Category Archives: Anecdotes

Genetically Modified Emerging Viruses: Debate over Gain-of-Function Research

Scientists generally loathe restrictions on their freedom to conduct research. Nonetheless, many virologists recognize the need to regulate studies that involve live, potentially pandemic, deadly pathogens, especially when those investigations involve modifying and even creating such pathogens.

We encountered this issue in an earlier posting (1), which told how in 2012 Yoshihiro Kawaoka and Ron Fouchier independently created variants of the H5N1 avian influenza virus that could be transmitted between ferrets (2, 3). They did so by using site-specific mutagenesis to modify the HA protein of the avian virus. Ferrets were used because they are a good model for influenza transmission between humans.

Important to our story, avian influenza viruses are spread in bird populations by the fecal-oral route. And, while H5N1 avian influenza viruses can be extremely pathogenic in humans, they have not yet naturally acquired the ability to be transmissible through the air—an ability that is necessary for influenza viruses to be pandemic in humans. So, to ascertain whether H5N1 avian influenza viruses could adapt to become transmissible via aerosols under natural conditions, both Kawaoka and Fouchier passaged their genetically modified H5N1 avian viruses in ferrets. The genetically modified H5N1 viruses indeed acquired additional mutations during passage in ferrets, which enabled them to become transmissible via the respiratory route. Moreover, each research group reported that the airborne-adapted mutant H5N1 viruses caused lung pathology in the recipient ferrets, none of which died.

The NIH supports studies like the above because of their potential to shed light on the interactions between emerging deadly pathogens and humans, and because they might help to clarify just how threatening these emerging viruses actually are. But regardless of those considerations, the White House Office of Science and Technology Policy (OSTP) and the Department of Health and Human Services were concerned by the potential risks of the avian flu experiments. Consequently, in October 2014 those governmental agencies initiated an assessment of gain-of-function research—that is, studies in which pathogens are manipulated to alter their virulence and transmissibility. Biosafety and biosecurity were the government’s key concerns. Possible risks included the prospects of a potentially pandemic modified virus either escaping from the laboratory, or being stolen from the laboratory and being misused to threaten public health and national security.

With those concerns in mind, the government imposed a temporary suspension of funding of new gain-of-function projects. The OSTP announced that effected studies would include those which “may be reasonably anticipated to confer attributes to influenza, MERS, or SARS viruses such that the virus would have enhanced pathogenicity and/or transmissibility in mammals via the respiratory route.” In addition, the government requested that researchers already carrying out gain-of-function projects should “voluntarily” postpone their studies until the risks might be evaluated by the National Science Advisory Board for Biosecurity (NSABB) and the National Research Council (NRC) of the National Academies.

As expected, these government-imposed restrictions caused quite a bit of controversy in the research community. Some scientists expressed concern that a ban on gain-of-function experiments might be applied too broadly, to include less dangerous types of work, such as development of seasonal influenza vaccines. [In that instance, gain-of-function research might be useful to evaluate the transmissibility of particular influenza strains, and to asses how those strains might mutate to evade candidate vaccines.] The government responded to this concern by modifying its review protocols in order to take public health considerations into account. Yet some researchers still feared that valuable research time could be lost while waiting for an exemption.

But what of experiments such as those of Fouchier and Kawaoka, which do entail a clear and present risk to public safety? The key question in those instances is whether the knowledge gained from the experiments might afford a benefit that is significant enough to justify the danger. Unfortunately, the answer is not always clear, as thoughtful individuals on each side of the debate make valid arguments. And, even if there were a consensus on the merit of a project, the delay in funding imposed by the review process might again cause valuable research time to be lost, or perhaps even destroy the research program, or cause outstanding young scientists to turn to other areas of inquiry, or even end their research careers.

The debate over government imposed restrictions on gain-of-function research waned somewhat after they were first announced. However, it was reignited last month by the announcement by Ralph Baric and co-workers at the University of North Carolina that they had created a chimeric SARS-like virus, which expresses the spike (attachment protein) of a bat coronavirus in a mouse-adapted SARS-CoV backbone (4). As in the cases of the genetically modified H5N1 avian influenza viruses, the newly generated SARS-like virus is potentially an extremely dangerous, possibly pandemic pathogen.

Coronaviruses, showing their characteristic spikes, which give them their characteristic “crown-like” (coronal) appearance
Coronaviruses, showing their characteristic spikes, which give them their characteristic “crown-like” (coronal) appearance

Baric’s team generated the chimeric SARS-like virus using the SARS-CoV reverse genetics system. The justification for the project was to evaluate the risk of SARS coronaviruses emerging from coronaviruses currently circulating in bats. Apropos that, the origin of the SARS coronavirus is not known for certain. However, the genetic diversity of coronaviruses in bats, in which they are avirulent, is consistent with the possibility that bats are a reservoir for SARS-coronaviruses.

The North Carolina group reported that their hybrid SARS-like virus could indeed bind to, and replicate efficiently in human airway cells in vitro. In fact, the chimeric virus replicated as well as epidemic strains of SARS-CoV in the human cells. Moreover, the chimeric virus replicated in, and caused severe pathogenesis in mouse lung in vivo.

Baric and co-workers began their project before the government announced the moratorium. Yet the work was allowed to continue because it was judged not risky enough to be bound by the restrictions; a decision that has since provoked quite a bit of controversy. Moreover, the North Carolina researchers themselves acknowledged the risk of their studies, noting, “Scientific review panels may deem similar studies building chimeric viruses based on circulating strains too risky to pursue…(4)”

Still, the key question is whether Baric’s experimental findings are important enough to justify their risk. At least some in the science community contend that they do not meet that test. In any case, Baric intends to study his new SARS-like virus in non-human primates, for the purpose of better understanding the potential threat of bat coronaviruses to humans.

References:

1. Opening Pandora’s Box: Resurrecting the 1918 Influenza Pandemic Virus and Transmissible H5N1 Bird Flu, posted on the blog April 15, 2014.

2. Imai M, Watanabe T, Hatta M, Das SC, Ozawa M, Shinya K, Zhong G, Hanson A, Katsura H, Watanabe S, Li C, Kawakami E, Yamada S, Kiso M, Suzuki Y, Maher EA, Neumann G, Kawaoka Y. 2012. Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets. Nature 486:420-428.

3. Herfst S, Schrauwen EJ, Linster M, Chutinimitkul S, de Wit E, Munster VJ, Sorrell EM, Bestebroer TM, Burke DF, Smith DJ, Rimmelzwaan GF, Osterhaus AD, Fouchier RA. 2012. Airborne transmission of influenza A/H5N1 virus between ferrets. Science 336:1534-1541.

4. Menachery VD, Yount Jr BL, Debbink K, Agnihothram S, Gralinski LE, Plante JA, Graham RL, Scobey T, Ge XY, Donaldson EF, Randell SH, Lanzavecchia A, Marasco WA, Sh ZL, Baric RS. 2015. A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence. Nature Medicine doi:10.1038/nm.3985

Mao Zedong and the Resurgence of Traditional Chinese Medicine

Our last posting told of how Chinese pharmacologist Tu Youyou was awarded a share of the 2015 Nobel Prize for Physiology or Medicine for discovering an anti-malaria medicine called artemisinin (1). A key feature of Tu’s story is that her discovery happened only because she could read 1,500-year-old Chinese medical texts.

Tu’s discovery caused some individuals to comment that Western medicine needs to pay more attention to traditional Asian therapies. In contrast, others noted that while traditional medicine has provided some potentially useful leads, it also has been the source of many useless and even harmful treatments. In any case, the current version of traditional Chinese medicine circulating in the West is not nearly as ancient as many might presume. The story is as follows.

Western medicine was brought to China in the 1880s by missionaries whose activities were protected by the trade deals forced on China by the Western powers. Initially, Western medicine spread only slowly through the country. But, during the first half of the twentieth century the Chinese people were suffering from epidemics of plague, smallpox, diphtheria, malaria, tuberculosis, and other illnesses, which traditional Chinese medicine was ineffective against. Consequently, by the late 1920s the Nationalist government, headed by Chiang Kai-shek, adapted the view that important principles of Chinese medicine, such as Yin Yang theory and the five elements (see below) were not based on any empirical reality, and, in fact, were fraudulent (See Aside 1). Thus, the government began assuming a preference for Western medicine. Moreover, implementing Western medicine was seen as an important step towards modernizing China.

[Aside 1: The Nationalist Kuomintang party was founded by Sun Yat-sen in 1912, and was led by Chiang Kai-shek after 1925. It came into power in China in 1927 and, for a time, unified the country. The Chinese Communist Party, established in 1921, was the major political force opposing the Nationalists. Following a long civil war, in 1949 the Communists, led by Mao Zedong, established control over all of China.]

Interestingly, the person most responsible for the resurgence of interest in traditional Chinese medicine, both in China and in the West, was Chairman Mao Zedong. Mao promoted traditional Chinese medicine in the 1950s, ostensibly to alleviate the shortage of health care providers among his under-served populations. However, his advocacy may also have been motivated by a political agenda. He may have hoped that exporting traditional Chinese medicine might improve relations with the West. And, he may have hoped that an indigenous Chinese medicine might diminish China’s reliance on its Communist rival, Russia, for medical equipment and drugs. Moreover, Mao considered traditional Chinese medicine to be a symbol of China and, in fact, a national treasure. Apropos that, in 1954 he wrote: “Chinese medicine [itself] should be well-protected and developed. Our country’s Chinese medicine has a history of several thousand years, and it is an extremely valued asset of our homeland (2).”

Chairman Mao Zedong declaring the establishment of the People's Republic of China during a ceremony at Tian'anmen Square, October 1, 1949
Chairman Mao Zedong declaring the establishment of the People’s Republic of China, Tian’anmen Square, October 1, 1949

Mao continued, “[Traditional] Chinese medicine has made great contributions to the people of our country. With a population of six hundred-million, China is the most populace country in the world. Of course, there are many factors contributing to the ability of our country’s people to propagate and prosper day by day, but of these, the part played by health care must be one of the most important. In this respect, we must give credit first to [traditional] Chinese medicine.”

“If we compare Chinese and Western medicine, [traditional] Chinese medicine has a history of several thousand years, whereas Western medicine was introduced into China only a few decades ago. To this day, there are still more than 500 million people in the entire country who rely on [traditional] Chinese medicine for the diagnosis and treatment of their illnesses, while those who depend on Western medicine number only in several tens of millions (and most are located in big cities). Therefore, if we speak of China’s health care since the beginning of history, the contributions and accomplishments of [traditional] Chinese medicine are very great.”

Notwithstanding Mao’s enthusiasm for traditional Chinese as expressed in the above writings, he was well aware that much of what he wrote was propaganda and that the then existent traditional Chinese medicine, with its implausible theories and untested doctrines, would not be accepted by empirically-minded people abroad, or indeed at home. Thus, Mao took several steps to facilitate the acceptance of traditional Chinese medicine. Among these steps, he ordered the clinical testing of traditional Chinese herbal remedies. And, he advocated the unification of traditional Chinese medicine with Western medicine. Interestingly, he stated, “the most important thing is to first ask practitioners of Western medicine to study [traditional] Chinese medicine, and not for practitioners of [traditional] Chinese medicine to study Western medicine (2).”

In order to make traditional Chinese medicine accessible to both foreigners and native speakers, Mao ordered that the ancient texts be translated into modern Chinese. [Recall that Tu Youyou’s discovery of artemisinin happened only because of her rather unique ability to read 1,500-year-old Chinese medical texts (1).] But, that was not all. There was no canon per se of traditional Chinese medicine. Instead, Chinese medicine was a hodgepodge of inconsistent ancient texts, implausible theories (e.g., five element theory), undetectable phenomena (e.g., qi, acupuncture meridians, yin yang imbalance), folk wisdom, and anecdotal accounts. Thus, to facilitate the acceptance of Chinese medicine by empirically-minded critics, Mao ordered the writing of new texts; ones that would present a standardized, internally consistent Chinese medicine. Additionally, notions such as “holism” and “preventative care” were incorporated into the new writings, no doubt to increase Chinese medicine’s appeal to Western sensibilities. Thus, while traditional Chinese medicine is generally thought to be an ancient opus (which many value precisely because it is presumed to be ancient), to a real extent it is the modern result of Mao’s advocacy.

Despite Mao’s efforts to foster acceptance of traditional Chinese medicine in the West, there are more than a few skeptics who consider its entire oeuvre, including its so-called “holistic” approach, to be unsupported quackery. Many also contend that it is unfair to disparage Western medicine for being reductionist, since Western medicine most certainly knows that human diseases involve complex interactions between many factors. Others worry that hyping traditional Chinese medicine might undermine confidence in conventional Western medicine (the issue of vaccine non-compliance comes to mind). Then again, to dismiss all of Chinese medicine out of hand is not evidence-based either.

In any case, Mao was clearly successful in getting many in the West to embrace traditional Chinese medicine. Just this past October, a U.S. Senate resolution sponsored by Sen. Barbara Mikulski designated October 7-13 as Naturopathic Medicine Week. One reason cited in the Senate resolution is that naturopathic physicians (including those who practice traditional Chinese medicine) can help address the shortage of primary care providers in the United States.

It is interesting that the U.S. Senate’s justification for officially incorporating traditional Chinese medicine into our health care (i.e., the shortage of primary care providers) reiterates one of Mao’s seventy-year-old reasons for preserving traditional Chinese medicine. But, can traditional Chinese medicine, which many consider to be unsupported quackery, alleviate our situation?

Addendum: In traditional Chinese medicine…“the internal organs are divided into two groups: the five Yin or solid organs, and the six Yang or hollow organs. Each of the Yin and Yang organs are identified with one of the five elements; fire, earth, metal, water, and wood. The heart (Yin) and small intestine (Yang) are associated with fire, the spleen (Yin) and stomach (Yang) with earth, the lungs (Yin) and large intestine (Yang) with metal, the kidney (Yin) and bladder (Yang) with water, and the liver (Yin) and gallbladder (Yang) with wood…The five-element medical model stresses interrelationships among the internal organs rather than their individual functioning. Using principles of mutual creation and mutual destruction (as contained in five element theory), Chinese medicine explains that both an excess as well as deficiency in one organ may affect another organ. Consequently, problems with one organ may be cured by the treatment of one or more related organs (3).”

