Paring pair frequencies pares virus aggressiveness

Okay, a bit late with this as it came out in Science about a month ago, but it's a cool paper & illustrates a number of issues I've dealt with at my current shop.. Also, in the small world department one of the co-author's was my eldest brother's roommate at one point.

The genetic code uses 64 codons to code for 21 different symbols -- 20 amino acids plus stop. Early on this was recognized as implying that either (a) some codons are simply not used or (b) many symbols have multiple, synonymous codons, which turns out to be the case (except in a few species, such as Micrococcus luteus, which have lost the ability to translate certain codons).

Early on in the sequencing era (certainly before I jumped in) it was noted that not all synonymous codons are used equally. These patterns, or codon bias, were specific to specific taxa. The codon usage of Escherichia is different from that of Streptomyces. Furthermore, it was noted that there is a signal in pairs of successive codons; that is that the frequency of a given codon pair is often not simply the product of the two codon's individual frequencies. This was (and is) one of the key signals which gene finding programs use to hunt for coding regions in novel DNA sequences.

Codon bias can be mild or it can be severe. Earlier this year I found myself staring at a starkly simple codon usage pattern: C or G in the 3rd position. In many cases the C+G codons for an amino acid had >95% of the usage. For both building & sequencing genes this has a nasty side-effect: the genes are very GC rich, which is not good (higher melting temp, all sorts of secondary structure options, etc).

Another key discovery is that codon usage often correlates with protein abundance; the most abundant proteins show the greatest hewing to the species-specific codon bias pattern. It further turned out that highly used codons tend to be most abundant in the cell, suggesting that frequent codons optimize expression. Furthermore, it could be shown that in many cases rare codons could interfere with translation. Hence, if you take a gene from organism X and try to express it in E.coli, it would frequently translate poorly unless you recoded the rare codons out of it. Alternatively, expressing additional copies of the tRNAs matching rare codons could also boost expression.

Now, in the highly competitive world of gene synthesis this was (and is) viewed as a selling point: building a gene is better than copying it as it can be optimized for expression. Various algorithms for optimization exist. For example, one company optimizes for dicodons. Many favor the most common codons and use the remainder only to avoid undesired sequences. Locally we use codons with a probability proportional to their usage (after zeroing out the 'rare' codons). Which algorithm is best? Of course, I'm not impartial, but the real truth is there isn't any systematic comparison out there, nor is there likely to be one given the difficulty of doing the experiment well and the lack of excitement in the subject.

Besides the rarity of codons affecting translation levels, how else might synonymous codons not be synonymous? The most obvious is that synonymous codons may sometimes have other signals layered on them -- that 'free' nucleotide may be fixed for some other reason. A more striking example, oft postulated but difficult to prove, is that rare codons (especially clusters of them) may be important for slowing the ribosome down and giving the protein a chance to fold. In one striking example, changing a synonymous codon can change the substrate specificity of a protein.

What came out in Science is using codon rewriting, enabled by synthetic biology, on a grand scale. Live virus vaccines are just that: live, but attenuated, versions of the real thing. They have a number of advantages (such as being able to jump from one vaccinated person to an unvaccinated one), but the catch is that attenuation is due to a small number of mutations. Should these mutations revert, pathogenicity is restored. So, if there was a way to make a large number of mutations of small effect in a virus, then the probability of reversion would be low but the sum of all those small changes would be attenuation of the virus. And that's what the Science authors have done.

Taking poliovirus they have recoded the protein coding regions to emphasize rare (in human) codon pairs (PV-Min). They did this while preserving certain other known key features, such as secondary structures and overall folding energy. A second mutant was made that emphasized very common codon pairs (PV-Max). In both cases, more than 500 synonymous mutations were made relative to wild polio. Two further viruses were built by subcloning pieces of the synthetic viruses into a wildtype background.

Did this really do anything? Well, their PV-Max had similar in vitro characteristics to wild virus, whereas PV-Min was quite docile, failing to make plaques or kill cells. Indeed, it couldn't be cultured in cells.

The part-Min part wt chimaeras also showed severe defects and some also couldn't be propagated as viruses. However, one containing two segments of engineered low-frequency codon pairs, called PV-MinXY, could but was greatly attenuated. While its ability to make virions was slightly attenuated (perhaps one tenth the number), more strikingly about 100X the number of virions was required for a successful infection. Repeated passaging of PV-MinXY and another chimaera failed to alter the infectivity of the viruses; the attenuation stability through a plethora of small mutations strategy appears to work.

When my company was trying to sell customers on the value of codon optimization, one frustration for me as a scientist was the paucity of really good studies showing how big an effect it could have. Most studies in the field are poorly done with too few controls and only a protein or two. Clearly there is a signal, but it was always hard to really say "yes, it can have huge effects". Clearly in this study of codon optimization writ large, codon choice has enormous effects.

