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April 29th, 2015

Ridley Scott Never Explained His Aliens

John

Are the bugs carbon-based? Do the bugs have DNA? Is their genetic code the same? How do the they generate chemical energy? Are their proteins made up of L- or D-amino acids? They never seem to eat.

In basic chemistry we have something called “chirality” which refers to a molecule with two possible non-superimposable configurations. One way to picture this is to look at your hands and place one on top of the other (not palm to palm) – your left and right hands are essentially the same shape but their shape is reversed. At the molecular level we can use one of the main building blocks of all proteins and all life – the amino acid alanine, depicted in the image below – to examine handedness.

The diagram shows the arrangement of atoms of two alanine molecules, both of which exist in nature, arranged so that they are mirror images. They are the same molecules but if you turn the one on the right around so that it is facing in the same direction as the one on the left, the R (a single carbon atom in alanine with three bonded hydrogen atoms) on this alanine molecule faces toward the palm of the hand and the COOH moiety (a carboxyl group) and the NH2 moiety (an amino group) face outward away from the palm.

No matter how you rotate the alanine on the right, you can’t get the three moieties attached to the central carbon to line up in the same position as the alanine on the left. Likewise, you can’t get those hands to super-impose each other no matter how much you twist and turn them. So the alanine on the left is called L-alanine (levo- for the direction the molecule rotates photons) and the alanine on the right is called D-alanine (dextro- for the direction the molecule rotates photons). They are called “enantiomers,” or chiral forms, of alanine, and both exist in nature with identical chemical properties except for the way that they rotate polarized light.

There are twenty natural amino acids comprising the building blocks of all proteins. Of these twenty, only glycine is symmetrical around a central carbon atom and therefore glycine has no enantiomers. The other nineteen can exist in the L- and D-conformation.

Funny thing though, only the L-enantiomer is used to make proteins by the protein synthetic machinery of all life-forms, from single-cell organisms up to humans. It’s quite easy to understand why one enantiomer is used in life over random use of either enantiomer. In explaining this, note the pictures below, which show the three-dimensional globular structure of human beta-actin on the left and, on the right, the architectural arrangement of this actin in the cytoplasm of a cell (I cloned this gene in 1982).

The protein composed of 374 amino acids has an intricate folding pattern with coils which would not be possible if both amino acid enantiomers for the nineteen amino acids were randomly incorporated into the protein. This three-dimensional structure has to be preserved in order for actin to perform its dynamic architectural function inside living cells, as shown in the picture on the right. The coils are possible because the amino acids are all L-amino acids and glycine is neutral; otherwise the protein would behave like a wet noodle. The precise structure of the actin protein determines its function, which has been preserved and conserved since the beginning of all eukaryotic life-forms (that is, cells with a cytoplasm and a nucleus). Understanding the atomic forces that fold proteins in a unique shape is part of the reason why Linus Pauling received the Nobel Prize for Chemistry in 1954.
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Aside from those who closely follow this blog, it is not well known that Linus Pauling was an avid reader of science fiction. In a 1992 interview with biographer Thomas Hager, he described his motivation to write a science fiction novel. The story line was to be the discovery of a human-like race from another planet that had evolved to use only D-amino acids (D-humans) rather than the L-isoform (L-humans). He explained that he never got around to writing this novel because the real science he was doing took all of his time.

If our L-humans met up with those D-humans, what consequences would there be? Well, what we would see in D-humans are people virtually indistinguishable from ourselves – barring, of course, the possibility that these extraterrestrials evolved out of some unearthly environmental niche. However, no mating, blood, or tissue sharing would be possible between these two races.
To explain this, consider the experience you have had when you accidently put your hand in the wrong glove. As you know, this doesn’t work well. All protein interactions and reactions catalyzed by enzymes require a direct fit to work. Substrates of enzymes have to fit precisely into the catalytic active site of the enzyme, like your hand fitting into the correct glove. Since L-humans have a different chirality from D-humans, nothing would fit or be transferrable, because of asymmetric incompatibility between L- and D- macromolecules. Even the food on our planet would not likely be nutritious for D-humans because all living things on Earth are L-organisms. In D-lifeforms, the actin coils would coil in the opposite direction and the DNA double helix would have to spiral in the opposite direction as well; otherwise the analogous D-proteins would not bind or fit on the chromosomal DNA.

It seems reasonable that D-humans might be found on other planets if you consider how life got started. By a quirk of nature on Earth, L-amino acids got a head start and self-assembled into peptides (small proteins) when this essential process for life as we know it got started. The assembly of only one enantiomer isoform into a peptide may have been favored thermodynamically over co-random assembly of L- and D-isoforms. This essential process evolved into a well-organized, membrane-protected and energy-driven protein synthetic machinery in single cell organisms like bacteria. Today, humans have essentially the same protein synthetic machinery that evolved in primordial bacteria and all life-forms on Earth have the same genetic code.
There are two essential enzymes that work together to catalyze protein synthesis in all living cells. One enzyme, called aminocacyl-tRNA synthetase, binds the amino acid to a transfer RNA molecule (there is one of these enzymes and a specific tRNA for each of the twenty amino acids). The second enzyme, peptidyl transferase, catalyzes the formation of a peptide bond linking two amino acids at the start of a chain and does this over and over again until the full length protein is synthesized and folded into its functional conformation. These two essential enzymes do not recognize the D-isoforms of the nineteen asymmetric amino acids. Thus, our chiral L-specificity has been preserved since the beginning of life.

I can’t think of any reason why the D-amino acids would not support life, but it has to be one isoform or the other, not both. Apparently Pauling felt the same way. Should it ever come to pass, D-humans will be interesting to meet and they will be equally interested to meet us, hopefully without mutual disappointment.

April 24th, 2015

Cloning of Human Beings – What Could Go Wrong?

From John

This article on human cloning has perhaps become more relevant with the success of the British series “Orphan Black” which tracks all of the problems faced by a set of clones (showing Saturday night at 9 on the BBC channel). The first chapter of this new series was shown last Saturday and can be seen for free here.

Yesterday we read in the NYTimes of a successful cloning of human embryonic stem cells by Oregon Health and Sciences University researchers led by Professor Shoukhrat Mitalipov. This accomplishment was also published yesterday in the acclaimed journal Cell. The synchronous timing of the news release and Cell article hints that a patent may have been filed a day earlier on this method of cloning.

This is the third time since 2005 that this achievement has been announced through a news release. This latest claim sounds more credible because a somatic skin cell nucleus was taken from an 8-month old infant with defined genetic markers; so the proof of this cloning will be in observing these markers as in the embryonic stem cells that are expanded in culture. My caution stems from the two previous announcements in 2005 and 2008.

In May of 2005 a Korean scientist reported a false claim of his cloning accomplishment, a claim that was later disproved as other scientists attempted to verify the Korean result.

On January 18, 2008, we learned in a news release of the successful cloning of human embryonic stem cells representing a unique human individual by scientists at the small private company, Stemagen in La Jolla California. We have not heard from Stemagen since except for a statement at the company’s website stating that it has refrained from publishing its results.

Professor Mitalipov assured readers that embryos containing a transplanted somatic nuclei would not be implanted in the uterus of a woman to grow cloned human beings but, of course, someone will eventually try this and a cloned human being will be born.

At this stage we really know nothing about the quality of life of a clone. Thousands of cloned animals have been produced but we have yet to be able to interview them. The cloned sheep, Dolly, would not speak to the press either.

In considering the prospect of cloning a human being, I believe that it is important to go beyond philosophical and religious arguments. One must build their case for or against cloning of human beings based upon science. While the odds appear to be extremely low for the near future that a human being will be born from the transfer of a somatic nucleus into a human oocyte — it is imperative that we consider the risks not only to prepare ourselves but to warn the perpetrators of the inherent dangers. Regardless of our feelings about human cloning, if a clone is born, we must treat any clone as a human being. We must greet this individual with support and empathy.