References:

1. 2015 Nobel Laureate Tu Youyou and Ancient Chinese Medicine, posted on the blog October 12, 2015.

2. Directive on Work in Traditional Chinese Medicine (July 30, 1954), pp. 464-467 in The Writings of Mao Zedong, 1949-1976: Volume I September 1949 – December 1955. Michael Y. M. Kau, John K (eds), M. E. Sharpe Inc., 1986.

3. The Tao of Tai-Chi Chuan, by Jou, Tsung Hwa, Shoshana Shapiro (ed), Charles E. Tuttle Co, 1980.

2015 Nobel Laureate Tu Youyou and Ancient Chinese Medicine

Three researchers shared the recently announced 2015 Nobel Prize for Physiology or Medicine, for their discoveries of therapies against parasites. Microbiologists William Campbell at Drew University in Madison, New Jersey, and Satoshi Ōmura at Kitasato University, Japan, received their share of the award for their discoveries of a therapy against infections caused by roundworm parasites. Chinese pharmacologist Tu Youyou received her share for discovering an anti-malaria medicine called artemisinin.

Tu’s discovery is especially interesting since it happened only because she could read 1,500-year-old Chinese medical texts. It isn’t virology, but it is a good tale. It came about as follows.

In the late 1960s Chinese soldiers, and their Vietnamese allies, were fighting against the United States armed forces in the jungles of Vietnam, where they were being decimated by malaria. Chloroquine and quinine—the main treatments for the disease—were losing their efficacy. Thus, in 1967 Mao Zedong decided that China urgently needed to find a cure for malaria. So, in 1967 he launched a secret research unit for that purpose. In 1969, Tu, then a researcher at the Academy of Chinese Traditional Medicine, became the group’s leader. [The United States too was working on a malaria therapy for the same reason.] See Aside 1.

[Aside 1: Malaria is a life-threatening disease caused by mosquito-borne parasites of the genus Plasmodium. Although malaria is now a preventable and treatable disease, in 2013 it caused an estimated 367,000 to 755,000 deaths, mostly among African children. See the CDC website.]

Tu’s team followed the unusual (by Western standards) route of perusing ancient Chinese texts for clues to historical methods for treating malaria. After screening more than 2,000 traditional Chinese herbal remedies for their effectiveness against malaria, the team came upon a short reference to sweet wormwood, used by the ancient Chinese as an anti-malaria therapy around 400 AD.

In 1972 Tu’s team isolated a compound, artemisinin, from a wormwood plant extract, which seemed to be effective against malaria parasites. Nevertheless, the compound was not effective at eradicating malaria in animals. So, Tu carefully reread the original ancient text, in which she discovered that the secret to the drugs efficacy was to heat the wormwood extract, without allowing it to reach the boiling point. When Tu followed that practice, artemisinin indeed was effective in mice and monkeys. Next, to ensure the safety of the new drug, Tu volunteered to be its first human recipient. Artemisinin still remains the best therapy against malaria.

Tu Youyou, now 84-years old, is the first Chinese woman to win a Nobel Prize. Earlier, in 2011, she won a Lasker prize. Yet, she has neither a medical degree nor a PhD. Instead, she attended a pharmacology school in Beijing and, shortly afterwards became a researcher at the Academy of Chinese Traditional Medicine. She never worked outside of China.

Tu Youyou, now 84-years old, is the first Chinese woman to win a Nobel Prize, awarded for her discovery of an anti-malaria medicine called artemisinin. Tu’s discovery happened because she was able to read ancient Chinese medical texts.
Tu Youyou, now 84-years old, is the first Chinese woman to win a Nobel Prize, awarded for her discovery of an anti-malaria medicine called artemisinin. Tu’s discovery happened because she was able to read ancient Chinese medical texts.

Tu has never won a major award in China, nor is she a member of the Chinese Academy of Sciences. This may be in part because she never received a formal doctoral degree. Moreover, as Tu herself says, “Chinese awards are always given to teams, but foreign awards are different. This honor belongs to me, my team and the entire nation (1).”

Some have commented that Tu’s story points up the need for Western medicine to pay more attention to traditional Asian therapies. Others noted that while alternative medicine has provided some potentially useful leads, it also has been the source of many useless and even harmful treatments. In any case, Tu’s Nobel Prize-winning discovery, linked to her ability to read ancient Chinese texts, is notable.

Reference:

1. http://sinosphere.blogs.nytimes.com/2015/10/09/tu-youyou-nobel-prize-malaria/

ALS (Lou Gehrig’s Disease) and a Human Endogenous Retrovirus

Our last posting was a review of sorts of The Theory of Everything, a movie biography of Stephen Hawking and his wife Jane Wilde (1). Hawking was the first to put forward a cosmology based on an attempt to combine general relativity and quantum mechanics. However, in 1963, before Hawking began his groundbreaking studies, he was diagnosed with the neurodegenerative disease, amyotrophic lateral sclerosis (ALS; commonly known as Lou Gehrig’s disease). He was 21 years old and given a life expectancy of only two years. Even after Hawking become a world-renowned scientist, he was totally dependent on Jane at home.

ALS has been a poorly understood and incurable disease, involving the death of neurons that control voluntary movements, speech, and breathing. The illness is usually fatal within three years of the onset of symptom, thus accounting for Hawking’s grim initial prognosis. Now, new evidence, from a research team at the NIH, headed by Avindra Nath, shows that a human endogenous retrovirus, HERV-K, likely plays a key role in the pathology of ALS (2).

Endogenous retroviruses probably arose millions of years ago, when retroviruses first began inserting their provirus (DNA) genomes into the genomes of germ line cells (see reference 3). Astonishingly, eight percent or more of the human genome is comprised of retroelements. Most of these are defective because of the accumulation of numerous mutations over time. Nearly nothing had been known with certainty about their relevance to human disease, although they had been implicated in a variety of illnesses, including cancer, inflammatory disorders, and neurodegeneration.

The new NIH study was prompted by several earlier observations. First, ALS can present as a rare complication in AIDS patients. Second, the ALS symptoms of at least some of these AIDS patients were alleviated by anti-retroviral therapy against their HIV infections. Third, reverse transcriptase activity was detected in the blood of some ALS patients. Fourth, despite an extensive search for exogenous retroviruses in ALS patients, none has ever been detected.

HERV-K, like other retroviruses, has three major structural genes, gag, pol, and env, which encode the viral capsid protein, reverse transcriptase, and envelope proteins, respectively. Nath and co-workers detected transcripts of each of these genes in postmortem brain tissue samples from ALS patients, thus implying that the entire HERV-K genome was expressed in these patient samples. No significant expression of other HERVs was detected in these patient samples. Nor was HERV-K expression detected in brain tissue from healthy individuals, nor was it seen in brain specimens from Alzheimer’s and Parkinson’s disease patients.

ALS patient samples were immunostained for the HERV-K env protein in order to identify the cell types in which the endogenous retrovirus was expressed in those individuals. Env was detected only in large pyramidal neurons in the cortex, and in anterior horn neurons of the spinal cord.

Next, the entire HERV-K genome and env alone were transfected into human neuronal cell cultures to evaluate whether HERV-K expression might be neurotoxic. Env alone, as well as the entire HERV-K genome, caused a decrease in cell numbers and a retraction of axons. Thus, the HERV-K env protein alone causes neurotoxicity and neuronal death in vitro.

The authors then assessed whether the HERV-K env protein might be neurotoxic in vivo; first by in utero electroporation of the env gene into embryonic mouse brain, and then by generating env-expressing transgenic animals. Expression of the HERV-K env in vivo indeed caused degeneration of motor neurons. And, as in ALS patients, only those motor neurons in the transgenic mice that control movements were damaged. Moreover, the transgenic animals developed progressive motor dysfunction, and 50% of the animals died by 10 months of age. The authors note that the mechanism by which the HERV-K env protein leads to neuronal injury is not yet known.

More than 12,000 Americans are currently living with ALS, and there is not yet any effective treatment for them. The current study offers hope that antiretroviral therapy, similar to that used to treat AIDS patients, might benefit at least some individuals suffering from this tragic illness. Be on the lookout for follow-up studies to this report, which are sure to occur.

Lou Gehrig's iconic speech, July 4, 1939.
Lou Gehrig’s iconic speech, July 4, 1939.
“Today I consider myself the luckiest man on the face of the Earth…”

References:

1. “The Theory of Everything,” Posted on the blog September 15, 2015

2. Wenxue Li, Myoung-Hwa Lee, Lisa Henderson, Richa Tyag, Muzna Bachani, Joseph Steiner, Emilie Campanac, Dax A. Hoffman, Gloria von Geldern, Kory Johnson, Dragan Maric, H. Douglas Morris, Margaret Lentz, Katherine Pak, Andrew Mammen, Lyle Ostrow, Jeffrey Rothstein and Avindra Nath. Human endogenous retrovirus-K contributes to motor neuron disease, Science Translational Medicine, 7:307ra153 (2015).

3. Howard Temin: In From the Cold, Posted on the blog December 14, 2013.

“The Theory of Everything”

HBO has recently been broadcasting the 2014 movie The Theory of Everything; the biographical film about the relationship between Stephen Hawking and his wife Jane Wilde.

The real Hawking and Jane in the 1990s
The real Hawking and Jane in the 1990s

The movie is adapted from Jane’s memoir Traveling to Infinity: My Life with Stephen and, consequently, it is supposed to accurately depict key moments of their life together (1). But, how accurate is the movie in depicting Hawking’s science? Since I am not a physicist, my answer is based largely on Hawking’s book, A Brief History of Time (2).

But first, consider that the movie is meant to entertain a general audience, which is not likely to want to sit through the intricacies of general relativity and quantum mechanics. Hawking himself tells us that when he was writing A Brief History of Time, he accepted the advice that every formula he published would halve his sales.

We begin with a scene from the movie, in which Hawking is shown having a sudden “aha” moment that leads to his major scientific achievement—his discovery that black holes emit particles and radiation. [The myth of Isaac Newton and the apocryphal apple is perhaps the most famous example of this cliché.] Hawking is seen looking at burning coals in his fireplace, through a sweater that he is struggling to pull over his head. Jane comes in, and Stephen announces, “I have an idea.”

Did Hawking indeed have the “aha” moment depicted in the movie? Hawking does not talk about it in A Brief History of Time. Nor do I recall mention of such a moment from any other source.

Hawking explains the thinking that led to his breakthrough in A Brief History of Time. He begins by making the case for entropy within black holes. Next, “If a black hole has entropy, then it ought to have a temperature. But a body with a particular temperature ought to emit radiation at a certain rate. It is a matter of common experience that if one heats up a poker in a fire it glows red hot and emits radiation.”

Did Hawking’s reference to a “poker in a fire” in A Brief History of Time inspire the movie’s producers to portray his key breakthrough as coming from staring at the coals glowing in his fireplace? Regardless, representing Hawking’s discovery in this way is a disservice to the science because it disregards the intense effort that lay behind it. Hawking worked strenuously, over a period of months, to prove his case; which he did with mathematical rigor. Moreover, since he remained troubled by the prevailing view that “by their very definition, black holes are objects that are not supposed to emit anything (2),” he spent more months trying to figure out where he might have gone wrong. But, he hadn’t gone wrong. In brief, the explanation stems from the uncertainty principle of quantum mechanics, which predicts that certain pairs of quantities, such as the position and velocity of a particle, cannot both be known with complete accuracy. [The uncertainty principle, formulated by Werner Heisenberg, is a cornerstone of quantum mechanics. For more on Heisenberg, see reference 3, in particular Asides 6 and 7.] See Aside 1.

[Aside 1: Hawking’s “aha” moment in The Theory of Everything reminds me of a similar moment portrayed in the earlier (2001) movie A Beautiful Mind, about mathematician John Forbes Nash Jr. In that movie, Nash’s “aha” moment—which led to his Nobel Prize winning work in economics—happened when a nasty rejection from a blond in a bar led Nash to suddenly realize that pursuing one of the more numerous brunettes was much more likely to lead to a successful outcome.]

The movie depiction of Hawking’s signature discovery also feeds the cliché that great scientific breakthroughs are the products of eccentric geniuses working in isolation. Actually, Hawking’s breakthrough was inspired by his 1973 meeting in Moscow with two leading Soviet black hole experts; Yakov Zeldovich and Alexander Starobinsky, who convinced Hawking that “according to the quantum mechanical uncertainty principle, rotating black holes should create and emit particles (2).”

Hawking also admits to being motivated by physicist Jacob Bekenstein; at the time a graduate student at Princeton. Bekenstein suggested that the area of a black hole’s event horizon (i.e., the black hole’s boundary) is a measure of the black hole’s entropy. And, as noted above, if a black hole has entropy, it has temperature, and thus must emit radiation (2).

The Theory of Everything shows Hawking introducing his discovery, in public, for the first time, in front of a small audience, in a small lecture hall, while seated in his wheelchair. The blackboard behind him is blank. When he finishes speaking, someone in the audience jumps up and declares that the theory is “complete nonsense,” and then storms out. Hawking impishly says to the departing individual, “Was it something I said, Professor?” Next, a Russian physicist stands up and announces that “the little one has done it (i.e. succeeded).” With that endorsement, Hawking becomes world famous, and his face adorns the cover of Nature.

Although aspects of the depiction of Hawking’s lecture seemed unrealistic to me, the incident actually did occur, and it was not entirely unlike its portrayal in the movie. It was during a conference at the Rutherford-Appleton Laboratory near Oxford. Hawking relates, “At the end of my talk the chairman of the session, John G. Taylor from Kings College London, claimed it was all nonsense (2).” The Russian physicist who commended the discovery was Isaac Kalatnikov, who earlier showed that the universe could have had a singularity (see below).