The youngest DNA author?

Earlier this year an interesting opportunity presented itself at the DNA foundry where I am employed. For an internal project we needed to design 4 stuffers. Stuffers are the stuff of creative opportunity!

A stuffer is a segment of DNA whose only purpose is to take up space. Most commonly, some sort of vector is to be prepared by digesting with two restriction enzymes and the correct piece then purified by gel electrophoretic separation and then manual cutting from the gel. If you really need a double digestion then the stuffer is important so that single digestion products are resolvable from the desired product; the size of the stuffer causes single digests to run at a discernibly different position.

Now, we could have made all 4 stuffers nearly the same, but there wasn't any significant cost advantage and where's the fun in that? We did need to make sure this particular stuffer contained stop codons guarding its frontiers (to prevent any expression of or through the stuffer), that it possess the key restriction sites and that it lack a host of other sites possibly used in manipulating the vector. It also needed to be easily synthesizable and verified by Sanger sequencing -- no runs of 100 As for example. But beyond that, it really didn't matter what went in.

So I whipped together some code to translate short messages written in the amino acid code (obeying the restriction site constraints) and wrap that message into the scaffold framework. And I started cooking up messages or words to embed. One stuffer contains a fragment of my post last year which obeyed the amino acid code (the first blog-in-DNA?); another celebrates the "Dark Lady of DNA". Yet another has the beginning of the Gettysburg Address, with 'illegal' letters just dropped. Some other candidates were considered and parked for future use: The opening phrase to a great work of literature ("That Sam I am, That Sam I am" -- the title also work!), a paen to my wagging companion,.

But the real excitement came when I realized I could subcontract the work out. My code did all the hard work, and another layer of code by someone else would apply another round of checks. The stuffer would never leave the lab, so there was no real safety concern. So I offered the challenge to The Next Generation and he accepted.

He quickly adapted to the 'drop illegal letters' strategy and wrote his own short ode to his favorite cartoon character, a certain glum tentacled cashier. I would have let him do more, but creative writing's not really his preferred activity & the novelty wore off. But, his one design was captured and was soon spun into oligonucleotides, which were in turn woven into the final construct.

So, at the tender age of 8 and a few months the fruit of my chromosomes has inscribed a message in nucleotides. For a moment, I will claim he is the youngest to do so. Of course, making such a claim publicly is the sure recipe to destroying it, as either someone will come forward with a tale of their toddler flipping the valves on their DNA synthesizer or will just be inspired to have their offspring design a genome (we didn't have the budget!).

And yes, at some future date we'll sit down and discuss the ethics of the whole affair. How his father made sure that his DNA would be inert (my son, the pseudogene engineer!) and what would need to be considered if this DNA were to be contemplated for environmental release. We might even get into even stickier topics, such as the propriety of wheedling your child to provide free consulting work!

The challenge of promulgating bear facts.

I had an opportunity this evening to briefly review the impact of DNA research on the taxonomy and conservation of Ailuropoda melanoleuca which also made me reflect on the frustrating struggle of scientific fact to struggle to the public forefront. Put more simply, we had a bedtime discussion of pandas, DNA, relatedness & poop.

As I've mentioned before, some innocent parental actions resulted in the strong imprinting of pandas on my greatest genetics project, so that our house is now filled with various likenesses of the great bicolor Chinese icons. That I can see only 3 where I am sitting now is surprising -- and partly reflects the fact it is dark outside. We have numerous books on giant pands and the school & public libraries have supplied more, and tonight a new little book from Scholastic arrived mysteriously on TNG's pillow. He was eagerly reading it when he came to the fateful passage "It says they're not bears!". But 'The Boy' knows better, and he knows why.

This is a recurring theme in panda books. For a long time the taxonomic placement of pandas was a matter of great dispute, with some assigning them bearhood, some placing them with raccoons, and some allotting pandas a unique clade. A related question concerned the affinity of giant pandas for red pandas and red pandas with the other carnivores. Finally, in the late 1980's the problem yielded to molecular methods, with the clear answer that pandas are bears, albeit a the root of the ursine tree.

What's surprising is how slowly this information has moved into the world of children's books. Of course, the public & school libraries often have books which predate the great resolution, so they are forgiven. Some explain that pandas are bears, but fail to give the evidence. And a few have caught up. But this Scholastic book wasn't one of them, despite having an original copyright solidly after the molecular studies AND a bunch of professors listed as advisors.

Given that TNG is so fond of pandas, and it is no secret, there are those (often adults) who will attempt to dissuade them in their bearness. So I've tried to coach him in how to go beyond simply asserting that they are bears, but explaining why science classes them so. And for an eight year old, he can give a pretty good 1-2 sentence summary.