What are the actual risks faced by the cloned child due to birth by this procedure? One area that most embryologists are concerned about is the concept of reprogramming of the foreign somatic nucleus used to “fertilize” a human egg. “Somatic” refers to the terminally differentiated state of a cell in contrast to the reproductive potential of a germ cell. “Reprogramming” is a concept that I first learned during a seminar by John Gurdon, an esteemed embryologist from Oxford and recent Nobel Prize winner, at Johns Hopkins University in 1975. At the time Gurdon was conducting research on “nuclear transfer” except that he was using frog oocytes and thus his work was not considered controversial. Much of his work was dedicated to understanding how an enucleated egg could reprogram a foreign nucleus such as a skin cell or a tumor cell nucleus to produce, in his case, tadpoles. Read the rest of this entry »

March 28th, 2015

More on Graduate Studies – “Walk a Mile in My Shoes and then…”

The benefit of the college experience and then graduate experience comes from what you make of it, not where you have it. JL

from Con

It’s ironic that, of most serious (and seriously difficult and time-consuming) endeavors and achievements, obtaining certain Graduate Degrees continues to be viewed in many derogatory ways and for as many reasons as their are negative opinions. Of course, that is all they amount to: Mere Opinions, yet it seems one area where no one calls out the advice to “Walk a Mile in their shoes and then you’re Opinion mayn’t be so bloody ignorant”.

After all, among the many reasons there are so few Americans earning Graduate Degrees (cost being the primary and valid reason) seems to be purely a matter of Ability – we are guaranteed Equal Treatment under the Constitution, but as conservatives love to point out ‘Not all Americans are in fact Equal’ – I mean something different. Not everyone has the actual Ability to even compete at that level, never mind complete all the massive work required to finish and earn the actual Degree.

One aspect that does seem similar (almost Universal) among and between those striving to earn a Graduate Degree is that their (our) initial and strong desire to, say, attend Harvard or the like to earn such a degree is often NOT what happens and that what does end up happening is likely to have been for the best; that if we had gotten our wish and gone to Harvard, for many reasons things mayn’t have worked out as well.

I, too, had my sights set on attending Harvard (when first Denied Admission – it is NOT a ‘Rejection’, but a mere denial) I pled my case through their ‘second chance’ Admission Policy; in my second letter pleading that they were making a mistake and should admit me, I quoted St. Augustine: “Do not despair, one thief was saved, do not presume, one thief was damned” (regarding the two thieves crucified another side of Jesus Christ – the one ‘saved’ is purported to be the only human whom Jesus promised would reach Heaven). Still, they lacked the insight to admit me.

The point, aside from my immature attempt at sophisticated double-entendre, was that soon after I was offered a Fellowship at an excellent school for my Degree and that Fellowship included Full Tuition Remission PLUS $1,000 a month living stipend – for all 3 years of Law School. Imagine the savings; imagine how long my student loans would have taken to repay (after already borrowing for Undergraduate School).

I worked with Harvard Law Graduates at my Law Firm in Boston (at the time one of the largest with 300+ Attorneys) and though he was the nicest guy in my incoming ‘class’ (firms divide by years and thus experience), he was not magic, had obtained the very same position and, once we were all at work for a few years, Harvard and all other schools attended by fellow Attorney’s became both forgotten and relatively meaningless.

Sure, I would have loved hanging a Harvard Degree on my wall and would consider it a high achievement that could never be taken away, but practically it would have given me no other real advantage and would have sunk me far into the deep debt I’d have been forced to carry for 20 years – it would not have been worth it, most especially compared to coming out with a J.D. and NO associated Debt – THAT was the true Head Start that I reckoned a Harvard degree would give me.

I don’t speak for you, obviously, and doubt you were as selfish and childish-thinking as I when I set my sights on Harvard, but examining the true reasons for such a desire leaves me abashed at the irony of such shallowness when dealing with a supposed pre-eminent Institution and what it had to offer me. Having not performed well in High School, forced then to truly over-achieve while trying to make up for that and earn my way into the very best school I could, the focus became all about the Prestige and showing the world by that particular (and almost undeniable) accomplishment. That is not enough and thankfully it did not work out that way; if it had, I’m pretty sure things would not have worked out as well for me for years to come.

I have contempt for those who have contempt for anyone who did attend Harvard and earn an advanced degree there. It’s not something that anyone can honestly scoff at – granted that often their scoffing is about how the person ‘wears’ their achievement and not the achievement itself, but no matter how it’s analyzed, it absolutely is an incredible achievement that cannot be taken away. It’s also not for everyone, even those who can actually Do It Successfully.

That said, my Father attended Johns Hopkins for both his Undergraduate AND Medical Degree and, far more so than other institutions, Johns Hopkins for both Science and Medical Science of all forms IS the pre-eminent Institution and perhaps unique in the sense that knowledge, skills, thinking ability, etc. acquired there may not be obtained at most any similar Institute. That place is unique and those associated with it, whether as Student, Researcher, etc., frankly to acquire something unique and special. It seems the exception to my own theory of no place being truly exceptional to the exclusion of all other Institutes.

Some people do consider that anyone who earns certain Graduate Degrees and accompanying work/experience at certain high level Institutions (as widely recognized) must have a massive ego, a sense of self-importance and even a haughty view of the world (‘Internally Referenced’), yet ironically they refuse to even acknowledge the fundamental fact that it truly IS extremely difficult and most likely, that at which they like to poke fun remains something they could not achieve themselves. More irony – especially given that most such people have achieved and/or do today that which I find almost magical in it’s high difficulty. Really it’s the lack of honest and realistic credit (if they have to comment at all) that I find most hypocritical and, like myself, extremely childish.

But the main lesson I would teach my daughter or son is that the kind of almost overwhelming desire I had for years and years regarding attending Harvard did not, in the end, turn out to be nearly as important as I thought and was led to believe through various socialization, often targeted akin to Marketing or Advertising. There is much to be said for the cliche’s of “Go with the Flow” in conjunction with “Regardless of circumstances or surroundings, always do your very best and the results will reflect this, as will unlooked-for recognition.”

March 25th, 2015

In Defense of the Ph. D.

from John

I once was told by a Navajo Indian that “Around here, Ph.D. means ‘post-hole-digger’.”

Over the course of my career after receiving my Ph.D. in Biochemistry I have heard many derogatory comments about the degree. I can’t remember ever hearing something positive about getting the degree or having it. I can say unabashedly that receiving my Ph.D. was the most important experience in my adult life because everything of importance that came after came as a result of getting the degree.

There are indeed many examples of Ph.D. disappointment and tragedy. I can remember talking to a grad student in his seventh year at Johns Hopkins. I’m not even sure that he finished but after seven years he left me with the impression that he was broken in spirit. Then there was a colleague of mine who had received her Ph.D. doing research on her problem in a fairly notable lab who whined a lot about wasting four or five years getting her degree; then she went on to get an MBA. I wondered why she didn’t know what to do with either degree. Then, there was Ted Streleski in the math department at Stanford. In his 19th year as a grad student he bludgeoned to death the chairman of the math department with a hammer. He was driven temporarily insane by the inconsiderate treatment by that faculty.

What experiences earlier in life compelled me to work toward my Ph.D? I was very dyslexic. Even in college I could not read very well out loud in class and I did my best to conceal this. During the summer after 8th grade I took a course that was meant to improve my reading skills. A series of photographs of my eye movements obtained while reading revealed that my eyes moved helter-skelter around the page as I read. My mediocre performance in school was probably due largely to this problem. Nevertheless, I had some gratifying results in school. Toward the end of 7th grade, Fred Newburg and I were hauled off to a meeting with our adviser who was also our math teacher. The purpose of the meeting was to discuss her feelings that we should be doing a lot better in school according to our IQs. In the two years around that grade I probably read 60 books – history books and Hardy Boys books – which made me feel like I wasn’t a total intellectual loser. On the last day of school after I got on the bus to go home, my homeroom teacher, Mr. Nartof, jumped on the bus and came down the aisle to congratulate me personally because I had gotten high honors for the year – a complete surprise to me. This was a precious moment never to be forgotten.

