continue to be published. Someone hand the deniers a copy of "Searching PubMed for Dummies".



Rodney Richards: Richards is a biochemist who founded the biotech company AMGen (though, I am assuming this is true – the only references to him I could find on Google all come from AIDS denial websites…). Richards begins talking about Peter Duesberg, the granddaddy of the AIDS denial movement (more on him later). He recalls his time at AMGen when Duesberg was invited to give a seminar. According to him, Duesberg's talk was boycotted by the other scientists working at AMGen. He notes that he thought this was odd and it prompted him to "go to the library". What did he find in his research? He doesn't say. Richards actually does not make an argument at all. He just says "that was 12 years ago and I've been studying this issue ever since". What Richards is doing is a subtle argument from authority; he is basically saying "I'm a scientist who has studied AIDS for 12 years, and I don't believe in it, therefore you can be confidant that HIV does not cause AIDS". He doesn't need to present an argument because his sycophants will simply hide behind his status as a scientist with a PhD.

He also implies that the truth about AIDS is being covered up by some scientific conspiracy, a notion that is common with alt-med movements. Such an argument, however, is laughable, considering that in the past, the editor-in-chief of Science has explicitly stated his support for Duesberg's right to air his views and to do his research, going so far as to voice his support for Duesberg's grant applications³ (he was not, of course, agreeing with Duesberg's views). It is not surprising, though, that scientists don't want to listen to Duesberg's claims. For scientists, the issue is settled – AIDS is caused by HIV. There is ample evidence built up over the last 30 years that shows this to be the case. It is the same reason why evolutionary biologists don't invite creationists to speak at evolutionary biology conferences; their claims are not supported by the scientific evidence and are a waste of time to deal with. This should not be confused with some sort of conspiracy to silence the anti-AIDS crowd. They have every right to express their views – and maybe when they have some real evidence, we'll listen.



Christian Fiala: As I watched Fiala's portion of the video I quickly began to ask myself "How on Earth did this man ever get a PhD?" Fiala's argument is so incredibly wrong that it is hard to believe that his segment isn't satire. In case you didn't catch his argument due to a massive brain haemorrhage from stupid overload, it goes like this: HIV spread to the heterosexual population in the 80s from the homosexual population. This is unlikely, he says, because the virus' method of transmission is predetermined and does not change easily. How did the virus know, he muses, that the risk group was almost all affected and it had to "break out" into a new population? Fiala claims that transmission from the homosexual population to the heterosexual population would require the virus to gain some sort of sentience and realize it needs to move out. This is impossible so, he claims, the appearance of AIDS in heterosexuals could not have been caused by HIV.

Are you done laughing?

The virus, of course, does not need to have some kind of sentient knowledge of its situation to transfer from one population to the other. In fact, we know EXACTLY how the virus moved into the heterosexual population. Blood samples used for blood transfusion are now closely screened for HIV, but back in the 80s, before we really knew much about the virus, it wasn't. Many members of a subset of the homosexual community were frequent blood donors, and their samples would have all been tainted. For people with haemophilia, blood transfusions can be an important life saving procedure, and in many instances, blood from HIV positive samples was used. It wasn't long until AIDS began to show up in haemophiliacs, and then in the general population. At no point did the virus knowingly decide to move into another population, and at no point did the mode of transmission change. The movement of the virus through populations is well known and well documented, so Fiala's argument falls flat on its face.

Peter Duesberg, Kingpin of Denial: Rodney Richards, in his video appearance, mentions a man by the name of Peter Duesberg. Anyone who looks into the AIDS denialist issue is bound to come across references to him; his name has practically become synonymous with the AIDS "skeptic" movement. In 1987, Duesberg published a paper in the journal Cancer Research⁴ where he claimed that HIV is simply a harmless passenger virus⁵. At that time, there was much not known about how the HIV virus causes AIDS, and Duesberg could perhaps be excused for being skeptical at the time. Nevertheless, the uncertainty in what mechanism the HIV virus utilized was dwarfed by the voluminous epidemiological data that suggested HIV as the cause. In the years following, the precise molecular mechanism of HIV's virulence has been elucidated, and there really is no longer any cause for skepticism. Duesberg nonetheless clings to his beliefs.

So what does Duesberg attribute AIDS to, if not to HIV? He and his followers believe that AIDS is actually caused by drug use – particularly intravenous drugs and nitrite inhalants – as well as malnutrition. There are many reasons why this cannot be the case. If AIDS is caused by drug use, then why is it that all drug users do not contract AIDS? And of the subset of drug users that do get AIDS, why do they all test positive for HIV? Why do we only find HIV in people who have AIDS or eventually progress to AIDS, if it is just a harmless virus with no real pathology? Why does AIDS pass from mother to child if it is not due to a transmissible pathogen? Surely newborn infants are not heavy drug users. These questions need to be addressed by Duesberg if he wishes his ideas to have any kind of validity, and to date, neither Duesberg nor his followers have been able to adequately provide a response.