The movie intermixes the lecture scene with another scene, in which one of Hawking’s friends is explaining to others how a black hole can eventually go poof. The purpose of the intermixed scene may have been to provide a context for Hawking’s discovery. In any case, Hawking himself comments on the implications of the discovery as follows: “The existence of radiation from black holes seems to imply that gravitational collapse is not as final and irreversible as we once thought (2).” He goes on to explain that Einstein’s theory of general relativity, taken alone, predicts that any matter falling into a black hole would be destroyed at the singularity (a region of zero volume in which the density of matter and the curvature of space-time become infinite), while the gravitational effect of the black hole’s mass would continue to be felt on the outside. But, “when quantum effects were taken into account, it seemed that the mass or energy of the matter would eventually be returned to the rest of the universe, and that the black hole, along with any singularity inside it, would evaporate away and finally disappear.”

Another of Hawking’s discoveries—that the universe may have come into existence from a singularity—is also highlighted in the movie. That discovery happened before his finding that black holes emit radiation. In fact, it was the subject of his doctoral thesis. The seed for the discovery was planted by physicist Roger Penrose’s proposal that a star collapsing under its own gravity eventually shrinks to a singularity. The movie indeed acknowledged Penrose’s contribution. What’s more, Penrose is also shown serving on Hawking’s dissertation committee.

Importantly, Penrose’s theorem applied only to collapsing stars. Hawking’s innovation was to ask whether the entire universe was a singularity in the past. “I soon realized that if one reversed the direction of time in Penrose’s theorem, so that the collapse became an expansion (2),” the conclusion would be that an expanding universe must have begun as a singularity. An important corollary is that the universe had a beginning. [Time and space too were created in the transition from nothing to something. There was no time before the big bang and, consequently, the big bang didn’t actually take place in time. Another interesting notion: since time came into existence at the moment of the big bang, there was never a moment in time when the universe did not exist.]

Penrose’s theorem about stars collapsing into black holes influenced Hawking in yet other ways. Hawking explains: “… at the time that Penrose produced his theorem, I was a research student desperately looking for a problem with which to complete my Ph.D. thesis. Two years before I had been diagnosed as suffering from ALS, commonly known as Lou Gehrig’s disease, or motor neuron disease, and given to understand that I had only one or two more years to live. In these circumstances there had not seemed much point in working on my Ph.D.—I did not expect to survive that long. Yet two years had gone by and I was not that much worse. In fact, things were going quite well for me and I had gotten engaged to a very nice girl, Jane Wilde. But in order to get married, I needed a job, and in order to get a job, I needed a Ph.D…The final result was a joint paper by Penrose and myself in 1970, which at last proved that there must have been a big bang singularity provided only that general relativity is correct and the universe contains as much matter as we observe (2).” And, as we know, Hawking and Jane were married. See Aside 2.

[Aside 2: Almost coincident with The Theory of Everything, there was another movie biography about a British scientist—The Imitation Game, about British mathematician and computer pioneer, Alan Turing, and his work in breaking Germany’s Enigma code during World War II. Despite its excellence, The Imitation Game leaves the impression that Turing virtually single-handedly, and with no prior basis to proceed from, invented and built the machine (the bombe) that broke the German code. Yet a machine, similar to Turing’s, which used rotors to test different letter combinations, was invented earlier by Polish cryptographers. Turing’s very significant contribution was to modify the Polish machine to recognize and ignore letter combinations that were unlikely to yield a useful result, thereby greatly speeding up the screening process. Moreover, the movie does not even mention mathematician Gordon Welchman—he and Turing were among the four original recruits to Britain’s code breaking center at Bletchley Park,—who substantially improved Turing’s machine. Welchman’s improved version of the machine actually broke Enigma ciphers during the war. Incidentally, after the war, Welchman taught the first computer course at MIT. Turing is generally considered to be the father of computer science, and I certainly do not mean to disparage him. My point is that even very good movie biographies of scientists take license with the science to enhance the drama.]

The Theory of Everything may have left some viewers with the impression that the notion of an expanding universe originated with Hawking. Actually, in 1929 Edwin Hubble discovered that the universe is expanding in all directions. And, importantly: “The discovery (Hubble’s) finally brought the question of the beginning of the universe into the realm of science…Hubble’s observations suggested that there was a time, called the big bang, when the universe was infinitesimally small and infinitely dense…One may say that time had a beginning at the big bang…(2).”

The 1965 discovery of the cosmic microwave background radiation, by Arno Penzias and Robert Wilson, provided compelling evidence for the big bang. What’s more, Hawking and Penrose showed that Einstein’s general relativity implied that the universe had a beginning.

The Theory of Everything advances the thought that if the universe had a beginning, then it had a creator. Afterward, without much in the way of explanation, the movie shows Hawking recanting his belief that the universe had a beginning. Instead, he proposes that the universe has no boundaries in space or time—i.e. no beginning, and no creation. He tells Jane that God is now out.

A Brief History of Time confirms Hawking’s change in view—that the universe did not have a beginning. He explains that combining general relativity with the uncertainty principle of quantum mechanics leads to black holes not being black, and the universe not having any singularities. Moreover, the universe “would neither be created nor destroyed. It would just be…What place, then, for a creator? ”

The movie does not address what impact, if any, Hawking’s new outlook may have had on his earlier work. Fortunately, Hawking explains in A Brief History of Time that his new proposal did not undo his earlier work on singularities. Rather, the real importance of the earlier singularity theorems was in showing that quantum gravitational effects could not be ignored in any grand unified theory. “…it seems that the uncertainty principle is a fundamental feature of the universe we live in. A successful unified theory must therefore necessarily incorporate this principle (2).”

Jane is deeply religious. Indeed, her faith helps to sustain her in caring for Stephen. [Despite Hawking’s fame and public acclaim, he was completely dependent on Jane at home.] In contrast, when Stephen refers to God, he seems to be making fun of Jane’s faith. Yet, Hawking does mention God often in A Brief History of Time. Moreover, the final words of the book are: “However, if we do discover a complete theory…then we should know the mind of God.”

In the movie, Jane discovers the above passage in Stephen’s manuscript. She then asks Stephen if he means it, adding, “Are you going to let me have this moment?” Stephen answers “yes” and “your welcome,” but he then adds, “However…”

Neither the movie, nor A Brief History of Time, tells us for sure what Hawking really believes about God. In any case, Hawking never suggests that he believes in a kind of supernatural creator that one might worship. So, it is likely that he refers to God in much the same spirit as Einstein did when he famously quipped, “God doesn’t play dice with the universe.” Einstein uses God as a religious metaphor, and I suspect that Hawking is doing the same.

Despite Hawking’s apparent agnosticism, he nevertheless seems uncertain as to whether science can ever explain existence. “What is it that breathes fire into the equations and makes a universe for them to describe? Why does the world go through all the bother of existing? Is the unified theory so compelling that it brings about its own existence? Or does it need a creator, and, if so, does he have any other effect on the universe? And who created him (2)?”

The following is from a piece by Caroline Graham and Gabrielle Donnolly in the Daily Mail (http://www.dailymail.co.uk/femail/article-2826974/Anguish-scientist-s-dumped-wife-revealed-star-Felicity-Jones-s-playing-movie.html#ixzz3lCV8caM4):

“British actress Felicity Jones – best known as the voice of Emma Grundy in The Archers, but whose film credits include Brideshead Revisited and the romcom Chalet Girl – plays the discarded wife and Eddie Redmayne, of Birdsong fame, plays Hawking.

During filming, Hawking and his ex-wife (Jane) both turned up on set. It was a daunting moment.

Felicity says: ‘Out of the corner of one eye I saw Jane and her new husband and out of the other eye I saw Stephen. It was probably one of the most intimidating moments of my life.

It must have been so bizarre for them to watch us playing them. It certainly felt awkward for me.’

Hawking and Jane watched a sequence during which Felicity and Redmayne danced together. After the director yelled ‘Cut’, Hawking – who communicates through a computer-based speech generator – asked: ‘Would you ask Felicity if she will come and give me a kiss?’

Felicity Jones and Eddie Redmayne in the dancing scene
Felicity Jones and Eddie Redmayne in the dancing scene

For 31-year-old Felicity, that moment was a revelation. ‘It shows his rather flirtatious nature and this amazing capacity he has not to take himself too seriously,’ she explains. ‘I embraced him and told him, “You’re amazing!” ’

References:

(1) L.V. Anderson, How Accurate Is The Theory of Everything?, Slate’s culture blog, November 7, 2014.

(2) Stephen W. Hawking, A Brief History of Time, Bantam Books, 1988.

(3) “The Upright Thinkers”, Posted on the blog, August 19, 2015.

“The Upright Thinkers”

The Upright Thinkers by Leonard Mlodinow—former professor of Physics at Caltech and author of several other best selling books on science—tells his version of “the human journey from living in trees to understanding the cosmos.” The story is epochal. We begin by noting several of Mlodinow’s general themes.

upright thinkers

Mlodinow asserts that our odyssey of discovery has been driven by our inborn and virtually insatiable curiosity; our propensity to ask “why?” “Human children all around the world ask their first questions at an early age, while they are still babbling and don’t yet speak grammatical language…Chimpanzees and bonobos, on the other hand, can learn to use rudimentary signing to communicate with their trainers, and even answer questions, but they never ask them. They are physically powerful, but they are not thinkers.”

We also are reminded that our astonishing progress was facilitated by a unique characteristic of our species—we add to knowledge already in existence. Recall the famous quote of Isaac Newton, “”If I have seen further than others, it is by standing upon the shoulders of giants.”

Related to the above, there is archaeological evidence that schools existed as early as 2,500 B.C. “…the idea that society should create a profession devoted to passing on knowledge, and that students should spend years acquiring it, was something entirely new—an epiphany for our species.”

Another of Mlodinow’s themes is that the major players in our quest to understand the cosmos (e.g., Newton, Mendeleev, Darwin, Einstein, Bohr, and Heisenberg) were not merely brilliant. They were also stubbornly persistent individuals. Contrary to popular belief, the groundbreaking discoveries they made did not result from sudden “aha moments” but, instead, came about only after years of dogged hard work. There was no apocryphal apple that serendipitously landed on Newton’s head.

Mlodinow also reminds us that scientists, as well as laypeople, do not easily to let go of the conventional beliefs of their day, even when faced with strong evidence to the contrary. Consequently, new scientific paradigms, even when supported by incontrovertible evidence, are often initially rejected by the establishment. And since the revolutionary breakthroughs that Mlodinow highlights in his tale indeed challenged the established wisdom of their times, the scientists who put them forward had to posses more than a little courage and even audacity to complement their doggedness. [Howard Temin provides a recent example of a virologist who persevered in the face of scorn and ridicule from his colleagues. See Howard Temin: “In From the Cold, posted on the blog December 16, 2013.]

Mlidinow allocates several pages to the origin and acceptance of the concept of universally applicable natural laws. The idea of natural laws already existed in ancient Greece. However, Greek natural laws were each specific to a particular situation. Our modern concept of universally applicable natural law came into being only in the early seventeenth century, as prompted by the discoveries of Kepler, Newton, and their contemporaries (see below).

Why might it have taken mankind so long to recognize that nature acts according to certain regularities? The answer may lie in the fact that when primitive humans were faced with seemingly inexplicable droughts, floods, plagues, earthquakes, and so forth, it was difficult for them to view the world as anything but chaotic. Remarkably, even today there still are those who reject the idea of a universe ruled by natural laws. [Consider this. Mlodinow relates that Einstein was astonished by the fact that nature has order. Einstein wrote: “one should expect a chaotic world, which cannot be grasped by the mind in any way… the most incomprehensible thing about the universe is that it is comprehensible.”]

Returning to the theme of our difficulty transitioning from judging the truth of a statement according to how well it fits convention or religious belief, to how well it is supported by empirical evidence, note that for nearly two millennia scientific progress was impeded by the conventions of ancient Greek science. Contrary to popular belief, the Greeks did not invent the scientific method (2). Nor were their theories developed with the intent of experimental verification. Aristotle’s science held that nature did what pure logic suggested it should do. For example, Aristotle said that heavier objects fall faster than lighter objects, because it is their purpose to do so. Neither Aristotle nor his contemporaries actually looked to see if heavier objects indeed fall faster than lighter ones. In fact, the first individual known to have actually tested this premise was Galileo (1564-1642). Only afterwards did observation and experimentation become the basis for western science.

Despite the fact that Aristotle’s non-quantitative search for purpose impeded scientific progress for nearly two thousand years, Mlodinow credits him with applying reason in the pursuit of understanding and, also, for the idea that nature acts according to certain regularities. Mlodinow also notes that Aristotle was hamstrung by the technology of his day. In particular, since Aristotle did not have a stopwatch, concepts such as velocity and acceleration—keystones of Newtonian physics—may have been beyond his reach. But, “More important was the fact that Aristotle was, like everyone else, simply not interested in quantitative description.”

Notwithstanding the above, why did the leap from Aristotelian physics to the breakthroughs of Galileo and Newton take 2,000 years? According to Mlodinow, the conquest of Greece by Rome was a key reason. The Romans were superb engineers, but they had little interest in the pursuit of knowledge for its own sake. Thus, the Roman conquest of the Greeks resulted in the fading out of the Greek scientific heritage from Western culture.

At the end of the eleventh century, a Benedictine monk, Constantinus Africanus, translated ancient Greek medical texts (preserved for centuries by Muslim court scholars) from Arabic to Latin. This was a first step in the revival of the Greek scientific heritage in Europe. Moreover, it was a step towards the eventual resumption of Western science (see Aside 1). Additionally, the development of European universities, such as Oxford (by 1250), facilitated scientific progress in the West, since—then as now—universities are a place that brings people together, where they might interact with and stimulate one another. [Most scientific progress still emanates from universities.] The emergence of moveable-type printing at around 1450 was another key development, since it enabled the widespread dissemination of new ideas.

[Aside 1: Intellectual progress in the Islamic world and in China often surpassed that in Europe during the Middle Ages. Why then did modern science not emerge in the Islamic world, or in China, instead of in Europe? For an answer, I turn briefly to Why the West Rules—For Now by Ian Morris (3). Morris tells us that the resurgence of science in Western Europe was largely driven by Europe’s “new frontier” across the oceans, and the need it created for the precise measurement of time and space. Moreover, “…by the point that two-handed clocks had become the norm Europeans would have to have been positively obtuse not to wonder whether nature itself was not a mechanism.” Additionally, the industrial revolution in the West, which was largely driven by the Atlantic economy, stimulated further mechanical invention and scientific development.]