Then, I took myself into 8th grade and regressed to, at best, a B-minus student. I was also very shy and remember turning down an invitation to a pretty eighth-grader’s birthday party. That year we all had to take multiple aptitude tests to see what we were good at so that we could begin planning our careers (prematurely). I was always competing with my best friend, Paul, who was straight-As and never did as well with one exception. I got 99%ile in abstract reasoning to his lowly 90%ile. After the test scores were in, the school guidance counselor came into our class to demonstrate how accurate the testing forecast our futures. Without knowing who the student was she indicated that Joan Kuchman had fine clerical skills and predicted that Joan would be a fine secretary. She described finely dressed and manicured Joan to a T. Then, she described a student that was likely a high honors student with high aptitude and great promise for the future. Boy was she disappointed when I burst her bubble.

One more anecdote. Seven years later in the fall of my senior year in college we had to take graduate record exams in our major field so I took the much feared Chemistry exam. It was almost as though the test scores were as much a measure of the quality of the faculty of the various academic departments than these test scores were for the students. Since the Chemistry Department had about 15 Dean’s list students, most of whom didn’t drink beer, I had no great expectation. But the time came when I had to meet with Chairman Dr. Draper to discuss my scores. I entered his office and sat down in front of his desk oblivious of the outcome. Then he looked at me eye-to-eye and thanked me profusely for my scores. While only a few of the conceited Dean’s List students had barely surpassed the 60th %ile nationwide, I had scored 82nd %ile. This, of course, proved that Dr. Draper’s department was academically top-notch. I recall that the English Department had one student who was average and all the rest were below the 20th %ile including my best friend Keats Friedman. From that point on Dr. Draper worked tirelessly on letters of recommendation because I had suggested that I should probably go to graduate school since I was interested in the modern genetics of DNA, RNA, and the newly discovered genetic code.

I got into a handful of grad schools (but not Harvard) and chose between the University of Pittsburgh School of Medicine which offered me a full scholarship, and Ohio State which offered me a teaching assistantship. While the Ohio State offer gave me a little more money I abhorred the idea of teaching, so I took the Pitt offer which paid for everything including $200 per month living expenses with no work obligations. Believe it or not, $200 was enough to live on in the fall of 1966 – $65 apartment rent, food, etc. And I got a $200 raise each year. Since I was in grad school I escaped the draft and Vietnam by passing a deferment exam which became unconstitutional a few months later.

After a summer roaming around Europe on my own, I started at Pitt in September 1966 and took this phase of my education very seriously for a change. For the first time I was taking courses on subjects that fascinated me. I chose to work in the lab of the chairman of the Department of Biochemistry, Kivie Moldave, in Scaife Hall, the Medical School, across the street from the football stadium. A few blocks away from the Med School was the baseball Pirate’s Forbes Field where Roberto Clemente and Willie Stargell entertained me for four years.

I basically gave Moldave no choice but to accept me into his lab because I selected no second or third option for an adviser. I was given a research problem that was a prominent issue in those years. What role did ribosomes, an organelle component of the protein synthesis machinery, play in the coupling of gene transcription with protein synthesis in E. coli? Moldave was well-known internationally for his work on the mechanism of mammalian protein synthesis but I chose to work separately on the RNA transcription-protein synthesis coupling in this bacterium being more interested in how genes were transcribed into RNA by the enzyme DNA-dependent RNA Polymerase. This was the hottest area of research back in the late 1960s with significant programs of investigation at Harvard and Stanford. For the next four years, I would envision the interactions of DNA, RNA polymerase, and ribosomes conducting RNA transcription and protein synthesis in my mind’s eye as an artist would envision an image in front of a canvas. I had embarked on the life of a monk that would span for eleven years from Pitt through Johns Hopkins to stepping onto the campus of the National Institutes of Health.

While at Pitt I was very fortunate to have as mentors two other more accessible professors, Dai Nakada who came from Brandeis, and Garrett Ihler who came from Harvard after I arrived. Ihler had been written up in the NYTimes for being the first to isolate a gene. To make a long story short it took me four and a half years to solve my problem, complete the work, and receive my Ph.D. in Biochemistry. I was given a problem to solve that was initially a black box to me. This was how I learned to do independent and creative research and gain confidence that I could actually do this. I was challenged to explain why ribosomes stimulated the rate of transcription of DNA by RNA polymerase which had been demonstrated previously in support of Gunther Stent’s hypothesis that transcription (RNA synthesis) was coupled with translation (protein synthesis). I started by teaching myself how to purify RNA polymerase and ribosomes from E. coli and isolating the DNA genome from a bacteriophage virus to use as a template for synthesis of RNA. By the summer of 1970 I had figured out that I could separate the stimulatory activity of the ribosome from the core of the ribosome by putting them in a high density cesium chloride solution and spinning the mixture in an ultracentrifuge until the ribosomal cores banded at a higher density separately from the split(off) proteins at the top of the density gradient. The split proteins were a group of acidic ribosomal proteins from the surface of the ribosome. I separated these proteins from each other by column ion-exchange chromatography and added the individual proteins to the DNA-dependent RNA polymerase reaction and found that some of these proteins stimulated RNA polymerase activity but the core of ribosomes did not.

When I mentioned to Moldave what I had found, he was disappointed because one could interpret this finding as a red herring – that the stimulatory activity of ribosomes on transcription was an in vitro artifact. Whether or not this is true, I had basically solved the problem that I was given. Shortly after I embarked on this research Moldave showed me a letter from Paul Berg at Stanford warning him not to pursue this area of research because he was doing this in his lab. We had a chuckle over this and moved forward. Berg got the Nobel Prize in 1980 for his work related to genetic engineering but he was viewed somewhat at Stanford as an ogre.

I defended my thesis in front of the professors from several departments at the Medical School. I don’t remember being very nervous because I was to talk about something that I was the foremost expert on – my own research. While standing at the front of the room getting ready to present, Professor McNary suggested that we play some squash right after I finished. We had played a lot of squash but this was his way of putting me at ease.

I went on to accept two successive postdoctoral fellowships at Johns Hopkins and received my first grant from the March of Dimes which had switched to funding research on birth defects. In 1972 I published my Ph.D. work in the top Journal of Molecular Biology and went on to publish about 70 research papers most of which were in top biomedical journals. With two papers published in Nature I was able to win a position at the National Institutes of Health working for the FDA’s Bureau of Biologics. This was a Senior Fellow position and then a career civil service position as a Research Biochemist. Later I moved away from government and became a senior scientist at the Pauling Institute in Palo Alto CA. Our invention of a powerful gene promoter was licensed by Stanford to the Biotech industry for the full 17 years of patent life. Two of our papers published in 1987 in the Proceedings of the National Academy of Sciences received >1000 citations. One night a block away from the Pauling Institute. I met Becki Calkins who asked what I did. I said that I was a “molecular biologist.” Thus, the Ph.D. paid off big time. :-)

The fact of the matter is that I would not have been able to obtain any of the jobs that I have had since 1971 or do the work that I have today if I had not first had the experience that I had working toward a Ph.D. This includes receipt of more than 10 federal and private grants. More importantly, I would not have known how to, or that I even could, conjure up and conduct the research that I completed which is still being extended today.

March 10th, 2015

Four Giant Steps for Mankind and the Importance of Gene Silencing in Drug Development

from John

During a two hour session with a Boston Globe reporter, when we were discussing the importance of litigation surrounding the ownership of the invention of “short interfering RNA” for gene silencing, I made the statement that ‘gene silencing is the most important biomedical development since gene cloning (e.g. recombinant DNA).’ I have thought about this a lot since making that statement and thought I should back this up with some facts.