Why does Duesberg continue to be a boil on the face of AIDS research if his ideas are patently ridiculous? Perhaps it is due to the false sense that his ideas are taken seriously by other members of the scientific community. Many AIDS "skeptics" will frequently cite numbers of scientists that supposedly support Duesberg. But these are often lists of scientists that do NOT believe Duesberg is correct on the AIDS/HIV issue; they merely support his right to investigate the issue and to voice his opinions. The credibility of his scientific views is falsely inflated this way.



These are the prominent faces in the AIDS denialist world. Continuing on into Part II, I'll look at Koch's Postulates, how they relate to AIDS, and how they confirm that HIV really is the cause.

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1. Those papers are as follows:
   -Fauci A.S. "Multifactorial nature of human immunodeficiency virus disease: implications for therapy". Science 1993.262:1011-1017.
   
   -Weiss R.A. "How does HIV cause AIDS?" Science 1993.260:1273-1278.
   
   -Gallo R.C., Satin P.S., Gelmann E.P., Roberto Gumff M. "Isolation human T-cell leukemia virus in acquired immune deficiency syndrome (AIDS)". Science 1983. 220:865-867.
   
   -Gallo R.C. et al "Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk from AIDS". Science 1984. 224:500-503.
   
   - Schechter M.T. et al "HIV-1 and the aetiology of AIDS". British Columbia Centre for Excellence in HIV AIDS, Vancouver, Canada, Lancet 1993. 341:658-659.
   
   - Weiss R.A., Jaffe H.A. "Duesberg, HIV and AIDS". Nature 1990. 345:659-660.
   
   
   
2. Actually, with a little math, we can see that all of these papers were available to Mullis, at least at the time his clip was filmed. As you will see in the clip with Rodney Richards, Richards claims that he has been researching AIDS and HIV for 12 years. Robert Gallo's papers which linked HIV with AIDS was published in 1983. If Richards began his research that very same year, then his clip was filmed 12 years later, in 1995 (that is, of course, the earliest it could have been filmed – it may have been filmed at an even later point). It is not unreasonable to think that Mullis' part was filmed around the same time. So, at the very earliest, Mullis' clip was filmed in 1995, after all of the papers had been published. He has no excuse for making the claims he does.
   
   
3. Cohen J. "The Duesberg phenomenon". Science. 1994 (Issue 266, 1642-1644)
   
   
4. Deusberg P. "Retroviruses as carcinogens and pathogens: Expectations and reality". Cancer Research Issue 47 (1987)
   
   
5. Gallo has actually challenged Duesberg to infect himself with HIV, if he really believes so strongly that it is harmless. Duesberg has, of course, backed down from this challenge, saying he could never get the proper approval of an ethics board to do it.

HIV and Aids Denialism: Intense Dumbosity

So I'm working on a new post about HIV and AIDS denialism (yes, AIDS denialists are real!). It's taking me a while to do it though, but I will have it up eventually.Really.

EDIT: It's finished. I split it into three parts to keep it from being too long to read all at once.

Part I: Deadly Ignorance
Part II: Koch's Postulates, AIDS and More Wackaloonery
Part III: Fuzzy Math and Distorted Reality

Arsenic Life - ZOMG! The straight dope on extremophiles and arsenic.

By now you may have heard today's big announcement in Science¹ of the discovery of extremophile bacteria that use arsenic in their DNA rather than phosphorous. This discovery has been played up by the media (as usual) as being a major discovery that will cause scientists to "rewrite the story of life" or other such hyperbole-based headlines. And while definitely interesting, this discovery, unfortunately, is just not a major finding.

What the discovery is:

The team of researchers were concerned with a lake in California called Mono Lake. This lake itself is particularly interesting: it is highly alkaline, with a pH at around 10, so it is very basic (remember that water has a neutral pH of 7, and that the pH scale is logarithmic, so the lake is 1000 times more alkaline than a regular freshwater lake). The lake also contains quite high levels of arsenic, an element that is highly poisonous to life. The scientists, aware that extremophile bacteria just love to live in what we would normally consider absolutely hostile conditions, were curious about what kind of microbial life lived in the lake.