Galileo was one of the most influential scientists who ever lived and a key figure in the resurgence of European science. One reason for his fame was his experiment at the Leaning Tower of Pisa, which showed that light objects fall as rapidly as heavy ones. Yet while he was disenchanted by the lack of experimentation in Aristotelian physics, he did not invent experimental physics per se. However, his quantitative approach to experimentation was indeed revolutionary. Moreover, “when he got a result that surprised him or went against conventional thinking, he didn’t reject it—he questioned convention and his own thinking.”

One of Galileo’s most insightful and important contentions was that objects in uniform motion tend to remain in that state of motion. That is, an object does not require the continuous application of a force to remain in motion; an assertion that was contrary to the prevailing Aristotelian view that objects require a continuing reason for their motion. If Galileo’s assertion sounds oddly familiar, it is because Newton later adapted it as his first law of motion. [“A few pages after stating the law, Newton adds that it was Galileo who discovered it—a rare instance of Newton giving credit to someone else.”]

Galileo is also famous for his conflict with the Catholic Church over his claim that the Earth is not at the center of the universe. But the idea of a heliocentric (i.e., sun-centered) universe did not originate with Galileo. In fact, it existed as early as the third century B.C. in Greece, and a “modern” European version can be attributed to Copernicus (1473-1543). But Galileo was the first to offer irrefutable proof that the Earth is not at the center of the universes. What’s more, Galileo made his case compelling only after he perfected the telescope, which enabled him to achieve unprecedented levels of power..

Isaac Newton was born in 1642; the year that Galileo died. He is commonly said to have invented the calculus, and to have discovered his laws of motion and universal gravitation, all in the single year, 1666. The last discovery supposedly followed his serendipitous encounter with a falling apple. The reality was a bit different. First, the calculus was concurrently and independently invented by Leibnitz. Second, Newton labored continuously for a period of three years—1664 through 1666—in order to discover his laws of motion. Yet even after accomplishing that feat, Newton was still not a Newtonian. “He still thought of uniform motion as arising from something internal to the body, and by the term ‘gravity’ he meant some inherent property arising from the material an object is made of , rather than an external force exerted by the earth.”

After discovering his laws of universal motion, Newton spent additional years acquiring information, largely compiled by others, regarding planetary orbits. Kepler’s finding—that planetary orbits are ellipses, with the sun at one of the foci—would be especially significant, as follows.

Edmond Halley (the astronomer who computed the orbit of the comet bearing his name), Robert Hooke (the discoverer of cells and perhaps the greatest experimentalist of the seventeenth century), and Christopher Wren (best known as an architect, but also an accomplished astronomer) gleaned from Kepler’s data a premise of singular importance; that the pulling force of the sun diminishes in proportion to the square of the planet’s distance from it. Yet Halley, Hooke, and Wren were unable to prove their conjecture.

Halley, happening to be in Cambridge, took the opportunity to visit Newton. During his visit with Newton, Halley informed his host of the conjecture concerning the pulling force of the sun. Imagine Halley’s surprise when Newton claimed that he had already discovered the premise and, indeed, had already proven it. Newton then set about looking for his proof. But when he found it, he discovered that it was in error. He then set about reworking his proof and, in so doing, he confirmed that Kepler’s planetary orbits indeed are explained by an inverse square law of attraction.

Newton’s proof of the inverse square law of gravitational attraction was followed by more hard work that resulted in “what is perhaps the most significant intellectual discovery that had ever been made”—Newton’s universal theory of motion, which showed that free fall and orbital motion are instances of the same laws of force and motion. Newton set down his laws of motion, and his law of universal gravitation, in his famous Principia; three books published in 1687, which are regarded as the foundation of classical mechanics and, thus, as one of the most important works in the history of science. Halley personally paid the cost of publishing the Principia.

Newton died in 1726. “Newton’s life and Galileo’s had together spanned more than 160 years, and together they witnessed—and in most respects accounted for—most of what is called the scientific revolution.”

A revolution in chemistry was occurring concurrent with the ones in physics and astronomy. The key breakthrough in chemistry would be the discovery that some substances—the elements—are fundamental, and that all else is made from them. Actually, the concept of elements dates back to Aristotle and the ancient Greeks. However, Aristotle’s chemistry had but four “elements”—earth, air, fire, and water.

The experimental results of Robert Boyle (1627-1691) were the first to free chemistry from Aristotelian convention. Mlodinow relates how in 1642, the 15-year-old Boyle was taken with science after reading Galileo’s book on the Copernican system. Afterwards, the adult Boyle (incidentally assisted by Robert Hooke) investigated respiration and combustion. His findings led him to conclude that air is not an element but, instead, is made up of different substances. Moreover, Boyle’s quantitative approach set in place a new convention in chemistry of careful experimental analysis.

Boyle’s experiments were followed by those of Joseph Priestly (1733-1804) and of Antoine Lavoisier (1743-1794). The relationship between these two men makes for an interesting tale, one that is too long to review here. But, together, their researches showed that respiration and combustion removed something from the air. Priestly may have discovered it first, but Lavoisier gave it its name, oxygen. Lavoisier went on to reveal one of chemistry’s major principles—the conservation of mass. That is, the total mass of the products produced in a chemical reaction must be the same as the combined mass of the initial reactants.

Lavoisier had been a tax collector in Paris in the days preceding the French Revolution. His day job provided financial support for his experiments, but it did not endear him to the revolutionaries who overthrew the French monarchy. He was arrested in 1793 during the Reign of Terror and sentenced to death. The presiding judge is said to have told him, “The Republic has no need of scientists.” “By the time of his execution, Lavoisier had identified thirty-three known substances as elements. He was correct about all but ten.”

The next important advancements would involve the nature of atoms. Although Lavoisier identified the elements as the reactants in chemical processes, he chose not to think in terms of atoms. His reason was simply that he did not know of any way by which something as tiny as an atom could be observed or experimented on. John Dalton (1766-1844) took the first step towards solving that problem when he discovered how to determine atomic weights by applying Lavoisier’s law of conservation of mass. [Mlodinow details Dalton’s approach.]

Dmitry Mendeleev (1834-1907) made a crucial breakthrough that would lead to an understanding of the relationship between the weight of an atom and its chemical properties. [The relationship of course involves atomic numbers, rather than atomic weights. Please be patient.] How that development came to pass was one of my favorite anecdotes in Mlodinow’s book. It was as follows. In 1866, while Mendeleev was a chemistry professor in St. Petersburg, he could not find a first-rate, up-to-date chemistry book, written in Russian, that he might use in his teaching. So, he decided to write his own. Mendeleev’s book was a labor of love that he worked on for years. Early on, he wrestled with the question of how to organize the book. His solution was to arrange the elements and their compounds into groups, as defined by their properties. Then, after much arranging and rearranging of the elements, he discovered a pattern that we know today as the “periodic table;” a discovery of singular importance that Mlodinow calls “chemistry’s version of Newton’s laws.”

Much important work remained to be done. Because there were as yet undiscovered elements, there were gaps in the Mendeleev’s table. Moreover, some of the assigned atomic weights were wrong and, crucially important, the chemical properties of an element are determined by the as yet undiscovered atomic numbers, rather than by atomic weights. Notwithstanding that, to Mendeleev’s credit, he accurately predicted the existence and chemical properties of the elements which corresponded to the gaps in his table.

The twentieth-century revolution in physics led to profound insights into the nature of space and time, and to a deeper “understanding” of the atom. Mlodinow tells that story only after he reviews several key breakthroughs in biology. Keeping with his format, he notes the early influence of Aristotelian convention on biology. Two Aristotelian beliefs were especially important. One was the belief that a divine intelligence designed all living beings. The other was the belief in spontaneous generation.

Readers of the blog are likely familiar with Louis Pasteur’s late nineteenth-century experiments, which laid spontaneous generation to rest once and for all (4). Many readers may also know that Francesco Redi experimentally challenged spontaneous generation more than two centuries earlier. In 1668, Redi’s microscopic observations of minute organisms showed that they are much more complex than had been imagined. In particular, these organisms had reproductive organs, which called into question one of Aristotle’s arguments for spontaneous generation; that lower organisms are too simple to reproduce.

The notion, that a divine intelligence was necessary to design all living beings, was a foundation stone of nineteenth-century thinking. Under that circumstance, Charles Darwin (1809-1882) gave the world a scientific theory—evolution by natural selection— that explained how species came to have their particular characteristics. Darwin’s theory directly challenged the biblical account of creation, which still has a very significant number of adherents. Thus, it is all the more remarkable that his grave occupies hallowed ground under Westminster Abbey.

Legend has it that Darwin had an “aha moment” while observing the beaks of finches on different islands of the Galapagos. The reality is that Darwin only gradually came to believe that species are not unchanging forms designed by God but, instead, evolved over time. And Darwin devoted many more years to discovering the mechanism behind evolution—natural selection. Moreover, knowing that his findings would expose him to ridicule and attack, he spent more years amassing incontrovertible evidence in support of his theory.

Darwin did not know of genes and mutations. Consequently, he was unaware of how genetic variability might provide the raw material for adaptation. His contemporary, Gregor Mendel (1822-1884), made the breakthrough in genetics. Mendel, carried out his experiments while a monk at a monastery in what is now the Czech Republic, perhaps explaining why Darwin never knew of his work.

One way or another, serendipity is usually a factor in all great scientific discoveries. Apropos the theory of evolution, Darwin likely would have remained an unknown English churchman were it not for the unlikely and unexpected letter inviting him, at twenty-two years of age, to sail around the world as a naturalist on the Beagle. The position on the Beagle was beforehand offered to a number of others, none of whom were willing to spend two years at sea.

Mlodinow devotes the last 100 or so pages of his book to the twentieth-century revolution in physics. Considering the stunning discoveries that were about to unfold, it is more than a bit ironic that the prevailing belief at the start of the century was that everything in physics was already known and that nothing else remained to be done.

Key individuals who fill Mlodinow’s final pages include Max Planck (see Aside 2), Albert Einstein (see Aside 3), Ernest Rutherford (see Aside 4), Niels Bohr (see Aside 5), and Werner Heisenberg and Erwin Schrodinger (see Aside 6).

[Aside 2: Max Planck was a German physicist who deduced the relationship between the frequency of radiation and its energy. Moreover, he proposed that electromagnetic energy could only take on discrete values, or quanta. In so doing, he originated quantum theory.

Mlodinow tells the following quip: “Since my name is so hard to spell and pronounce, when I make a restaurant reservation, I often do it under the name Max Planck. It’s very rare that the name is recognized, but one time when it was, I was asked if I was related to the “guy who invented quantum theory.” I said, “I am that man.” The maitre d’, in his early twenties didn’t believe me. He said I was too young. “Quantum theory was invented around 1960,” he said. “It was during World War II, as part of the Manhattan Project.”]

[Aside 3: In the single year 1905, while Einstein was employed as a third-class clerk at the Swiss patent office in Zurich, he published three singularly important, ground-breaking papers. The first paper explained Brownian motion, the second the photoelectric effect (in which light impinging on a metal causes it to emit electrons), and the third, his discovery of special relativity. Although each of the three papers dealt with a different topic, astonishingly, each was important enough to merit a Nobel-Prize. And, since Einstein is best known for his theories of relativity—which revolutionized our concepts of space and time—it may surprise some that his Nobel Prize was actually awarded for his explanation of the photoelectric effect. Einstein’s key insight was to treat light as a quantum particle, rather than as a wave. Consequently; his analysis of the photoelectric effect would significantly advance further developments in quantum theory; which Einstein eventually came to abhor (see below). His explanation of Brownian motion provided the most convincing evidence of the day for the existence of atoms. After years more of work, Einstein put forth his theory of general relativity, which incorporated gravity into relativity theory.]

[Aside 4: Rutherford discovered of the structure of the atom, with its positive charge concentrated in the nucleus. Interestingly, this key advancement  began fortuitously, as a “small research project,” for a young undergraduate student named Ernest Marsden; for him “to get his feet wet.”]

[Aside 5: Bohr applied quantum mechanics to the whole atom, asking what it might mean if the atom, like light quanta, could have only certain energies. His premise, that electrons occupy only certain allowable orbits, provided an explanation for why electrons don’t spiral into the atom’s nucleus. And his proposal that the atom’s outer orbits determine its chemical properties, gave new insights into why Mendeleev’s heretofore mysterious periodic table worked.]

[Aside 6: Heisenberg and Schrodinger proposed dissimilar quantum theories, each of which left behind Newton’s conventional description of reality. In Newton’s physics, position and velocity are independently measured quantities, one event causes another, and the world exists independently of our observation of it. In quantum theory, reality is based on probabilities and uncertainties, and reality does not exist independently of our observations. At a scientific conference, when Einstein famously attacked the indeterminacy and probabilistic nature of quantum physics, saying “God does not play dice with the universe,” Bohr famously replied, “Einstein, stop telling God what to do (5).” Yet even Schrodinger eventually turned against quantum theory, offering up his famous fable of the cat that was neither alive nor dead, to point up the seeming absurdity of the theory. Still, quantum theory remains as the most predictive of all scientific theories.]

The Nazis came to power in Germany in 1933. They hated the new physics for being surreal and abstract but, more importantly, because they considered it to be the work of scientists of Jewish heritage (e.g., Einstein, Born, Bohr, Pauli). [Incidentally, the new physics was also condemned in the Soviet Union in the early 1920s; in that instance for not conforming to the principles of Marxist-Leninist ideology. The Soviets similarly banned Mendelian genetics for the same reason.]

Einstein was still living in Germany when the Nazis took control. But, fortunately, he was visiting Cal Tech on the very day in January 1933 when Hitler became Chancellor. He never returned to Germany. His property in Germany was confiscated, his notes on relativity were burned, and a five-thousand-dollar bounty was put on his head.

Heisenberg tried to accommodate himself to the Nazis, but he was nevertheless harassed by them for having worked on “Jewish physics” with Jewish physicists, and for trying to have Max Born (incidentally Max Delbruck’s mentor) exempted from the Nazi non-Aryan work prohibition. Under pressure, Heisenberg disavowed the Jewish physicists and “their” science.