“Gene cloning”, which is synonymous with “recombinant DNA”, began in the early 1970s with the discovery of restriction enzymes (restriction endonucleases like EcoR1) by Hamilton Smith at Johns Hopkins University. These enzymes cut double-stranded DNA at discrete recognition sites (short DNA sequences of four to eight nucleotide base pairs). Thus, with any particular restriction enzyme you could cut double stranded DNA into reproducible size pieces depending on the location of these restriction sites in the sequence to map the organization of the genes in a DNA molecule like, for example, the DNA genome of a virus.

I was lucky to be working as a postdoc across the street at Johns Hopkins doing experiments to see if I could find evidence that bacteriophage DNA could replicate in mammalian cells and that the bacterial gpt gene could be expressed in a mammalian cell. My answer was negative for the latter. I was interested in the work that was being done in Dan Nathans’s lab with the slicing up and splicing of the oncogenic monkey virus DNA (SV40) and was invited to talk about my relatively naive research in Nathan’s and Smith’s department in their weekly seminar series. While I had virtually nothing to contribute to their work, I sensed that I was contributing to their imagination.

While Hamilton Smith was purifying restriction enzymes, Nathans was applying these enzymes to study the structure and organization of SV40 DNA. I remember having a conversation with one of Nathans’ postdocs who told me that he was going to work at New England Biolabs near Boston. This company was founded in 1974 with the specific purpose of manufacturing and selling quality restriction enzymes. For molecular geneticists, including the founders of New England Biolabs, it was easy to predict the explosion of molecular genetics research and the advent of recombinant DNA as a research tool and an approach to developing therapeutic biomolecules. In fact, when I joined the Bureau of Biologics (FDA) in the fall of 1977, the therapeutic value of recombinant proteins like insulin had already been recognized along with the need of regulatory guidelines for this industry to avoid escape of recombinant DNA into the environment.

I can think of four developments over the last 40 years in biomedical science that stand out and have dominated medical science up to today. A fifth that might be considered is the slower emergence of stem cell research. The four developments are as follows:

  • DNA cloning, e.g. recombinant DNA;
  • Hybridoma-mediated antibody cloning, e.g. monoclonal antibodies;
  • The polymerase chain reaction, e.g. PCR;
  • Gene silencing with interfering RNA, e.g. RNAi or siRNA or microRNA.

These developments do not preclude the fact that they were built on the shoulders of hundreds of thousands of individual researchers, most notably scientists like Albert Szent-Györgyi (oxidation, energy transfer, and vitamin C; Nobel Prize in Medicine, 1937), Linus Pauling (chemical bond-mediated folding of proteins and prediction of the molecular genetic nature of sickle cell anemia; Nobel Prize in Chemistry, 1954), and Watson and Crick (structure of DNA; Nobel Prize in Medicine, 1962) just to name a few. Even my own research papers have been cited >4000 times since their publication.

The learned ability to splice and rearrange DNA molecules, which started to take place across the decade of the 1970s, led to genetic engineering of new life forms – recombinant bacteria, bacterial viruses, plants and algae, mammalian cells which produced therapeutic biomolecules as drugs and vaccines and even whole mammals.

  • In 1978 the Nobel Prize in Physiology or Medicine 1978 was awarded jointly to Daniel Nathans, Hamilton Smith, and Werner Arber “for the discovery of restriction enzymes and their application to problems of molecular genetics.”
  • In 1980 the Nobel Prize in Chemistry 1980 was awarded to Paul Berg “for his fundamental studies of the biochemistry of nucleic acids, with particular regard to recombinant DNA” culminating with the repair of a genetically defective gene in Lesch-Nyhan Syndrome fibroblast cells with the bacterial gpt gene – yes, the same gene I was examining in 1976. The difference was that Berg placed this gene next to a eukaryotic gene promoter derived from SV40 virus to express the gpt gene which was missing in Lesch-Nyhan patients. But, it was Stan Cohen and Herbert Boyer who patented the invention of genetic engineering based on experimental cloning of frog ribosomal DNA into a bacterial plasmid which was then transferred into bacterial cells and expressed in bacteria in 1973. Their work led to the founding of biotech companies such as Genentech, Inc (founded by Boyer in 1976). Their “genetic engineering patent” issued in the US in December 1980 and raised hundreds of millions in royalties for Stanford Univ. over the ensuing 17 years.
  • In 1984 the Nobel Prize in Physiology or Medicine 1984 was awarded jointly to Niels K. Jerne, Georges J.F. Köhler and César Milstein “for theories concerning the specificity in development and control of the immune system and the discovery of the principle for production of monoclonal antibodies“. But, it was Hillary Koprowski at the Wistar Institute who got the patents for application of monoclonal antibodies to treat disease like cancer. Recombinant techniques such as cloning and humanizing of the amino acid sequences of these antibodies are required to produce an effective antibody drug.
  • In 1993 the Nobel Prize in Chemistry was awarded to Kary Mullis at formerly at Cetus Corporation “for his patented invention of the polymerase chain reaction (PCR) method. This technology revolutionized molecular genetic research in the lab and became an important diagnostic tool.
  • In 2006 the Nobel Prize in Medicine was awarded jointly to Andrew Z. Fire and Craig C. Mello “for their discovery of RNA interference – gene silencing by double-stranded RNA”. But, it was Thomas Tuschl and colleagues at Max-Planck Institute in Goettingen who reduced to practice “small interfering double-stranded RNAs” for gene silencing and got the patent for this invention which was licensed exclusively to Alnylam Pharmaceuticals.

So how do we rank these developments in terms of their importance? One way to do this is to examine their direct contribution to treatment of human disease. First one must recognize these developments were all part of a sequence of advancements of knowledge in the general field of life science – each dependant on preceding break-throughs. Many aspects of drug development depend on the use of restriction enzymes to clone genes and conduct genetic experiments. Using my own experience as an example, my colleagues at Stanford and I (at the Pauling Institute) quickly cloned the human beta-actin gene by December 1982 using recombinant DNA techniques and then we developed and patented an expression vector using Paul Berg’s PSV2neo and PSV2gpt plasmids with the human beta-actin gene promoter to engineer human cells. Also, the invention of PCR revolutionized biomedical research from 1986 on so that virtually every lab began using this method to examine gene expression and its regulation or to detect viral and bacterial pathogens; but this technique was exclusively diagnostic. Luc Montagnier eventually got the Nobel Prize in Medicine in 2008 for his discovery and isolation of human immunodeficiency virus (HIV) which led to his earlier patenting of a PCR test to detect the virus in blood. So, recombinant DNA techniques and PCR were indispensable advances for the life science.

If we measure these advances by looking at drug development we can measure their impact on human well-being based on number of approved drugs and drugs in development. The first recombinant therapeutic protein approved by the FDA was insulin in 1982 three years following the filing of the Boyer-Cohen genetic engineering patent by Stanford Univ. Genentech followed with growth hormone and then the clot buster TPA in the ensuing years. There are now over 400 recombinant proteins on the market for treatment of diseases and over 900 therapeutic recombinant proteins still in development. Some classes of these recombinant proteins are listed in the table below with some of these categories overlapping.

While therapeutic monoclonal antibodies were conceived in about 1978 it took 20 years before the FDA approved Herceptin. Herceptin now generates about $7 billion in sales with the top 10 therapeutic antibodies ranging from $1 to $8 billion in revenues. There are several RNAi drugs near market (in clinical phase III development) right now. To date there are 67 therapeutic (recombinant) monoclonal antibodies on the market that have followed the launch of Herceptin in 1998 with another 510 such antibodies in clinical development. Many of these antibodies are highly specific anti-tumor drugs which have been conjugated with anti-cancer small molecule drugs for specific delivery to the tumor – a kind of double whammy therapy.