What they found were microbes that not only lived happily in the highly alkaline, arsenic-laden conditions, but actually utilized arsenic in place of phosphorous in their biochemistry: their DNA contained arsenic where it normally would have a phosphate backbone and even some of their amino acids contained arsenic instead of phosphorous. They did this by growing the bacteria in media containing radiolabelled arsenic, and after allowing the bacteria time to utilize the arsenic as they pleased, found that the radiolabelled atoms ended up in the DNA, amino acids and other normally phosphorus-containing molecules.

What this DOESN'T mean:

This does not mean we have discovered "alien" life, like some media outlets have been saying. Such life easily fits in with what we know about extremophile bacteria - they are resilient enough to find a way to use toxic environments to their advantage.

It also doesn't mean that we need to re-examine the way we think early life evolved. It is unlikely this discovery says anything about evolutionary history (though it might say volumes about how evolution can be incredibly innovative!).

What it DOES mean:

It does mean that evolution can work in wondrous ways that we hadn't even imagined. Make no mistake, however - this finding surely fits within current evolutionary theory.

It also means, on a more hypothetical level, that astrobiologists may need to broaden their definitions of "life" when searching for the signs of life outside of our planetary system.

Why this discovery isn't as big of a deal as it first seems:

Although at first the announcement of life that uses arsenic as a building block is exciting, a little critical thinking shows that there is reason for skepticism. The researchers studied the microbes by slowly increasing the level of arsenic in the media they were grown in. After multiple rounds of this, they were left with microbes that were found to have arsenic in their DNA and amino acids. However, this method leaves open the possibility that they were simply selecting for microbes that could use arsenic under high arsenic/low phosphorous levels. In other words, this experiment does not show that using arsenic is the natural way these microbes live. It is entirely possible that they use phosphorous for their important biochemical molecules like ever other microbe, and under stressful conditions, switch to different mode where they use arsenic instead. It's even possible that after successive rounds of increasing arsenic content, the team artificially selected for microbes that had this ability: in effect, they may have forced the microbes to evolve a new pathway that does not exist in nature.

This finding doesn't say alot about evolutionary history either: Lake Mono is a relativley recent geological feature, so it's likely the microbes adapted to such an environment relatively recently also.

Furthermore, the team did not attempt to determine HOW the bacteria incorporate arsenic into their DNA and amino acids: what biochemical pathways are involved? Do they use NTAs instead of NTPs? How stable would such molecules be - phosphate plays an important role in stabilizing the structure of DNA, so would the use of arsenic affect this? Are such molecules recognised by the microbes' DNA polymerase? Or by any important enzymes, for that matter? Arsenic is a larger atom than phosphorous - will this alter the shape and size of the major and minor grooves and if so, how does this affect the binding of proteins? Is arsenic ALWAYS used by these microbes, even in low arsenic/high phosphorous levels? These questions are just begging to be answered, and until they are, I think the vast majority of molecular biologists and biochemists will remain skeptical.

An interesting find, for sure - the very fact that organisms can use arsenic to live is exciting - but a "new form of life" or even "arsenic-based life"? Well, there's really no evidence to support such claims at the moment.

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PZ over at Pharyngula has a great rundown of why calling this "arsenic based life" is silly, echoing many of the points I made above.

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1. Wolfe-Simon F, Blum JS, Kulp TR, Gordon GW, Hoeft SE, Pett-Ridge J, Stolz JF, Webb SM, Weber PK, Davies PCW, Anbar AD, Oremland RS (2010) A Bacterium That Can Grow by Using Arsenic Instead of Phosphorus. Science DOI: 10.1126/science.1197258.

Next-Generation DNA sequencing: The Future is Now! Part 1: Pyrosequencing

Are you tired of sequencing your gene of interest 800bp at a time? Sick of straining your eyes staring at a fuzzy chromatogram? Fed up with waiting hours for your PCR to finish only to realize you forgot to add ddNTPs to your reaction mix? Well, nex-gen DNA sequencing is for YOU!

Cheesy sales-schtick aside, next-generation DNA sequencing technologies are on the rise (and are poised to soon become current-gen technologies - some already are!). The days of loading your PCR'd sequencing mix onto an automated capillary sequencer may soon be numbered. So, to make the change of power of our mighty sequencing overlords a little easier, this series of posts will be dedicated to how the upcoming technologies work, their advantages over conventional sequencing technologies, and their problems.

Today's post: Pyrosequencing

Pyrosequencing is perhaps the one Next-Gen sequencing technology that is the most like current generation automated sequencing (if you need a reminder on how that works, I've written about it in the past). It still requires primer design, and rounds of PCR, but the method of detection of incorporated nucleotides differs.