Max Born, with the help of Pauli’s refugee organization, escaped to Cambridge. Schrödinger, an Austrian who had been living in Berlin when Hitler came to power, was an outspoken anti-Nazi. He left Berlin to take a position at Oxford. Bohr’s real-life exploits during the Nazi era are more fascinating than fiction, and are covered in some detail in reference (5).

In 1933, Max Planck met face-to-face with Hitler to dissuade him from carrying out the anti-Jewish policies that were causing top Jewish scientists to leave Germany. The meeting came to nothing, and Planck quietly went on with his work. However, Planck’s youngest son was a member of the failed plot to assassinate Hitler on July 20, 1944. He was arrested by the Gestapo and was executed along with the other conspirators. Earlier, in 1907, when women were nearly entirely barred from receiving advanced education, Planck invited Lisa Meitner (only the second woman to receive a physics doctorate from the University of Vienna) to carry out postdoctoral studies under his guidance (5). Mlodinow includes Meitner among the short list of physicists who he believes deserved, but did not receive the Nobel Prize. See Aside 6. [Reference 6 notes the wartime experiences of Andre Lwoff, Francois Jacob, Jacques Monod, and Elie Wollman. Reference 7 describes a wartime experience of Renato Dulbecco.]

[Aside 7: By 1938, Lisa Meitner’s situation in Nazi Germany had become desperate. So she fled to Holland, aided by Dutch physicists who persuaded their government to admit her on her Austrian passport, which was no longer valid after the Anschluss (5). She next moved to Sweden, where Niels Bohr found for her a laboratory where she could continue her work. While in Sweden, she received a correspondence from her former collaborator in Germany, chemist Otto Hahn, from which she and her nephew Otto Frisch calculated that Hahn had unknowingly witnessed nuclear fission (5). Meitner passed that information on to Niels Bohr, which Bohr took to America and made public at a January 1939 conference at George Washington University. Leading proponents of the new physics immediately realized that a nuclear bomb was now possible. The famous Einstein letter of August 1939, which warned Franklin Roosevelt of that possibility, led to the American Manhattan Project to develop an atomic bomb. German scientists, led by Heisenberg, likewise began work on nuclear energy. In September 1939, Germany invaded Poland and World War II was underway. By September, 1941, Britain was fighting alone against Germany in the west, and the Soviets were reeling under the German onslaught in the East. Those were the circumstances under which Heisenberg attended a German-sponsored conference in Nazi-occupied Copenhagen. Bohr boycotted the conference in protest against the Nazis. However, Heisenberg sought out his former mentor in Copenhagen for a private meeting. While it is known that Bohr and Heisenberg discussed the nuclear weapon issue, the two men were never able to agree on exactly what was said, or even where their conversation actually took place, and to this day their meeting remains shrouded in mystery. Perhaps because Bohr was potentially a huge scientific asset to the Germans, or perhaps because his life was in danger because of his Jewish heritage, the British in 1943 carried out a harrowing rescue mission to smuggle him out of occupied Denmark. Details of the rescue are recounted in reference 5. Bohr then came to America, where he worked on the Manhattan Project and he later became an outspoken arms control advocate. Although it is not entirely clear, there are reasons to believe that Heisenberg may have surreptitiously impeded the German nuclear effort. Bohr and Heisenberg reestablished their relationship after the war, but their shaky friendship was sustained only by their mutual understanding not to revisit their 1941 meeting.]

Mlodinow asserts that “quantum theory was created by a concentration of brain power in Central Europe that surpassed or at least rivaled that of any of the intellectual constellations we’ve encountered in our journey through the ages.” Moreover, “It was a magical time in Europe, with burst after burst of imagination lighting the sky, until the outline of a new realm of nature began to appear.”

A photo in The Upright Thinkers, taken at the famed 1927 Fifth Solvay International Conference on Electrons and Photons, shows Erwin Schrodinger, Wolfgang Pauli, Werner Heisenberg, Paul Dirac, Max Born, Niels Bohr, and Albert Einstein among others; thus bearing out Mlodinow’s contention that it was a very special time in the history of science.

The “golden age of molecular biology” of the 1950s and 1960s is not discussed in The Upright Thinkers. Thus, I feel an urge to proclaim before closing that it too was a very special time in the history of science, when fundamental discoveries were made in biology that were comparable in significance to those made earlier in the century in physics . As a graduate student in the 1960’s I was privileged to experience the thrill of attending, and speaking at the summer conferences at Cold Spring Harbor, which were simultaneously graced by James Watson, Francis Crick, Max Delbruck, Sidney Brenner, Francois Jacob, Jacques Monod, Seymour Benzer, and Norton Zinder (all of whom are discussed elsewhere on the blog), as well as by the likes of Erwin Chargaff, Matthew Meselson, Frank Stahl, Sol Spiegelman, John Beckwith, and so forth.

This piece at last concludes with an item from The Upright Thinkers, which I found to be especially intriguing. Recent DNA analysis shows that around 140,000 years ago, a catastrophic event—possibly related to climate change—caused the entire human population to plummet to a mere several hundred individuals. We truly were an endangered species. “Isaac Newton, Albert Einstein, and everyone else you’ve ever heard of, and the billions of us who live in the world today, are all descendants of those mere hundreds who survived.”

References:

(1) Leonard Mlodinow, The Upright Thinkers, Pantheon Books, 2015.

(2) Thucydides and the Plague of Athens, posted on the blog September 30, 2014.

(3) Ian Morris, Why the West Rules—For Now, Farrar, Straus and Giroux, 2010.

(4) Louis Pasteur: One Step Away from Discovering Viruses, posted on the blog January 7, 2015.

(5) Max Delbruck, Lisa Meitner, Niels Bohr, and the Nazis, posted on the blog November12.

(6) Genealogies and a Selective History of Lysogeny: Featuring Friedrich Loeffler, Emile Roux, Andre Lwoff, Elie Wollman, and Francois Jacob, posted on the blog January 28, 2015.

(7) Renato Dulbecco and the Beginnings of Quantitative Animal Virology, posted on the blog December 3, 2013.

Frederick Li, p53, and the Li-Fraumeni syndrome

Frederick Li passed away on June 12 of this year. In 1969, Li and Joseph Fraumeni, working together at the U.S. National Cancer Institute, discovered a familial (inherited) cancer syndrome, known as the Li-Fraumeni syndrome. Members of Li-Fraumeni syndrome families have a greatly increased risk of developing several types of cancer; particularly breast cancer, but also brain tumors, leukemias, and other cancers as well (1).

Frederick Li and Joseph Fraumeni  in 1991

In 1990, Li and Fraumeni, in collaboration with Stephen Friend and coworkers at the Massachusetts General Hospital Cancer Center, discovered that all Li-Fraumeni syndrome families harbor germ line mutations in TP53; the gene which encodes the cellular p53 protein (2). This report was the first to document that a mutation in TP53 can be inherited. What’s more, the 1990 paper proved to a previously skeptical medical community that heredity can play a major role in some cancers. [Fraumeni says that environmental factors such as air pollution, occupational exposures, diet, and even viruses were, at the time, considered far more likely causes of cancer than genetic mutation (3).]

Although Li’s research focus concerned genetic mutations that might cause cancer, rather than virology, his story is relevant to the blog because p53 is a key factor in the life cycles of the DNA tumor viruses (i.e., the polyomaviruses, papillomaviruses, and adenoviruses). Moreover, p53 was discovered by virologists. So, we begin with a brief review of the discovery of p53 and its mode of action.

In 1979, the p53 protein was discovered independently by several research groups. The discovery happened when David Lane and Lionel Crawford at the Imperial Cancer Research Fund, and Daniel Linzer and Arnold Levine at Princeton University, unexpectedly discovered a non-viral protein of molecular mass around 53 in association with immunoprecipitates of the SV40 LT protein in SV40-transformed cells.

Importantly, the SV40 LT protein was already known to be a key factor in the ability of that virus to induce neoplastic transformation. What’s more, the papillomavirus E6 gene product likewise interacts with p53, as do the adenovirus E1B proteins. Furthermore, each of these viral proteins promotes transformation, and each does so by either inactivating p53 or by facilitating its degradation. Taken together, these facts strongly implied a role for p53 in transformation. See Aside 1.

[Aside 1: Our last posting featured Harald zur Hausen and his discovery that cervical cancer is caused by papillomaviruses (4). Recall that papillomavirus genomes are integrated into the cellular DNA of cervical cancer cells. Harald zur Hausen and coworkers found that while these integrated viral genomes often contain deletions, two papillomavirus genes, E6 and E7, are present and transcribed in all cervical cancer cells; a finding which implied that these viral genes act to initiate and maintain the neoplastic state. And especially germane to the current tale, Peter Howley and coworkers demonstrated that the interaction of the papillomavirus E6 gene product with p53 results in the degradation of p53.]

At the time TP53 was discovered, it was thought to act like the oncogenes carried by the retroviral RNA tumor viruses. Retrovirus oncogenes are actually captured cellular genes, which promote cancer when they are inappropriately expressed under control of viral promoter elements. However, clues eventually emerged which pointed to a very different understanding of p53’s function. The p53 protein is actually a tumor suppressor. Evidence in that regard included the mid 1980s findings of David Wolf and Varda Rotter at the Weizmann Institute, and others as well, who showed that cell lines derived from a number of sporadic (nonfamilial) cancers have TP53 genes that are dysfunctional by mutation. Importantly, it is the loss of p53 function, rather than its expression, which may lead to cancer. [Retroviral oncogenes act dominantly when introduced into non-malignant cells, whereas mutations in tumor suppressor genes are recessive to their wild-type alleles.]

Why, we ask, do the polyomaviruses, papillomaviruses, and adenoviruses inactivate p53? The answer reveals a key tumor suppressor function of p53. Basically, it is because these DNA viruses require the cellular DNA replication enzymes and substrates to support their own DNA replication. Since these cellular enzymes and substrates are available only in dividing cells, these viruses induce cells to bypass the complex circuits that regulate exit from the G0 or “resting” phase of the cell cycle. They do this by freeing the cellular E2F transcription factor from the blocking activities of the pRb family of tumor suppressor proteins, in that way enabling cells to enter into S phase. [The multifunctional SV40 LT protein, the papillomavirus E7 gene product, and the adenovirus E1A protein carry out this function for their respective viruses.] However, p53 remains as a crucial component of a cellular surveillance mechanism that prevents cells from undergoing unscheduled and potentially disastrous cell divisions. If the cell should enter an inappropriate S phase, p53 triggers apoptosis; a cell death program that can be activated by a variety of signals from within and outside the cell. [From the point of view of the host, cell suicide by p53-mediated apoptosis is preferable to the generation of rampant daughter cells that might produce full-blown tumors.] Consequently, the clever DNA tumor viruses (which comprise three unrelated virus families) undermine the normal regulatory functions of p53, as well as those of pRb. See reference 5 for details on these mechanisms.

Li, Fraumeni, and collaborator, Stephen Friend knew that they could not identify the genetic mutation underlying the Li-Fraumeni syndrome by conventional linkage analysis. That was so because Li-Fraumeni syndrome families are quite rare and, moreover, the cancer death rate among affected family members is high (nearly all individuals who carry the mutation develop cancer). So, their strategy was to investigate plausible candidate genes. They chose TP53 because, in their words: “Inactivating mutations of p53 have been associated with sporadic osteosarcomas, soft tissue sarcomas, brain tumors, leukemias, and carcinomas of the lung and breast. Together, these tumors also account for more than half of the cancers in selected series of LFS families (2).” Furthermore, evidence was emerging that TP53 actually encodes a tumor suppressor protein.

The finding by Li, Fraumeni, Friend, and their coworkers, that the TP53 mutation is present in the normal cells of Li-Fraumeni syndrome individuals, as well as in their tumor cells, proved that the mutation is passed down through the germ line. Yet these findings raise the following interesting question. If the TP53 mutation is present in all cells of an affected individual, why does that individual have “only” one or a few tumors? The reason, at least in part, is that progression to full blown cancer requires additional genetic changes. [Apropos that, Li helped discover that people with the Li-Fraumeni syndrome are particularly prone to developing additional tumors when given radiation therapy to treat their cancers.]

Li and his collaborators closed their 1990 paper as follows: “In conclusion, we have shown that alterations of the p53 gene occur not only as somatic mutations in human cancers, but also as germ line mutations in some cancer-prone families (3).” With that paper, the three researchers, and their collaborators, became the first to demonstrate a genetic condition in which a predisposition to cancer is passed from one generation to the next.

Li was born in Canton, China, in 1940. His father was a general in the Chinese Army (Kuomintang), who fought against the Japanese domination of China during the Second World War. The Li family immigrated to the United States in 1947, and opened a Chinese restaurant in White Plains, N.Y.

At 16 years of age, Li matriculated at NYU, where he majored in physics. He earned his MD from the University of Rochester.

Li joined the NCI in 1967, but spent the last 30 years of his career at the Dana-Farber Cancer Institute in Boston, where he also held appointments as a professor at Harvard Medical School and at Harvard’s School of Public Health. In 1991, he was appointed head of Dana-Farber’s Division of Cancer Epidemiology and Control. David G. Nathan, a former president of Dana-Farber, said that Li had been recruited to Dana-Farber to bring more scientific rigor to cancer research there (3). In 1996, Li was appointed to the NCI’s National Cancer Advisory Board by President Bill Clinton.

Li founded a clinic for immigrants in Boston’s Chinatown, where he frequently treated patients at night for free. He retired from his medical activities in 2008 because of dementia resulting from Alzheimer’s disease.

In July 2012, Joseph Fraumeni celebrated his 50th anniversary as a scientist at the NCI. He observed the occasion by stepping down as the NCI’s Director of the Division of Cancer Epidemiology & Genetics. He continues to serve as a senior investigator and adviser at the NCI and NIH, where his major research contributions concerned the environmental and genetic determinants of cancer. Fraumeni is an elected member of the US National Academy of Sciences.

Stephen Friend was on the Harvard Medical School faculty from 1987 until 1995, when he joined the Fred Hutchinson Cancer Research Center as chairman of Pharmacology. His research focused on genomic analysis of large patterns of gene expression. In 1997, Friend and Leroy Hood co-founded the company, Rosetta Inpharmatics, which specialized in genomic approaches to drug discovery. When Rosetta was acquired by Merck in 2001, Friend served as a Merck Senior Vice President and led the parent company’s Oncology Early Discovery and Development Divisions. Friend left Merck in 2009 to advocate for and promote open access biomedical research. Earlier, in 1986, Friend cloned the gene encoding pRB; the first tumor suppressor gene to be isolated.