Aside from the widespread application of RNAi as a research tool, several recent market reports project the therapeutic RNAi market for gene silencing to grow to over $1 billion by 2020 (only five years from now). Since therapeutic monoclonal antibodies are, in fact, engineered recombinant proteins, the development of small interfering RNAs as drugs represents a new paradigm in drug development because synthesis of these drugs is basic nucleic acid chemistry independent of recombinant techniques although recombinant methods are routinely used to measure the effectiveness of gene silencing. It is my belief that once the first RNAi drug is approved and launched, the flood gates will open in the same fashion as the launch of Herceptin.  Part of the excitement about gene silencing is that RNAi drugs can target previously undrugable protein targets that are inside cells like heritable mutant proteins that cause genetic diseases. Indeed, Alnylam’s lead RNAi drug in phase III clinical trials targets an inherited mutant protein (transthyretin) produced primarily in the liver. This defective protein causes amyloid deposits on peripheral nerves and in the heart resulting in peripheral sensory neuropathy, autonomic neuropathy, and/or cardiomyopathy.

February 26th, 2015

The Fight Over Who Invented siRNA Used for Therapeutic Gene Silencing

from John (I’ve been following this story since the beginning of 2010)

Prequel

The Tuschl II priority application describing the invention of siRNA for therapeutic gene silencing assigned to Max-Planck Institute was filed in the European Patent Office patent application number ‘325 to establish a priority date of the December 1, 2000. This filing date indicates that Thomas Tuschl and his two colleagues, Elbashir and Lendeckel, invented siRNA before that date. The Tuschl I (a second patent family) priority date was March 30, 2000. The Tuschl I interfering RNA invention assigned to Max-Planck, Whitehead Institute, MIT, and UMass   described siRNA vaguely in comparison with the Tuschl II invention which described dsRNA molecules of precise size with the required 3’-overhanging nucleotides. Max-Planck agreed to allow one Tuschl II experiment to be placed in the specification of a Tuschl I application. It was because of negligent management of the Tuschl I invention assignment in early 2000 by the Whitehead Institute that Philip Zamore, one of the four Tuschl I inventors, managed to assign his rights to the invention to the University of Massachusetts (UMass) in April 2000. On the record in court, this mistake led Judge Saris to call the Whitehead Technology Licensing office staff “airheads” in dismissing the assignment issue because, at the time of the initiation of the lawsuit by Max-Planck and Alnylam Pharmaceuticals in 2009, the statutes of limitation had expired on this assignment issue.

The size of the siRNA molecule is important for “best mode” practice of the art of therapeutic gene silencing. Large double-stranded RNA molecules will activate innate immune mechanisms such as the induction of Interferon which will diminish therapeutic potential whereas small siRNA molecules by-pass this adverse side effect.

In 2001 Whitehead Institute requested and received approval from Max-Planck to incorporate aspects of the Tuschl II filings into the specifications of the Tuschl I filings, but not as amended claims. Apparently an understanding (outlined in a 2001 document and an Information Disclosure Statement submitted to the USPTO in 2005) was struck that the rights to the Tuschl II invention would be owned exclusively by Max-Planck. In 2005, according to the complaint filed by Max-Planck and Alnylam in federal district court on July 2, 2009, Whitehead began a secret “scheme” to obtain those Tuschl II rights, essentially by incorporating important features of the Tuschl II invention into the claims of the Tuschl I filings. Alnylam Pharmaceuticals which had been founded by the fourTuschl I inventors – Tuschl, Zamore, Sharp, and Bartel – was supposed to have exclusive rights to Tuschl I (and Tuschl II), but UMass licensed its rights to Tuschl I to Sirna Therapeutics, a direct competitor of Alnylam, in 2003. After acquiring this license Sirna Therapeutics purported licensing of Tuschl II because of the Tuschl II features inappropriately placed in Tuschl I applications. An exhibit regarding this Sirna license (from UMass), “List of Patent Rights,” identified the earlier Tuschl II ’325 priority application as the priority document of the Tuschl I patent family. In 2008, Max-Planck and Alnylam Pharmaceuticals accused Whitehead, MIT, and UMass of taking affirmative steps to effectively gain title to the Tuschl II invention and destroy the property interests of Max-Planck Society and Alnylam in those inventions. Thus, in June 2009 Max-Planck and Alnylam sued Whitehead, MIT, and UMass to win back control of both Tuschl I and Tuschl II in licensing for therapeutic uses of siRNA.

Thomas Tuschl had moved from the Whitehead Institute to Max-Planck in 1999 and teamed up with Sayda Elbashir and Winfried Lendeckel. From late 1999 to the fall of 2000, these three inventors determined that the optimal structure of exogenous interfering RNA in mammalian cells was a 21-23 nucleotide dsRNA with one or two nucleotides at each 3’-strand end unpaired (3’-overhangs) which they synthesized de novo. This is the essence of the Tuschl II invention. These three inventors published this discovery on January 15, 2001 (Elbashir et al) that was submitted for publication on October 25, 2000, and in this paper the authors coined the term “siRNA” for “short interfering RNA.” The authors described 21-23 nucleotide synthetic dsRNAs with 3′-overhangs and demonstrated efficient gene silencing in a drosophila cell lysate and later for the first time in human cells four months later in Nature (Elbashir et al) without further processing of the siRNA molecule.

Here are the authors of the Nature paper (about 10,000 reference citations to date) published on May 25, 2001, left to right: Klaus Weber, Jens Harborth, Thomas Tuschl, Winfried Lendeckel, Sayda Elbashir, and Abdulla Yalcin in the lab at Max-Planck in Goettingen.

Merck bought Sirna Therapeutics for $1.2 billion in 2006 purportedly believing (wink, wink) that it had access to the Tuschl II invention. The lawsuit filed in June 2009 was settled in March 2011 with conditions favoring the Plaintiffs, Max-Planck and Alnylam. In return Merck was given several options to pursue siRNA therapeutics. This competitive situation was ultimately resolved when Merck shut down its RNA interference program and Alnylam was allowed to purchase all of Sirna Therapeutics RNA interference IP from Merck in an amicable agreement.

UUtah Sues Max-Planck, Alnylam, Whitehead, MIT, and UMass Claiming Inventorship of siRNA for Therapeutic Gene Silencing

In the week after settlement of the lawsuit described above in March 2011, UUtah sued all the Plaintiffs and defendants of this first case. UUtah alleged that Professor Brenda Bass invented the Tuschl II subject matter before the defendants’ inventors. UUtah alleged that the defendants “misappropriated” her invention stating that the Tuschl II named inventors (Tuschl, Elbashir, and Lendeckel) intentionally claimed Dr. Bass’s invention as their own. The defendants responded that Dr. Tuschl never discussed any joint research endeavors, or even plans for joint research endeavors, at scientific meetings at Cold Spring Harbor Laboratory and in Upsala Sweden which both he and Dr. Bass attended; and in fact, Dr. Bass and Dr. Tuschl never engaged in any such joint research. By contrast, UUtah alleged that Dr. Bass communicated her alleged conception of the Tuschl II invention to Dr. Tuschl in presentations in both meetings.

Dr. Bass had identified a gene “K12H4.8” in C. elegans (a nematode) that was known to produce an enzyme of a type known as “RNase III” colloquially known as “Dicer”. Unlike the Tuschl II inventors, Dr. Bass knew that Dicer cleaved dsRNA into 21-23 nucleotide pieces. As early as 1993, Dr. Bass understood that Dicer cleaved longer strands of dsRNA into short dsRNA causing staggered cuts that leave 3’ overhangs of about two nucleotides in length well before the RNAi phenomenon (gene silencing) was demonstrated. When RNA interference (RNAi) was first reported in 1998, the discoverers (Nobelists Andrew Fire at Stanford and Craig Mellow at UMass) were unable to describe the mechanism by which it functioned in a cell. When she learned of the RNAi discovery, however, Dr. Bass recognized that Dicer was likely to be involved. In 1998, she began developing experiments to test this conception that was ultimately carried out in her laboratory beginning in 1999. These successful experiments, completed in May 2000, reduced to practice Dr. Bass’s conception that Dicer was responsible for catalyzing RNAi; therefore, she concluded that short interfering dsRNA of about 21-23 nucleotides in length with 3’ overhangs were the mediators of RNAi in living organisms, and these short dsRNAs could be used to accomplish RNAi as a treatment for disease. This, later statement was not actually made in Dr. Bass’ insightful April 28, 2000 mini-review in Cell time.