Pyrosequencing starts by adding your PCR'd sequence to a reaction mix that contains DNA polymerase, and three important enzymes: DNA sulfurylase, apyrase and luciferase. DNA sylfurylase is an enzyme that converts a pyrophosphate molecule into ATP. Luciferase is an enzyme which uses ATP to convert luciferin into oxyluciferin, resulting in the emission of light. Apyrase's job is to degrade unincorporated nucleotides. Given that the addition of a nucleotide into a growing DNA strand results in the release of a pyrophosphate molecule, keen readers might already see how pyrosequencing works.

With conventional automated chain-termination sequencing techniques, we add all of our dNTPs to the reaction mix at once; they can easily be distinguished because each base has a different fluorescently labelled dye bound to it. With pyrosequencing, however, one cannot toss in all the nucleotides at the same time. Rather, we have to add each one sequentially for each nucleotide in the sequence. That is, we first do a reaction with A, then with T, then with C and lastly with G. We then repeat this cycle over and over until the sequencing reaction is complete (if this sounds confusing, hopefully the diagram and video below will clarify it). Why we do this will be apparent in a moment.

When a nucleotide is incorporated into the DNA strand, a pyrophosphate molecule is liberated¹. This pyrophosphate molecule can then be converted by the DNA sulfurylase into a molecule of ATP. Luciferase picks up this ATP² molecule and uses it to catalyze the conversion of luciferin to oxyluciferin. This chemical conversion results in the emission of a photon. This chain of events, then, means when a nucleotide is added by DNA polymerase to the growing DNA chain, we get the emission of light. A computer with a suitable detector could detect this light, and we would have an indication of when a nucleotide was added in our sequencing reaction.

But if we add all of our dNTPs at once, then how do we know which ones are being ? This is why we only add one nucleotide to the mix at a time. First the reaction is run using dATP. We then add apyrase to remove any remaining nucleotides and repeat with dCTP, and so on. If we add the nucleotides one at a time, then they will be incorporated (or not incorporated) into the sequence one at a time, and consequently we get one light signal at a time.

The light signals are recorded on a chart called a pyrogram. This chart records not only which nucleotides resulted in the emission of light but also the intensity of that signal. If three dTTP molecules were incorporated at once, then there would be three photons emitted and three times as much light; this would result in a triple peak on the pyrogram. From the pyrogram, then, one could easily read off the exact DNA sequence. The figure to the left shows one such pyrogram. The sequence in this example would be GCAGGCCT.




The following video puts the whole process together nicely.



So why would one choose pyrosequencing over automated chain-termination methods? Well, for one, it's cheaper (though, not as cheap as some of the other upcoming next-generation sequencing methods). Practically, it's easier to do, since it doesn't require running through gels or capillaries. Analyzing the resulting pyrogram is also easier than analyzing a chromatogram. Chromatograms can often be spotted with "N"s when the computer cannot tell if the wavelength of light from a dye is one color or the other; however, with pyrosequencing, detection is binary - either a photon is emitted or not - so results are more accurate and clearer.

Though, pyrosequencing does have it's drawbacks. It requires a greater number of PCR cycles than traditional sequencing does, so it may take longer to complete, especially for longer sequences. Currently, a typical read of sequence data from pyrosequencing is about 300 to 500bp - shorter than the typical 800 to 1200bp you get from chain-termination methods. This, however, is likely to improve as the technology advances and becomes more refined. The shorter reads, though, make it tough to sequence genomic regions containing high amounts of repetitive DNA.

So that is pyrosequencing in a nutshell. Next time: Helicos sequencing!

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1. NTPs are triphosphates (that's what the TP stands for!), meaning each nucleoside (base+sugar) is attached to a phosphate molecule. To add a nucleotide to a growing DNA strand, the reaction requires an input of energy. This energy comes from the breaking of the triphosphate chain in each nucleotide; two phosphates are broken off and released as a pyrophosphate (PP_i) molecule, and the remaining portion of the nucleotide is attached to the hydroxyl group on the 3' carbon of the previous nucleotide in the sequence.

2. Observant readers might be confused here. Since luciferase requires ATP to convert luciferin to oxyluciferin, won't this cause a problem when we add dATP to the sequencing reaction? Won't there be competition between DNA polymerase and luciferase? Well, if you thought that, then you'd be right! For this reason, we use dATPαS instead of dATP for pyrosequencing. This molecule has a sulfur atom attached to the α phosphate of the nucleotide, and cannot be used as a substrate by luciferase. Problem solved!

Dawkins' Answers Some Questions

His answers are interesting enough, but the best part starts at 11:42, when he reads out some of his hate mail.

"Science knows it doesn't know everything. Otherwise, it'd stop."

Dara O'Briain is great. Gotta love his takedown of homeopaths, nutritions and other snake oil salesmen.