References:

1. Li, F.P., and Fraumeni J.F. Jr. 1969. Soft-tissue sarcomas, breast cancer, and other neoplasms. A familial syndrome? Annals of Internal Mededicine. 71:747-752.

2. Malkin, D., F.P. Li, L.C. Strong, J.F. Fraumeni Jr, C.E. Nelson, D.H. Kim, J. Kassel, M.A. Gryka, F.Z. Bischoff, M.A. Tainsky, and S.H. Friend. 1990. Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science 250:1233-1238.

3. Grady, D., Frederick P. Li, Who Proved a Genetic Cancer Link, Dies at 75, N.Y. Times, June 21, 2015.

4. Harald zur Hausen, Papillomaviruses, and Cervical Cancer, Posted on the blog June 19, 2015.

5. Norkin, L.C. 2010. Virology: Molecular Biology and Pathogenesis, ASM Press.

Harald zur Hausen, Papillomaviruses, and Cervical Cancer

Harald zur Hausen (1936- ) was awarded a share of the 2008 Nobel Prize in Physiology or Medicine for discovering that papillomaviruses cause cervical cancer. He received the award jointly with Luc Montagnier and Françoise Barré-Sinoussi, who were given their portion for discovering HIV (1). Before getting on with zur Hausen’s story per se, we begin with bit of earlier history.

zur hausen Harald zur Hausen in 2008

Genital warts are benign epithelial tumors that have been known and associated with sexual promiscuity since the time of the ancient Greeks. In 1907 these lesions were unequivocally proven to be an infectious disease by Italian researcher, G. Ciuffo, who showed that they can be transmitted by filtered extracts of wart tissue; a finding which also implied that the etiologic agent is a virus. Ciuffo inoculated himself to advance his case.

Ciuffo’s finding is relevant to our story since members of the papillomavirus family of DNA viruses are the cause of warts. What’s more, and importantly, some papillomaviruses  also cause malignant cervical carcinomas.

In 1933 Richard Shope, at the Rockefeller Institute, became the first researcher to isolate a papillomavirus, the cottontail rabbit papillomavirus. Shope went on to show that this virus is the cause of skin papillomas in its rabbit host. This finding by Shope was the first to demonstrate that a DNA virus can be tumorigenic.

Years earlier, in 1911, Peyton Rous discovered that an RNA virus—the Rous sarcoma virus (the prototype retrovirus)—causes solid tumors in chickens. Peyton Rous was Richard Shope’s friend and colleague at the Rockefeller Institute. In 1934 Shope asked Rous to characterize the warts that the rabbit papillomavirus induces in jackrabbits. Rous found those warts to be benign tumors that could progress to malignant carcinomas.

Despite the earlier findings of Ciuffo, Shope, and others, the notion that genital warts in humans is a sexually transmitted malady was slow to gain acceptance. Oddly, perhaps, recognition of that truth was prompted by a 1954 report that American servicemen, who had been serving in Korea, were transmitting genital warts to their wives upon returning to the U.S (T. J. Barrett, et al., J. Am. Med. Assoc. 154:333, 1954). [Sexually transmitted diseases were a long-standing problem in the military. Servicemen were most often infected by sex workers who frequented the vicinity of military quarters.]

The key discoveries of this tale are Harald zur Hausen’s 1983 and 1984 findings that two human papillomavirus subtypes, HPV-16 and HPV-18, together account for about 70% of all cervical cancers. Considering that more than 120 distinct HPV subtypes have been identified, the high degree of association of cervical carcinoma with only two of these subtypes provided compelling evidence for the viral etiology of this malignancy. Later studies showed that HPV-31, HPV-33, HPV-45, HPV-52, and HPV-58 are responsible for another 20% of cervical cancers. Indeed, an HPV infection is present in virtually all cervical carcinomas. See Aside 1.

[Aside 1: Cervical cancer was once the leading cause of cancer-related deaths in women in the United States. However, the number of cervical cancer deaths in the industrialized world decreased dramatically over the last 40 years, largely because of the Pap test, which can detect pre-cancer cervical lesions in their early stages. The CDC website reports 12,109 cervical cancer cases and 4,092 deaths from cervical cancer in the U.S. in 2011 (the most recent year for which data are available). Worldwide, cervical cancer was responsible for 275,000 deaths in 2008. About 88% of these deaths were in developing countries (J. Ferlay et al., Int. J. Cancer, 127:2893, 2010).]

Harald zur Hausen was a child in Germany during the Second World War, growing up in Gelsenkirchen-Buer, which was then a center for German coal production and oil refining and, consequently, a major target for allied bombing. [The city also contained a women’s sub-camp of the Buchenwald concentration camp. The Nazis used its prisoners for slave labor.] All members of zur Hausen’s family survived the war. However, zur Hausen’s primary education contained significant gaps because schools were closed during the allied bombing (2).

Despite the gaps in zur Hausen’s early education, he was keenly interested in biology and dreamed of becoming a scientist. Yet at the University of Bonn he opted to study medicine, rather than biology. After zur Hausen received his medical degree, he worked as a medical microbiologist at the University of Düsseldorf, where he enjoyed the opportunity that the University gave him to carry out research on virus-induced chromosomal aberrations.

Although zur Hausen was fascinated by his research, he was soon aware of the deficiencies in his scientific background. So, in 1966 he looked to enhance his proficiency as a scientist by securing a postdoctoral position in the laboratories of Gertrude and Werner Henle at the Children’s Hospital of Philadelphia.

The Henles were a German-born husband and wife research team, known for their work on flu vaccines. More apropos to our story, they are also known for demonstrating the link between the recently discovered Epstein-Barr virus (EBV; a herpesvirus) and infectious mononucleosis, as well as for showing that EBV is the etiologic agent of Burkitt’s lymphoma; a cancer found in parts of Africa. EBV was, in fact, the first virus associated with a cancer in humans. [Gertrude Henle’s mother was murdered by the Nazis in 1943.]

Although zur Hausen took part in the Henles’ experiments involving EBV, he did so grudgingly because he was intimidated by his inexperience in molecular biology. In his own words: “I urged Werner Henle to permit me to work with a different agent, namely adenovirus type 12, hoping that this relatively well established system would permit me to become acquainted with molecular methods. He reluctantly agreed. I started to work eagerly on the induction of specific chromosomal aberrations in adenovirus type 12-infected human cells…and, to please my mentor, I demonstrated electron microscopically the presence of EBV particles directly in… Burkitt’s lymphoma cells (2).”

In 1969 zur Hausen returned to Germany to take an appointment as an independent scientist at the University of Wurzburg. His research was now focused entirely on EBV. Specifically, he wanted to challenge the prevailing view that Burkitt’s lymphoma tumors are persistently infected with EBV (i.e., that the tumors continuously produce low levels of the virus).

I presume that zur Hausen was interested in this issue because it was reasonable to believe that EBV gene expression is necessary to maintain the neoplastic state of the Burkitt’s tumor cells. Persistent infection would be one means by which viral genes could be carried by the cells. But zur Hausen believed that EBV DNA might be maintained in Burkitt’s lymphoma cells, even if they did not produce EBV particles.

Werner Henle in Philadelphia (and also George Klein in Stockholm) sent zur Hausen a large number of Burkitt’s lymphoma cell lines and tumor biopsies to aid in his study. One of those cell lines, the Raji line of Burkitt’s lymphoma cells, did not produce EBV particles. Nevertheless, zur Hausen isolated sufficient EBV DNA from the Raji cells to prove that multiple copies of EBV DNA were maintained in them. This was the first time that tumor virus DNA was shown to be present in malignant human cells that were not producing virus. See Aside 2.

[Aside 2: In 1968 Renato Dulbecco and co-workers were the first to discover viral DNA integrated by covalent bonds into cellular DNA (J. Sambrook et al., Proc. Natl. Acad. Sci. U S A. 60:1288, 1968). They were studying cells transformed by the polyomavirus, SV40. Integration explained how SV40 genes could be stably maintained and expressed in transformed cells, in the absence of productive infection. Integration is now recognized as a key event in cell transformation by members of several virus families, including the polyomaviruses, papillomaviruses, and the oncogenic retroviruses.

The situation in the case of EBV, a herpesvirus, is different, as herpesviruses are able to enter into a latent state in host cells. In the latent state the viral genome is maintained as an episome, and only a subset of the viral genes (i.e., those concerned with latency) are expressed. The episomal viral genome is replicated by the cellular DNA replication machinery during the cell cycle S phase, and a viral gene product, EBNA-1, ensures that viral genomes are equally partitioned between the daughter cells. In 1978 George Klein and co-workers were the first to demonstrate episomal EBV DNA in a cell line derived from a Burkitt’s lymphoma biopsy (S. Koliais et al., J. Natl. Cancer. Inst. 60:991, 1978).]

In 1972, while zur Hausen’s attention was focused on EBV and Burkitt’s lymphoma, his research direction took a providential turn that would lead to his most important discoveries. It happened as follows.

Recent seroepidemiological evidence was suggesting a link between herpes simplex virus type 2 (HSV-2), a well known genital infection, and cervical cancer. Since HSV-2, like EBV, is a herpesvirus, and since zur Hausen had already demonstrated that EBV DNA is present in Burkitt’s lymphoma tumor cells, zur Hausen believed he was well positioned to search for HSV-2 DNA in cervical cancer biopsies. However, in this instance, all his repeated attempts failed.

Harald zur Hausen then came across anecdotal reports of genital warts converting to squamous cell carcinomas. Importantly, those genital warts were known to contain typical papillomavirus particles. Taking these two points into account, zur Hausen considered the possibility that papillomaviruses, rather than herpesviruses, might be the cause of cervical carcinomas. Indeed, his initial thought was that the genital wart papillomavirus might also be the etiologic agent for cervical carcinomas.

Thus, Harald zur Hausen began his foray into papillomavirus research. His first experiments found papillomavirus particles in benign plantar (cutaneous) warts. His next experiments demonstrated that there are multiple papillomavirus subtypes. [In brief, zur Hausen used in vitro-transcribed plantar papillomavirus RNA as a hybridization probe against the DNA from various plantar and genital warts. Only some of the plantar wart DNAs, and none of the genital wart DNAs, reacted with his planter wart RNA probe. Restriction endonuclease patterns of a variety of human papillomavirus isolates confirmed that the HPVs comprise a heterogeneous virus family.]

Harald zur Hausen’s next experiments sought to detect papillomavirus DNA in cervical carcinoma biopsies. However, his initial trials in this crucial undertaking were unsuccessful.  He was using DNA from HPV-6 (isolated from a genital wart) as a hybridization probe in those failed attempts. But zur Hausen and co-workers had at hand a number of additional HPV subtypes, from which they prepared other DNA probes. DNA from HPV-11 (from a laryngeal papilloma) indeed detected papillomavirus DNA in cervical carcinomas.

In 1983, two of Zur Hausen’s former students, Mathias Dürst and Michael Boshart, using HPV-11 DNA as a probe, isolated a new HPV subtype, designated HPV-16, from a cervical carcinoma biopsy. In the following year, they isolated HPV-18 from another cervical carcinoma sample. Harald zur Hausen’s group soon determined that HPV-16 is present in about 50% of cervical cancer biopsies, while HPV-18 is present in slightly more than 20%. [The famous HeLa line of cervical cancer cells contains HPV-18 DNA.]

Additional key discoveries took place during the next several years, including the finding that papillomavirus DNA is integrated into the cellular DNA of cervical carcinoma cells. This finding clarified how papillomavirus genes persist in the cancers, and also revealed that the cancers are clonal (see Aside 2, above). Moreover, while the integrated viral genomes often contain deletions, zur Hausen’s group found that two viral genes, E6 and E7, are present and transcribed in all cervical cancer cells. This finding implied that E6 and E7 play a role in initiating and maintaining the oncogenic state. [In 1990 Peter Howley and co-workers demonstrated that the interaction of the E6 gene product with the cellular tumor suppressor protein p53 results in the degradation of p53. In 1992 Ed Harlow and coworkers showed that the E7 gene product blocks the activity of the cellular tumor suppressor protein pRb. Reference 3 details the mechanisms of papillomavirus carcinogenesis.]

The above findings led to widespread acceptance that cervical carcinoma is caused by papillomaviruses. Yet acceptance was not immediate. The prevailing belief, that herpesviruses cause cervical carcinoma, was well-entrenched and slow to fade away. It was based on the observation that many women afflicted with cervical carcinoma also had a history of genital herpes. But, individuals infected with one sexually transmitted pathogen are often infected with others as well. Apropos that, genital warts were long thought to be associated with syphilis, and later with gonorrhea. In any case, in 1995 the World Health Organization officially accepted that HPV-16 and HPV-18 are oncogenic in humans.

Harald zur Hausen was awarded one half of the 2008 Nobel Prize for Medicine or Physiology for proving that cervical cancer is caused by human papillomaviruses. By the time of his award, his findings had led to key insights into the mechanism of HPV-mediated carcinogenesis and, importantly, to the development of a vaccine to prevent cervical cancer. See Aside 3.

[Aside 3: The first generation of Gardasil, made by Merck & Co., helped to prevent cervical cancer by immunizing against HPV types 16 and 18, which are responsible for an estimated 70% of cervical cancers. Moreover it also immunized against HPV types 6 and 11, which are responsible for an estimated 90% of genital warts cases. Apropos genital warts, there are 500,000 to one million new cases of genital warts (also known as condylomata acuminate) diagnosed each year in the United States alone.

The original vaccine was approved by the USFDA on June 8, 2006. An updated version of Gardasil, Gardasil 9, protects against the HPV strains covered by the first generation of the vaccine, as well as five additional HPV strains (HPV-31, HPV-33, HPV-45, HPV-52, and HPV-58), which are responsible for another 20% of cervical cancers. Gardasil 9 was approved by the USFDA in December 2014.]