The Tuschl I patents presented an experiment that indicated that the short fragments were less active than larger dsRNA leaving open the discovery of the best mode for siRNA activity. The Tuschl I paper published on March 31, 2000, suggested that one could affect RNAi by using the short dsRNAs but this paper makes no mention of 3’ overhangs or the length of the overhangs. About that time Dr. Tuschl and the other authors admitted in email communications that they did not know how the short 21-23 nucleotide dsRNAs were produced in the drosophila lysate, and that they did not know the structure of the short dsRNA fragments. Upon reading a pre-publication version of this paper in early March (published on March 31, 2000), Dr. Bass recognized that the K12H4.8 gene she had identified had all the properties necessary for RNAi, and that the resulting short dsRNA fragments would have the 3’ overhang. On March 21, 2000, she began drafting her mini-review, titled “Double-Stranded RNA as a Template for Gene Silencing.” Her draft included information about the Dicer enzyme that cleaves dsRNA into short 21-23 nucleotide dsRNAs with 3’ overhangs.

It has never been clear to me that Dr. Bass ever reduced to practice short siRNA-mediated gene silencing although public speculation can be a problem in prior art. She knew very well what the end-product of an RNase III reaction was, but not what the active best-mode structure of siRNA was for achieving therapeutic intracellular RNA interference. In addition, there was the first-time demonstration in the May 2001 Nature paper that synthetic siRNA worked in human cells using Weber’s and Harroth’s elegant (though non-inventive) indirect immunofluorescence assay (Klaus’ contribution of this assay was communicated to me directly with the added note that they repeated the assay over and over again because he was astonished that gene silencing actually worked in living human cells). It seems to me that if Dr. Bass’ description in 2000 was persuasive and enabling, either Dr. Zamore or Dr. Tuschl or someone else who read her paper or heard her talks would have reduced the invention to practice within a month. Klaus Weber mentioned this later in a videotaped interview of him and Thomas Tuschl. In support of this concern there is that pesky patent Figure 12 in the Tuschl I application that indicated that short RNAi duplexes were far less effective in RNAi than the larger duplexes.

Where the UUtah Case Stands Now

This is Moakley Federal Courthouse in Boston where the case is being litigated. If UUtah and Dr. Bass win this case you can imagine that hundreds of millions in royalties might be claimed (royalties previously earned by the four University assignees). Although this UUtah case was initiated in March of 2011, proceedings have been delayed by multiple amended complaints and jurisdiction appeals. On February 4, 2015, the court reconvened to hear arguments for and against release of 18 “banker boxes” containing 20,000 pages of hard-copy documents to the Defense. UUtah believes that these documents are not responsive to the Defense’s requests for evidence and are not relevant to this case. In making their argument the Defense stated “That’s a small amount of documents – and the burden in producing them – is nothing compared to the hundreds of millions of dollars that they’re seeking in this case.” Apparently the Plaintiff lawyers have reviewed each of these documents. These documents include lab notebooks, descriptions of experimental protocols, recording of ideas for experiments, and possibly commentary about the Tuschl inventions mostly written down after the filing dates of the Tuschl patents in 2000 up to March 22, 2011. Early in the first case UUtah learned that the Defense had revealed a theory that Dr. Bass was the actual inventor of siRNA-mediated gene silencing and proposed a deposition of Dr. Bass.

The Defense would like to focus on whether Dr. Bass ever did any experiments with short siRNAs of the type produced by Dicer cleavage or described in the Tuschl II invention. The Tuschl II patents now number 12 issued patents and over 500 claims. The Defense points out that most, if not all, of Dr. Bass’ experimental research on gene silencing was with large dsRNAs of about 500 base pairs in the nematode model. The Defense asks the question “why did she wait for 10 years before she came forward as the purported inventor?”… and she never filed a patent on her concept of the invention – e.g. preparation and use of siRNAs. The Defense wants to know how Dr. Bass was characterizing the discovery of therapeutic siRNA right up to the filing of its lawsuit.

The Plaintiff’s lawyers insist that they have produced all documents relevant to “short double-stranded RNA molecules”. They pointed out that the reason Dr. Bass did most of her research with long double stranded RNA molecules which in theory become Dicer substrates was because her research was conducted in C. elegans which lacked the innate immune response of mammalian cells. The Plaintiffs further pointed to Bass’ 2000 mini-review that described the action of Dicer to produce the active siRNA entities. UUtah’s lawyers assert that the description of siRNA in the mini-review entitles Dr. Bass to become an inventor. The Defense was offered an opportunity to visit UUtah and review the 20,000 pages of documents on December 2nd, but the Defense elected not to take this opportunity asserting that the Plaintiff had already pre-determined that these documents were irrelevant based on their own criteria and therefore would not turn them over.

At the end of this hearing the Defense revealed that Dr. Bass was to be deposed the following Friday (February 7th) . We shall see how this case proceeds.

February 7th, 2015

Holmeslea Gardens is Where We Will Buy Our Veggies This Coming Season

From John

Holmeslea Gardens website

Click to enlarge

January 22nd, 2015

Rowayton, My Home Town

From John

I lived my first 18 years in Rowayton CT. I’ve attached below an inspiring picture of Tavern Island. This is the harbor and open waters where I took sailing lessons for five years.

In the Picture:

dark green arrow = Hickory Bluff dock, beach, and store.

dark blue arrow = Tavern Island custodian house and dock.

purple arrow = Our house and beach in the 1960s.

red arrow = Shelly T. and the Trubowitz house with beach and canoe.

gray arrow = Fashionable Thomas School for girls where Diane went along with other Rowayton girls.

green arrow = Wilson Point beach where I was lifeguard/beach boy/tennis teacher for two years.

light blue arrow = Where I capsized in Shelly’s canoe in March of 1964 and had to swim the canoe with the current and the wind to the Wilson Point Beach…burrr.

pink arrow= Bell(e) Island where Margo, Laurie, Pam, Bronwyn, Roussie, Linda, and my Aunt and Uncle Charlie and Kate Cornbrooks with Suzie and Nancy lived. No wonder the Island was often called Belle Island.

orange arrow = Bayley Beach around the corner from Bell(e) Island and Roton Point. I spent almost every summer day there playing tennis in the summers. Ran the beach concession for two summers.

source: http://www.norwalkcitizenonline.com/news/article/Island-oasis-for-sale-in-Norwalk-3974742.php#photo-3633268 

also see my brother Peter’s story about lobstering at Tavern Island at http://rowaytonkids.com/

From our terrace looking at Tavern Island at dusk.

January 10th, 2015

Abortion as an Expression of Religious Freedom

From Diane

They say, “Be careful what you wish for. You might get it.” Welcome to the 114th Congress.

First, for those not conversant with our US Constitution, when the First Amendment uses the phrase “an establishment of religion,” it is not talking about building a brick and mortar church. The phrase applies to ideas, some codified in named religions, some promulgated by philosophical constructs, and
some simply held by ordinary people as deep, personal convictions in how Life and the Universe work. However these beliefs come to exist, they, and the people who hold them, are protected by our US Constitution from laws that would prohibit “the free exercise” of those beliefs.

I am amazed at the arrogance and hypocrisy shown when one simultaneously says, “We must follow the Constitution” and then turns right around and says, “Except when it come to MY beliefs. I am RIGHT so MY beliefs trump everyone else’s, never mind that the Constitution expressly forbids me imposing MY beliefs on you.”