Harald zur Hausen reviewed the overall contribution of viruses to human cancer in his 2008 Nobel lecture (4). Some of his key points are as follows. HPVs were discussed above with respect to cervical carcinoma. HPVs also are associated with squamous cell carcinomas of the vagina, anus, vulva, and oropharynx. What’s more, 40% of the 26,300 cases of penile cancer reported worldwide in 2002 could be attributed to HPV infection.

Epstein-Barr virus too was discussed above. This member of the herpesvirus virus family causes nasopharyngeal carcinoma, as well as Burkitt’s lymphoma. Another herpesvirus, human herpesvirus 8, causes Kaposi’s sarcoma; the most frequent cancer affecting AIDS patients. Hepatitis B virus (HBV, a hepadnavirus), as well as hepatitis C virus (HCV, a flavivirus), causes hepatocellular carcinoma. The human T-lymphotropic virus 1 (HTLV-1), a retrovirus, induces adult T-cell leukemia. And the recently discovered Merkel cell polyomavirus (MCPyV) is responsible for Merkel cell carcinoma.

Harald zur Hausen estimated that viruses directly cause about 20% of all human cancers, and a similar percentage of all deaths due to cancer! And while 20% might seem to be a remarkably high figure for the extent of viral involvement in human cancer, zur Hausen suggests that it is actually a minimal estimate. That is so because it is difficult to determine that a particular virus is actually the cause of a cancer. Consequently, it is likely that other examples of viral involvement in human cancer will be discovered.

Harald zur Hausen gave two principal reasons for why it is difficult to establish that an infectious agent is the cause of a cancer in humans. First: “… no human cancer arises as the acute consequence of infection. The latency periods between primary infection and cancer development are frequently in the range of 15 to 40 years…” Second: “Most of the infections linked to human cancers are common in human populations; they are ubiquitous… Yet only a small proportion of infected individuals develops the respective cancer type.”

Viruses also contribute to the human cancer burden in an indirect way. For instance, HIV types 1 and 2 play an indirect role in cancer via their immunosuppressive effect, which is the reason for the extraordinarily high prevalence and aggressiveness of Kaposi’s sarcoma in AIDS patients.

Bacterial infections also contribute to the cancer burden. For example, Helicobacter pylori infections may lead to stomach cancer. What’s more, the parasites Schistosoma, Opisthorchis, and Clonorchis have been linked to rectum and bladder cancers in parts of Northern Africa and Southeast Asia, where they are prevalent.

Obviously, but important enough to state anyway, knowing that a particular cancer is caused by a particular infectious agent opens the possibility of developing a rational strategy to prevent that cancer. Gardasil is an exmple. A vaccine against HBV is also available, and one against HCV is under development.

References:

1. Who discovered HIV, Posted on the blog January 23, 2014.

2. MLA style: “Harald zur Hausen – Biographical”. Nobelprize.org. Nobel Media AB 2014. Web. 27 May 2015. <http://www.nobelprize.org/nobel_prizes/medicine/laureates/2008/hausen-bio.html&gt;

3. Norkin, Leonard C. (2010) Virology: Molecular Biology and Pathogenesis. ASM Press, Washington, D.C. See Chapters 15 and 16.

4. Zur Hausen, Harold, The search for infectious causes of human cancers: where and why. Nobel Lecture, December 7, 2008.

Felix d’Herelle, the Discovery of Bacteriophages, and Phage Therapy

Viruses infect all classes of cellular organisms. Those that infect bacteria are called bacteriophages, or phages for short. Some phages are referred to as “lytic,” since they cause the infected bacterium to lyse. In fact, the bacteriolytic effect of these phages actually led to their discovery by Frederick W. Twort in 1915, in London. Felix d’Herelle, made the discovery independently in 1917, in Paris, after he too observed bacterial lysis.

Twort noted that his “bacteriolytic agent” exhibited two of the defining properties of viruses; the ability to pass through filters that block bacteria, and the requirement for cells (bacteria in that instance) in order to proliferate. Nevertheless, whereas Twort considered the possibility that his bacteriolytic agent might be a virus, he seemed to favor the notion that it was an enzyme secreted by the bacteria.

In contrast to Twort, d’Herelle was quite certain that the phenomenon he observed was due to a virus; one able to infect bacteria. Moreover, unlike Twort, d”Herelle ardently followed up his initial observations. d’Herelle also gave bacteriophages their name; which means “bacteria eaters,” although this is not what actually happens. See Aside 1.

Felix d'Herelle
Felix d’Herelle

[Aside 1: As has happened in other instances—e.g., the discovery of HIV (1)—priority for a scientific discovery can be a matter of bitter dispute. In the case of Twort and d’Herelle, contention over priority for the discovery of bacteriophages eventually waned, as many in the scientific community came to accept that their discoveries were independent. However, the observable effect of lytic phages was reported even earlier, in 1896, by British bacteriologist Ernest Hankin, who found that an agent which lyses Vibrio cholerae can pass through filters. Two years later, the Russian bacteriologist Gamaleya noted a similar occurrence in Bacillus subtilis. Yet neither of these researchers pursued the phenomenon, which lay dormant until resurrected by Twort in 1915.]

d’Herelle’s 1917 report of his discovery contains several notable observations (2). First, he stated that the bactericidal microbe cannot be seen under his microscope: “From the feces of several patients convalescing from infection with the dysentery bacillus, as well as from the urine of another patient, I have isolated an invisible microbe endowed with an antagonistic property against the bacillus of Shiga.”

Next, d’Herelle noted that the invisible microbe can be isolated from patients only when the Shiga bacillus is present as well: “In convalescent cases…the antagonistic microbe disappears very soon after the disappearance of the pathogenic bacillus…I have never found this antagonistic microbe in…normal subjects.”

d’Herelle then asserted that the antagonistic effect of the antibacterial microbe is seen only in association with the Shiga bacillus. That is, the agent is host-specific: “I have never obtained an activity against other microbes: typhus and paratyphoid bacilli, staphylococci, and so on.”

d’Herelle went on to proclaim that the agent is indeed a “living germ.” That is, it can proliferate. Moreover, its proliferation requires the presence of the Shiga bacillus: “The antagonistic microbe can never be cultivated in media in the absence of the dysentery bacillus…This indicates that the antagonistic dysentery microbe is an obligate bacteriophage.” Note that this is the first known use of the term “bacteriophage.”

d’Herelle concluded his 1917 paper with a comment on the generality of his findings: “It is probable that the phenomenon is not restricted to dysentery, but that it is of general significance, because I have been able to observe a similar situation, even though weaker, in two cases of paratyphoid fever.” See Aside 2.

[Aside 2: Bear in mind how little was known about viruses in 1917, other than that they pass through filters that block bacteria, and that they require cells to proliferate. Our last posting featured the crystallization of tobacco mosaic virus by Wendell Stanley (3). In Stanley’s 1946 Nobel lecture, he recounted that when he began that project in 1932, “the true nature of viruses was a complete mystery. It was not known whether they were inorganic, carbohydrate, hydrocarbon, lipid, protein or organismal in nature.”]

d’Herelle’s next goal, which he pursued with a passion, was to develop therapies against bacterial infections that would be based on using bacteriolytic phages. He achieved his first success in 1919, when he stopped a typhoid plague in chickens, by means of a phage isolated from a fecal sample of a typhoid-infected chicken.

Later in that same year, d’Herelle attempted phage therapy for the first time in a human; a 12-year-old boy suffering from dysentery at the Hôpital des Enfants-Malades in Paris. d’Herelle successfully treated his young patient with a phage isolated from a fecal sample of another dysentery patient. He ingested the phage preparation himself, to test its safety, before administering it to the boy. Soon afterwards, he cured three additional dysentery patients, using the same phage preparation.

Phage therapy was soon booming, with thousands more successes being claimed. Remarkably, these successes were achieved despite the fact that neither d’Herelle, nor anyone else at the time, knew the actual nature of bacteriophages. See Aside 2 above, and Aside 3.

[Aside 3: The 1925 Pulitzer Prize-winning novel, Arrowsmith, by Sinclair Lewis, describes fictional efforts to develop phage therapy. I highly recommend it, if for no other reason than to get a glimpse of what it was like to do medical research in that earlier time. Interestingly, the idea for Arrowsmith was suggested to author Sinclair Lewis by his friend Paul de Kruif, then a scientist at the Rockefeller Institute, and later a well-known science writer, perhaps best known for his 1926 book, The Microbe Hunters.]

Some of the successes claimed by d’Herelle occurred in 1920 in Indochina, where he used phages from plague-infected rats to treat human plague patients. In 1927, in India, under the sponsorship of the British colonial government, he stopped a cholera epidemic by means of two procedures. In the first, he administered phage preparations directly to individual recipients. In the second, he seeded the wells of cholera-stricken villages with the phages. In both instances the phages were isolated from Indian cholera victims, and in both instances it was claimed that the epidemic was ended within 48 hours, whereas the conventional intervention procedures of day were said to achieve the same result only after 26 days.

Yet despite the thousands of successes claimed by d’Herelle and his adherents, many in the scientific community questioned the efficacy of phage therapy. A major reason was that d’Herelle’s clinical trials did not meet accepted standards of experimental rigor. Most importantly, he did not include appropriate placebo controls in those studies. Perhaps he did not want to deprive any of his subjects of the benefit of the treatment that he so strongly believed in. Yet he also failed to include placebo controls in his earlier studies in chickens.

Nonetheless, in the pre-antibiotic era, there were few alternative interventions against bacterial infection, other than vaccination (where apt) and raising standards of public hygiene. Consequently, many large drug firms, including American companies Eli Lilly, Parke-Davis, Abbott, and Squibb, produced phages to treat a variety of bacterial pathogens. d’Herelle himself founded a commercial laboratory in Paris to produce therapeutic phage stocks. He returned the profits from his commercial venture into the support his phage research.

Notwithstanding the often spectacular successes that could reasonably be attributed to phage therapy, it was not a panacea. For instance, the outcome of phage therapy could be inconsistent, in part because companies were producing phage stocks at a time when these agents were far from understood. Also, phages are highly host-specific. Thus, effective phage therapy depended on accurately identifying the infecting pathogen.

Interest in phage therapy waned in the United States and in Western Europe in the late 1940s. The major reason was the sudden availability of penicillin and other antibiotics, which were easier to use than phages—they could be effective even when the identity of the etiologic agent was not known—and they were more reliable as well. During the Second World War, supplies of penicillin were limited, and priority was given to the military. But, when the war ended in 1945, and the general public became aware of the new “miracle drug,” demand for it was huge and phage therapy quickly fell by the wayside.

But while antibiotics became readily available in the United States and in Western Europe after World War II, this was not the case in Eastern Europe and in the former Soviet Union, which were isolated from Western advances in antibiotic production. Consequently, interest in phage therapy remained high in the East after the war. In fact, hundreds of scientific papers concerning phage therapy were being published in Eastern Europe and in the former Soviet Union. Many of these papers were from the Eliava Institute in Tbilisi, in the former Soviet Republic of Georgia (see Aside 4). The Eliava Institute produced phages against numerous bacterial pathogens, including staphylococci, Pseudomonas, Proteus, and Shigella.

Eliava Institute Bacteriophages
Eliava Institute Bacteriophages

[Aside 4: Georgian microbiologist, Giorgi Eliava, met d’Herelle, and acquired his enthusiasm for phage therapy, while carrying out research at the Pasteur Institute in Paris between 1918 and 1921. In 1923, after Eliava returned to Georgia, he established a laboratory in Tbilisi (renamed the Eliava Institute in 1988) for the explicit purpose of researching phage therapy. In1937 Eliava invited d’Herelle to join him in Tbilisi, where the two might then collaborate. d’Herelle accepted, and after spending several months in Tbilisi, he decided to stay permanently. But, Eliava was arrested by the Soviet secret police and executed as a “People’s Enemy.” One version of events has it that Eliava was executed because he was a rival of the Soviet secret police chief, Lavrenti Beria, for the affection of a woman. d’Herelle fled from Georgia for his own life.]

The Hirszfeld Institute of Immunology and Experimental Therapy, in Wroclaw, Poland, is another Eastern European organization actively engaged in phage therapy research. Work at the Institute has largely been motivated by “the emergence of bacterial strains, which are highly resistant to virtually all available antimicrobial agents.” That quoted phrase, as well as the following, is from the Institute’s web site: “Since 1980 the specific bacteriophages have been used in our Laboratory for the treatment of over 1500 patients with suppurative (i.e. pus producing) bacterial infections, in which a routine antibiotic therapy failed. The results obtained so far showed that phage therapy is safe and highly effective (the majority of patients were cured).” See Aside 5.

[Aside 5: The Hirszfeld Institute was founded by Ludwik Hirszfeld, perhaps best known as one of the co-discoverers of the inheritance of ABO blood type. Hirszfeld was Jewish by birth, but converted to Catholicism. Nevertheless, in 1939 the Nazis removed him from his scientific posts and banished him to the Warsaw Ghetto, where he organized underground anti-epidemic measures and vaccination campaigns against typhus, and also taught secret medical classes. In 1943, a day before he was to be sent from the Ghetto to an extermination camp, he and his wife escaped and found refuge in small Polish villages, using false names. Hirszfeld resumed his scientific career after the war. Incidentally, during the war, d’Herelle was kept under house arrest by the German occupiers of Vichy, France.]

Although enthusiasm for phage therapy has remained high in Eastern Europe and in the former Soviet Union, enthusiasm there did not forestall the declining interest in phage therapy in the West. The Eastern European studies were published in Russian and, as a result, were largely unavailable to scientists in the West. Moreover, those studies often did not meet Western standards of scientific rigor, as they often lacked proper controls and they did not adequately describe experimental procedures. [See reference 4 for a detailed review and assessment of the Eastern European phage therapy trials.]

The standing of phage therapy in the West was also undermined by d’Herelle himself, who hurt his cause by vigorously advocating his abiding belief that natural recovery from bacterial infection results from the bacteriolytic action of phages. He based this assertion on his early finding that he could isolate lytic phages only from those patients who were convalescing from a bacterial infection (see above and reference 2). He also believed that long-lasting immunity is the result of the phages present in the recovered patient.

d’Herelle’s assertions concerning recovery from infection and acquired immunity were at odds with key discoveries then being made by pioneering immunologists, such as Nobel laureates Elie Metchnikoff and Paul Ehrlich, who demonstrated the importance of leukocyte recruitment and phagocytosis in recovery from bacterial infection. But d’Herelle continued to assert that neither phagocytosis nor antibodies could produce bacteriolysis, which he thought was necessary to completely destroy the bacteria. The scientific community responded to d’Herelle’s heresy as though support for phage therapy implied lack of support for key developments in immunology, serology, and vaccines.

Yet despite the scientific and social reasons for the decline of phage therapy in the West, phage therapy may yet undergo a resurgence here because of the ever increasing threat of antibiotic-resistant bacteria. For instance, the European Commission is now funding a research project, called “Phagoburn,” which involves collaboration between France, Belgium, and Switzerland, with the goal of evaluating phage therapy for the treatment of burn wounds infected with Escherichia coli and Pseudomonas aeruginosa.

Patients in the Phagoburn study will receive phage preparations made by the French company, Pherecydes Pharma, which now has more than 1,000 bactericidal phages in its collection. And while researchers in the West are eagerly awaiting results of Phagoburn, some EU citizens, who are suffering with antibiotic-resistant infections, have travelled to Georgia, to receive phage therapy there.

In March, 2014, the U.S. National Institute of Allergy and Infectious Diseases included phage therapy among the seven approaches it was considering to counter antibiotic resistance. Yet it remains to be seen whether American drug companies will invest in phage therapy. They might be hesitant because of a 2013 U.S. Supreme Court ruling that prevents patenting natural genes; a ruling that might also apply to phages isolated from nature. Obtaining a patent might also be impeded by the fact that the principle of phage therapy is now nearly a century old. What’s more, after the research, development, and patenting phases, the company must still secure approval from the U.S. Food and Drug Administration to treat humans with a self-replicating agent that has the ability to evolve.

Before moving on, somewhat more might be said regarding the promise of phage therapy in the era of antibiotic resistance. First, as stated above, the specificity of a phage for a particular host bacterium can be a disadvantage since; as a result, effective phage therapy requires accurate identification of the infecting pathogen. However, the host-specificity of phage therapy nonetheless provides a significant advantage, relative to broad-spectrum antibiotics. Phage therapy selects for resistant bacteria only among the targeted bacteria, while antibiotics select for resistant mutants of many bacterial species; not just for resistant mutants of the targeted bacteria. And, if a particular bacterium should become resistant to a particular phage, it is very much easier, and less expensive, to produce a new generation of phage mutants able to attack the phage-resistant bacterium than it is to develop new antibiotics to attack multi-drug resistant bacteria. Furthermore, antibiotics target the normal body flora, as well as infecting pathogens. Thus, antibiotics, but not therapeutic phages, may affect the overall microbial balance in a patient, which in turn may lead to serious opportunistic infections.

Interestingly, a 1945 paper in The Lancet, by F. Himmelweit, who was working at St. Mary’s Hospital in London (where penicillin was first discovered), reported that the combination of antibiotics and phage therapy gave positive outcomes in clinical trials, while greatly reducing the occurrence of penicillin-resistant bacteria. Yet this finding seems to have been completely ignored in the West, despite the fact that penicillin-resistant bacteria were emerging as soon as penicillin was put into practice.

Irrespective of the future of phage therapy in the West, Felix d’Herelle’s legacy would seem to be secure. In addition to pioneering phage therapy, he also firmed up the discovery of bacteriophages, and he was also the first to develop the plaque assay method for titrating and purifying a phage (2). Taken together, these additional achievements provided the foundation for the origin of molecular biology (5).

Remarkably, d’Herelle had only a high school education and he was self-taught in science. Yet in 1928, in recognition of his development of phage therapy, he was offered a tenured professorship by Yale Medical School, which he accepted. Earlier, in 1924, he received an honorary medical degree from the University of Leiden, as well as the Leeuwenhoek medal from the Royal Netherlands Academy of Arts and Sciences. The latter award is notable, since it is granted only once every ten years to the scientist deemed to have made the most significant contribution to microbiology during the preceding decade. It was especially meaningful to d’Herelle, since his idol, Louis Pasteur, received the same award in 1895.

In the 1960s d’Hérelle’s name appeared on a Nobel Foundation list of scientists who were deemed worthy of the Nobel Prize, but who did not receive it for one reason or another. d’Herelle is believed to have been nominated eight times for the Nobel award. So it is also remarkable that when d’Herelle died in Paris in 1949, he was virtually a forgotten man.

References:

1. Who Discovered HIV?, Posted on the blog January 23, 2014.

2. D’Herelle, F. (1917) Sur un microbe invisible antagoniste des bacilles dysenteriques (An invisible microbe that is antagonistic to the dysentery bacillus). Comptes rendus Acad. Sciences 165:373-375. [The English translation, here and above, appears in Thomas Brock’s Milestones in Microbiology, Prentice-Hall, 1961.]

3. Wendell Stanley: First to Crystallize a Virus, Posted on the blog April 23, 2015.4.

4. Sulakvelidze, A., Z. Alavidze, and J. G. Morris, Jr. (2001) Bacteriophage Therapy. Antimicrob Agents Chemother. 45: 649–659.

5. Norkin, Leonard C. (2010) Virology: Molecular Biology and Pathogenesis. ASM Press, Washington, D.C. See Chapter 1.

Wendell Stanley: First to Crystallize a Virus

In 1935 Wendell Stanley crystallized tobacco mosaic virus (TMV); an accomplishment for which he was awarded a share of the 1946 Nobel Prize in Chemistry. As a matter of history, Stanley’s Nobel award was the first ever bestowed on a virologist.

Wendel Stanley. 1946 Nobel Prize photo.
Wendel Stanley. 1946 Nobel Prize photo.

I have long considered Stanley’s achievement to be one of the most important developments in virology and, indeed, in biology in general. To appreciate why Stanley’s feat might have been so significant, we need to consider how little was known in the mid 1930s—and for the next two decades as well—about the chemical nature of genes. In fact, it was still widely assumed that genes are comprised of proteins. That was so because before James Watson and Francis Crick solved the structure of DNA in 1953, that molecule was thought to be structurally simple; rather like a starch. In contrast, proteins were structurally complex, and their wide variety seemed to provide for a virtually unlimited number of genes. But, as you might suppose, attempts to explain how proteins might be replicated led to rather unsatisfying models. Consequently, many serious biologists of the day adhered to the vitalist belief that life could not be explained by known laws of physics and chemistry.

The 1928 experiments of Frederick Griffith, involving the bacterium Diplococcus pneumoniae, provided the groundwork for later experiments by others that would cause some to consider that DNA indeed might be the genetic material. Griffith demonstrated that exposing live avirulent pneumococcal cells to an extract prepared from heat-killed virulent cells could transform the avirulent cells into virulent ones. [These experiments were part of Griffith’s efforts to create a vaccine against D. pneumoniae. He died in 1941, never knowing that his work would constitute one of the keystones of molecular biology.]

Griffith’s 1928 experiments were followed by the 1944 experiments of Oswald Avery, Colin MacLeod, and Maclyn McCarty, who identified the transforming activity in the extracts of virulent pneumococcal cells. Avery, MacLeod, and McCarty fractionated the extract into its various macromolecular constituents—protein, lipid, polysaccharide, and DNA. Next, they asked which of these fractions might have transforming activity. To the surprise of almost everyone, only the DNA fraction transformed avirulent, non-encapsulated pneumococcal cells into virulent encapsulated ones.

The remarkable findings from these transformation experiments were met with widespread skepticism. That was so because it was difficult for the classically trained geneticists of the day to accept these strange, seemingly bizarre experiments. Classical geneticists experimented by crossing organisms; not by transforming them with extracts. What’s more, they thought in terms of hereditary units called “genes,” rather than in terms of molecules of nucleic acid, or whatever other substance that genes might be comprised of. Moreover, as noted above, DNA was viewed as a rather uninteresting molecule. Thus, most biologists of the day continued to hold the view that genes are comprised of protein.

As to the state of virology in the mid 1930s, most interest in the field was concerned with medical and agricultural issues. Moreover, essentially all that was known about viruses per se was that they are smaller than bacteria and can propagate only within suitable host cells. Thus, virology had not yet advanced biological knowledge in general (see Aside 1.)

[Aside 1: Stanley relates in his 1946 Nobel lecture (1), “…when the work on viruses, which is recognized by the1946 Nobel Prize for Chemistry, was started in 1932, the true nature of viruses was a complete mystery. It was not known whether they were inorganic, carbohydrate, hydrocarbon, lipid, protein or organismal in nature. It became necessary, therefore, to conduct experiments which would yield information of a definite nature. Tobacco mosaic virus was selected for these initial experiments because it appeared to provide several unusual advantages…”]

Nonetheless, by the mid 1930s biochemists had made great strides in purifying and crystallizing proteins. [Solving the structure of proteins by crystallography was still well beyond the technology of the day.] Inspired by the success of the protein crystallographers, and encouraged by his evidence that TMV is at least partly a protein, Stanley proceeded to crystallize TMV (see Asides 2 and 3).

[Aside 2: Stanley’s evidence that viruses are comprised of protein was recounted in his Nobel lecture (1): “…in studies with pepsin it was found that this enzyme inactivated the virus only under conditions under which pepsin is active as a proteolytic agent… It was concluded in 1934 that “the virus of tobacco mosaic is a protein, or very closely associated with a protein, which may be hydrolyzed by pepsin.”’]

[Aside 3: The theme of this posting is the significance of Stanley’s feat of crystallizing tobacco mosaic virus. See Stanley’s 1946 Nobel lecture (1) for details on the heroic effort that went into that achievement.]

Importantly, and to the surprise of many, Stanley’s protein-containing TMV crystals retained the infectious activity of the actual virus! Also, it is crucially important that crystals are exquisitely pure. This key fact enabled Frederick Bawden and Norman Pirie in 1936 to demonstrate unequivocally that TMV is not a pure protein. Instead, TMV contains about 6% ribonucleic acid (RNA) (3). Consequently, whatever it is about TMV that enables it to produce copies of itself, that ability resides in its protein, or in its nucleic acid, or in a combination of its two macromolecular constituents (see Aside 4). [Aficionados might note that the ability of TMV to form crystals also implied that the virus has a regular structure.]

[Aside 4: Stanley may have initially believed that TMV is comprised entirely of protein. In his 1935 Science paper (2), he notes: “Although it is difficult, if not impossible, to obtain conclusive positive proof of the purity of a protein, there is strong evidence that the crystalline protein herein described is either pure or is a solid solution of proteins.”]

The finding that TMV is comprised of protein and RNA also gave rise to the notion that a virus is more complex than a mere chemical, even if not quite an organism. But note that Max Schlesinger in 1933 was actually the first one to find nucleic acid in a virus. Making use of new high-speed centrifuges, Schlesinger purified a bacteriophage to high purity and demonstrated that it is comprised of 50% protein and 50% DNA. However, Schlesinger did not study crystalline material, as Bawden and Pirie had done. Moreover, no one at the time knew quite what to make of Schlesinger’s findings. Consequently his work did not get as much attention as that of Bawden and Pirie.

Although Stanley’s work would eventually be recognized by the Nobel committee, when it first appeared many scientists could not accept that a crystal might actually possess a key property that we associate with life—the ability to replicate. And other researchers failed to see how an infectious mottling illness in tobacco plants could be relevant to disease in humans. [This point is reminiscent of the medical community’s disinterest in Peyton Rous’ 1911 discovery of a transmissible cancer in chickens. Medical researchers of the day could not see its relevance to malignancies in humans (4).]

Recall that many serious biologists and chemists in those earlier years still adhered to the belief that some “vital” force outside the known laws of chemistry and physics would be needed to explain the phenomenon of life. Yet if viruses are so simple that they that they could be crystallized like table salt, and still express that most fundamental property of living systems—the ability to replicate—then there might be reason to believe that the nature of biological replication indeed might be understandable in terms of conventional chemistry and physics. Moreover, note that crystallography is a very precise science. Thus, taken together, the facts that TMV could be crystallized, and yet retain biological activity, strongly implied to at least some scientists that conventional physics and chemistry would suffice to explain life.

Spurred on by this line of thought, a somewhat atypical group of investigators sought to understand the nature of genes. These researchers were atypical in that they generally had little or no knowledge of traditional genetics, or of biochemistry, or, in fact, of biology of any sorts. Many were physicists by background. But, they had a single goal in mind: to understand the physical basis of the gene. What’s more, several of these investigators recognized the advantages of focusing their research efforts on viruses.

This odd group’s interest in genes, and its focus on viruses, would lead to discoveries of singular overwhelming importance. Indeed, their research approaches and the results they generated gave rise to molecular biology. Thus, Stanley’s achievement would mark the death knell of vitalism and spur the beginning of the field of molecular biology (see Aside 5). And, when Watson and Crick solved the structure of DNA in 1953, it became clear that the expression and replication of the genetic material would be accounted for by the known laws of physics and chemistry.

[Aside 5: Physicist Max Delbruck was a key player in this atypical group of researchers, and he is recognized as one of the principal founders of the new science of molecular biology. Yet it is ironic that Delbrück was initially drawn to biology by the belief that it might reveal new concepts of physics. For more on Delbruck and the “phage group” he founded at CalTech, see reference 5.]

Wendell Stanley carried out his ground breaking research on TMV at the Rockefeller Institute (now the Rockefeller University). He passed away at a scientific conference in Salamanca, Spain in June, 1971.

References:

(1) Stanley, W.M., The isolation and properties of crystalline tobacco mosaic virus,
Nobel Lecture, December 12, 1946

(2) Stanley, W. 1935. Isolation of a crystalline protein possessing the properties of tobacco-mosaic virus. Science 81:644-645.

(3) Bawden F.C., N.W. Pirie, J.D. Bernal, and I. Fankuchen. 1936. Liquid crystalline substances from virus-infected plants. Nature 138: 1051–1055.

(4) Howard Temin: “In from the Cold,” Posted on the blog December 14, 2013.

(5) Max Delbruck, Lisa Meitner, Niels Bohr, and the Nazis, Posted on the blog November 12, 2013.