So what do the new Republicans in Congress do on their very first day of office? They re-introduce as HR 36 the Pain Capable Unborn Child Protection Act that would criminalize abortions after 20 weeks of “probable” gestation. [BTW, note that the law includes this definition: “UNBORN CHILD- The term `unborn child' means an individual organism of the species homo sapiens, beginning at fertilization, until the point of being born alive as defined in section 8(b) of
title 1.” This definition is completely religious with no scientific basis whatsoever yet this proposed legislation includes this definition as fact.] The idea that a 20-week old embryo “feels” pain has been discredited by many scientists as well as both the AMA and the British Royal College of Obstetricians and Gynecologists. It would seem that what the conservative religious crowd BELIEVES trumps not only what you and I might believe but the
predominant scientific point of view as well. Are there scientists who state that a 20-week old embryo can feel pain? Yes. Are they in the majority? Do they
have peer-reviewed studies to back them up? No.

I can sympathize with those who oppose abortion. If your religion teaches that at the moment of conception, a one-celled zygote is a Human Being, an unborn Child, then clearly, for you and your religion, the premeditated “killing” of that zygote is first degree murder. However, the key phrase here is, “if your
religion.” Not all religions teach that a zygote is a Human Being. There are many different beliefs concerning when an embryo or a fetus becomes a “human
being,” none of which are allowed to be the basis of a law. Our Constitution CLEARLY states that Congress (and by amendment the States) shall not make laws based on religious beliefs. So although YOU sincerely believe that a zygote is a human being, UNDER THE CONSTITUTION you may not impose that belief through law on the rest of us who do not hold your beliefs. It does not matter if you “know” you are right. ALL religions “know” they and they alone are right.

What matters is that you cannot PROVE your beliefs so you take them on faith, the very definition of a religion.

January 7th, 2015

My Life With Influenza

From John

At a regular doctor’s appointment in the fall of 2012, I was approached by a third year medical student who was interning to learn private practice. He asked me if I would like to have the influenza vaccine and said “It’s got three antibodies!” I said no thanks thinking, this guy is going to be a doctor soon and he has no idea what a vaccine is. I think of that experience when I hear someone in a health profession tout the merits of getting the flu vaccine which happens ad nauseam every fall and winter. Scary articles are also published in newspapers by naive reporters predicting the worst for humankind each winter.

My first memory of the scourge of influenza was when I was in high school and my mother took me and my siblings (minus Andy) to Uncle Charlie’s office in Darien to get flu shots. Since I was significantly older and more mature, I was elected to go first to demonstrate that getting a flu shot wasn’t all that bad. Well, Charlie must have hit a nerve because I was racked with pain which shot up my arm and caused me to writhe in agony as I staggered out of Charlie’s office into the waiting room. I remember the horrified look on brother Peter’s face as he Phoebe and David prepared for the worst. It was obvious that I was not faking it ensuring that the experience would be most horrific for my younger siblings :-) .

I never got a flu shot again and have somehow made it to 70. Severe influenza should not be underplayed. I remember the two times I had it in my life – in January 1986 when I huddled in my one room apartment in Palo Alto, and then again in January of 2001 when both Becki and I were flat on our backs together. This disease can be horrible with a fever, extreme muscle soreness, and a hacking cough because our T-cell-mediated immune response in the lungs kills a lot of infected cells. When we were lying there, we both wondered if we were going to survive; so I gave Becki the upside that the virus was killing off cancerous and precancerous cells preferentially as it migrated through our bodies during the course of the infection. This testimony is the evidence that we survived to trudge on through life’s adversities.

I don’t mean to downplay the importance of vaccines like the polio vaccine, the smallpox vaccine, the diphtheria vaccine, and many others which have nearly wiped out these crippling or deadly diseases sparing many human lives. A paper published in November in the New England Journal of Medicine concluded that modern vaccines have prevented more than 100 million cases of severe disease (van Panhuis et al., N. Eng. J. Med. 269(22):2152-2158, 2013) caused by 12 major disease pathogens.

I know something about how these vaccines work having been hired by the FDA division that regulates government licensed vaccines. I also had learned how to grow the virus that causes diphtheria (a lysogenic bacteriophage) and how that disease works in Papenheimer’s lab at Harvard in 1974. I started to pay close attention to flu vaccine campaigns in 1976 when I was at Johns Hopkins in the fall of 1976 after the head of the CDC warned President Ford that the swine flu was coming (the same flu as the Spanish Flu that killed millions in the first quarter of the 20th century). Worried doctors at the hospital trekked across North Wolfe Street to warn us that we all had to get immunized or else… I remember thinking “me thinks not.” But my life changed dramatically for the better because of this national emergency. I had been hovering over the National Institutes of Health in Bethesda MD hoping to latch onto a job, preferably at the Bureau of Biologics which was the Division of the FDA that regulated vaccines and blood products. Then, the CDC’s concern was shared with Congress and, in their imminent wisdom, they voted to give the Bureau of Biologics extra funding to hire people to help deal with the impending swine flu crisis. The Bureau inaugurated a new class of temporary three-five year positions for Staff Fellows and guess who was the first to be hired – moi, as a Senior Staff Fellow no less. This was a wonderful break for me.

The epidemic never happened because the virus never showed up but some vaccinated people developed paralytic Guillain-Barre syndrome…and the head of the CDC was fired for embarrassing President Ford who had gone on nationwide TV to promote vaccination (Presidents giving health advice is a bad idea. Obama should have left promotion of Affordable Healthcare to the Head of NIH or the CDC :-) ). I first learned of this side-effect when I was invited to dinner at a local family’s house in Maryland. At the dinner table the patriarch of the family asked me where I worked. I described the Bureau as the division that regulated vaccines. He became very angry and described his debilitating Guillain-Barre syndrome in his vaccinated arm.

Early on I learned a lot about these viruses and how our immune system works with hands-on research. I persuaded my brother Andy to join me at the Bureau to help me with some experiments to show that we could type flu viruses by the electrophoretic properties flu virus proteins since there was some confusion about the serological identity of the vaccine H1N1 strain (“H1” stands for the haemagluttinin serotype and “N1”stands for the neurominidase serotype, the two surface antigens of the virus that can produce a humoral antibody immune response). In our study we included a strain of the invading H1N1 flu virus, the horrific Russian flu, that was isolated in mainland China that year which was going to wipe us out. Read the rest of this entry »

January 3rd, 2015

Remembering What It Was Like Living in Palo Alto CA

from John

Things were going quite well in my career in 1981 with a tenured civil service position with the FDA at the National Institutes of Health in Bethesda MD but for some reason I felt the need to move on. Twenty-two years ago on January 1, 1982, I loaded up my VW Rabbit with belongings, four immunized mice in a cage, and my liquid nitrogen tank containing all my frozen cell stocks and headed west from Marriottsville MD.

I had spent the previous month in Palo Alto scrutinizing my decision to leave the government, but returned before Christmas to submit my resignation. My colleagues at NIH said I was crazy to shed government security for a high risk position as a senior scientist at the Pauling Institute in Palo Alto which depended to some degree in me winning federal grants to fund my research. I was armed with a novel discovery and wanted to collaborate with a group at Stanford to get the jump on a possible competitor in Japan. My risk turned out well in retrospect. Moving to Palo Alto was like starting a new life on all fronts. No need to talk about the science side because that is a matter of record. This is more about the environment I created for myself in a delightful place to live.

(Click to enlarge map) So I embarked on that cold day, headed west on Route 70, then south at Hagerstown on Route 81 down the Shenandoah Valley to Tennessee and stopped in eastern TN for the night, don’t remember where. In the morning I got on Route 40 near Knoxville and made it to Henryetta OK by evening, a lonely night. The next morning I continued on Route 40 past Albuquerque. I remember looking in my rear view mirror to see the vast lights of Albuquerque on the western slope of the mountain range. I stopped at Grants NM on Route 40 where unknown to me, Becki had lived in a van and caught rattlesnakes for the local college. I was so eager to get to California that the following day I left Grants and drove 19 hours straight through a driving el nino rain storm to Palo Alto ending the trip at 4 AM in the morning in the rear parking lot of the Pauling Institute which was drenched and covered with tree debris. That winter was the wettest of the century for that area, but rainy days always included sections of blue sky in the distance. The hills to the west of Palo Alto were a verdant green, a shade of green that you never saw in the east. The summers were opposite as the open fields surrounding Palo Alto and Stanford turned golden because there was no rain from April to October or November. Except for unusual years the temperature rarely went below 50 degrees and summers were usually in the low 80s and a dry heat. In the Bay Area spring came around the third or fourth week of January with the yellow flowering of the Acacia trees and the popping of bulbs.

I had moved to a heavenly place and never looked back because I was challenged with survival but I survived because of momentum I quickly built through my research as a cell and molecular biologist. I quickly found Larry Kedes lab at Stanford, visited them, and told them that I needed their help in cloning a family of human genes. This was early in gene cloning, especially cloning of human genes. This collaboration moved forward rapidly and by late December I had cloned the human beta-actin gene although it took the better part of 1983 to prove it. At this point I had the ammunition to fund my lab and hire staff, but this story is not about the science.

I found a great coffee house/bookstore, Printers’ Inc. a few blocks away from the Institute which opened a 10 AM, so I would walk from my apartment in central Palo Alto to the coffee house arriving when it opened. There I would read the paper and gather my thoughts for the day. This is where I drafted grants and research papers for publication. I was surrounded by interesting people – writers, composers, and an ocassional Nobel Prize winner like Linus Pauling who I would address as Dr. Pauling. This where I started my coffee habit as well.

Palm Drive, the main entrance to Stanford, was only a few blocks away and I had the run of the campus using the Lane Medical Library and the university’s fields for our softball team, the Pauling Squeeze, which played various Stanford departments. There was also Stanford football, basketball, and top-notch college tennis to attend. One year my eldest daughter, Elizabeth (first marriage), visited and attended a tennis camp held on the campus by the tennis coach. Life was simple in that I walked or biked to work seven days a week working from about 11 AM to 8 or 9 PM each day. My apartment in the early days was a $200 a month single room with four closets – one for a fridge, one for clothes, one for a sink and shower, and one for a toilet. Walking through the neighborhoods was a delightful experience and I marveled that the owners usually had to have a 1st, 2nd, and 3rd mortgage in order to own, betting on their start-up company stock options. I ended up with one too. This was how scientists in the Silicon Valley got rich…that is if the business succeeded.

This was my playground (below). University Ave is the main street of downtown Palo Alto which crosses El Camino Real to Palm Drive, the main entrance to the campus. My small apartment was on Byron. Becki and I met one night at the Class Reunion a block way from the Pauling Institute. We ended up renting two side-by-side houses at the corner of Maureen and Rambow off of Cowper Ave. Mariah was born in October 1988 and we walked her to school a few blocks on East Meadow. The yellow lines are the routes I biked or walked from 1983 through 1995. The area encircled by the purple lines is Stanford Research Park which housed companies like Varian, Wall Street Journal, Xerox, Syntax (first birth control), Alza Corp, Roche, and many other notable companies. Eventually we were joined by Christina, Drew and Elizabeth who visited annually.

December 24th, 2014

Five Years Ago – Eleven Inches at Our House in North Woodstock

GOT PICTS? send them to woodstockctcafe@gmail.com.

We measured 11 inches. Below (1) our house, (2) John wrestling with the snow blower, and Becki pausing from shovelling on Route 197.

house-2.jpgsnow-blower-2.jpgbecki.jpg

December 7th, 2014

New Cell Towers to be Placed at the Fairgrounds

Formerly a Student: I just skimmed the proposal. The “new cell towers being placed” in the title of this article is misleading. To summarize, they’ll be replacing a few wood utility poles in the fairgrounds with steel ones “of equal height,” and adding a battery box and extra antenna (which itself looks like a box) to the poles. The existing antennas will be moved to the top of the poles. No extra access roads, no wetland disturbance, no ground disturbance except for the actual digging of the pole, little aesthetic change, especially from a distance (169).

Sounds good to me on the surface from how they’re presenting the info. But what makes me leery is that they propose that the new antennas will initially be 700 MHz (at 500 mW power). So do they plan on adding more antennas at different frequencies? Adding more poles in the future?

However, the other frequencies only go up — to 2100 MHz. Higher frequencies are lower range, so higher power, so they’re starting off with the best range. For comparison, the Wifi in your house is 2400 or, lately, 5000 MHz frequency but only 70 mW power. Lower MHz travel farther but have more interference (think AM vs. FM radio). Speaking of radio, think of how strong those signals are, compared to the cell towers, which are everywhere and thus have lower range.

Not sure how the numbers play in to health issues, but they say these things are mainly intended for the fairgrounds, and those are usually vacant. It doesn’t seem like the signal will be excessively strong outside the grounds (have a look at the coverage maps).

But again, all this is coming from the big business trying to weasel its way in, so take it with a grain of salt.

PETITION NO. 1119 - Cellco Partnership d/b/a Verizon Wireless petition for a declaratory ruling that no Certificate of Environmental Compatibility and Public Need is required for the proposed construction, maintenance, and operation of three pole-mounted small cell telecommunications facilities on the Woodstock Fairgrounds property located at 281 Route 169, Woodstock, Connecticut.   Field Review.

November 29th, 2014

Woodstock Academy Football 1 and 32 – September 2012 to November 2014 – Subtle Signs of Improvement

Woodstock football is 1-32 since two years and 3 months ago with this week’s loss of 8-41 to 9-1 Windham.

“Several years ago the Academy chose to invest in football with a new stadium and other unneeded athletic fields rather than invest in enhanced academic programs that would help students to develop professional careers.”

The average game score this year is 9 for WA vs 39 for the opponents compared to 7 for WA vs 42 for the opponents last year, and 5 for WA vs 43 for the opponents.

This year the Woodstock Academy football team ranks 156 out of 161 CT high school varsity football teams. The results for this season are:

Windham (9-1) – 41 Woodstock (1-10) – 8

Plainfield (9-1) – 47 Woodstock (1-9) – 7

Woodstock Academy  -   8  to Norwich Free Academy – 44

Woodstock Academy  – 3 to Waterford  – 35

Vinal RVT/East Hampton/Goodwin RVT -  16 to Woodstock Academy  -  13

Griswold   – 34 to Woodstock Academy  – 0

New London – 51 to Woodstock Academy  -  6

Woodstock Academy  – 0 to Montville  – 40

St. Bernard-Norwich RVT – 14 to Woodstock Academy  -  35

Woodstock Academy  – 7 to Bacon Academy -  52

Stonington – 48 to Woodstock Academy  – 7

“Headmaster Foye said that a successful football program would produce college scholarships for Woodstock Academy senior football players” Cafe, July 28, 2008. HERE

“The misguided priorities of the Academy leadership and its ‘track record’ does not bode well for the long term viability of its students’ futures.” Cafe, May 26th, 2008. HERE

By the way, I enjoy football and faithfully support losing programs like the ones at Norwalk High (late 50s to 1960; see the heroic story of Jerry Fishman), Bethany College (1961-1966), the University of Pittsburgh (1966-1972, 16-56)  just before Dan Marino and Tony Dorsett, and currently UConn at 2-7.  I sat virtually alone in driving rain in 1968 watching the lowly Pitt team come back from a 34-0 halftime deficit to beat West Virginia 35-34, the best game I ever saw.

Several years ago the Academy chose to invest in football with a new stadium and other unneeded athletic fields rather than invest in enhanced academic programs that would help students to develop professional careers. This was due to the low-level visionary thinking of Headmaster Foye and the Academy Board of Trustees.

November 26th, 2014

The Latest Developments with Ebola

From John

Yesterday I was interviewed by the Des Moines Register about Merck’s acquisition of NewLink’s Ebola vaccine. This graph shows that there is something different about this Ebola outbreak compared to previous outbreaks. A little bit of immunity caused by the vaccine should have a big impact on the spread of the disease. These numbers provided by the CDC are actual confirmed cases and deaths caused by Ebola. They under report the actual cases and deaths.

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