Which leads us to scat. He merges the two a bit, certainly because of the affinity of his age group for matters excretory (which, of course, his cunning father considered in introducing this topic!). A key question in panda conservation is how many are in the wild. Between their secretive habits and dense bamboo forest habitat, it is difficult to spot a panda in the wild, let alone make a census (nevermind those questionnaires!). So, as with many wild animals, DNA from panda scat is a convenient way to track individuals, and with this tracking the estimate of the number of pandas has shot up -- from the really depressing (to panda fans) ~1500ish to perhaps about a thousand more -- still in grave peril as a wild species, but a thousand more pandas napping in the woods is something to cheer. Unfortunately, the items on pandas in kids magazines & kids sections of newspapers still often quote the older figure.

A similar sort of experiment came up as an item of controversy earlier this year. There are many things I find admirable about John McCain (which is not synonymous to say I'm voting for him -- I haven't decided & I won't tell once I do!), but his pandering about a bear issue earlier this year wasn't one of them. In his fight against congressional earmarks (a good thing), he had singled out a study in Yellowstone National Park's which was sampling DNA from grizzly scat. Amongst his assaults on this study was the question asked loudly of what good this would do beyond setting up a bear dating service. Now, on the one hand I think scientists should be carefully thinking why this important study is apparently being funded by earmark and not peer review. But it is truly sad when you can explain population sampling to an eight year old, but not to someone older than his father who wishes to run the country. Yes, bear counting isn't quite on the same scale as some of the other great scientific issues which are being discussed this election year. But, given that the source behavior of that study is often cited as a benchmark for veracity ("Does a bear..."), it wouldn't be a bad one to get right.

Do we know how Velcade doesn't work?

In my recent piece on the proteasome inhibitor Argyrin A, a commenter (okay, so far THE commenter) noted something I can't argue with, that there is not a well nailed-down understanding of why proteasome inhibition is lethal to tumor cells. I probably should write up a further exploration of how they might work, but I really need to skim the literature for any new findings (so far, nothing stunning).

As Yogi Berra might say, Velcade (bortezomib) is effective in cancer except when it isn't. Indeed, in cell lines in culture the stuff is devastating, but that certainly isn't what's seen in the clinic. In that setting, there is this obnoxious tease of a signal in Phase I (remember, in oncology Phase I is tried in patients with the disease, not with healthy volunteers as in most indications) followed by cruel let-down in Phase II. Even in diseases where the drug works, such as myeloma, it doesn't work in all patients and some patients become resistant. Perhaps that resistance is the key to the puzzle: understand how tumors stop being sensitive and you'd understand the ones which are never sensitive to start with.

Three recent papers (in Blood, J Pharmacol Exp Ther & Exp Hematol) have found the same mechanism for this transition. Alas, none are free & I've only read the abstract (one of many reasons to swing by the MIT library soon All point to overexpression and/or mutation in PSMB5, the proteasome subunit which binds Velcade. Two of the papers report different point mutations, but both in the Velcade binding pocket and in at least one a reduced affinity for Velcade was demonstrated. Game, set & match?

Well, perhaps not. First of all, all three studies are in cell lines, two in closely related ones. As noted above, cell lines are highly imperfect for exploring proteasome inhibition in particular (and not uniformly reliable for oncotherapeutic pharmacology in general). Judging from the abstracts, none of them went fishing around in patient samples, or if they did they came up dry. Given that PSMB5 is an obvious candidate gene for bortezomib resistance, I'm pretty sure this one's been hammered on hard by my former colleagues. Nobody likes to publish the Journal of Negative Results, which I'm pretty sure is where it would end up. Almost certainly some patients will be found who went from sensitivity to resistance due to mutations in PSMB5, but at the moment it's not the long-awaited (and much desired/needed) central hypothesis of why proteasome inhibition works and which patients it should be used in.

Another missed Nobel

The newswires carried the story of Dr. Victor McKusick's passing today. McKusick was the first to catalog human mutations (as Mendelian Inheritance in Man, now better known as OMIM in its Online version), and can be truly seen as one of the founders of genomics. I won't claim to know his full biography, but compiling lists of human mutations way back when probably seemed like a bit of an odd task to a lot of his contemporaries.

This follows the sudden passing of Judah Folkman earlier this year in stealing from us a great light in biology, both of whom which the Nobel Committee failed to recognize.

Of course, there are only three Medicine awardees a year (sometimes the biologists sneak in on the Chemistry prize, but clearly McKusick & Folkman would have been in consideration for the Medicine prize). Nobel picking is a strange and unfathomable world. I'm not complaining about anyone unworthy getting it (though the Nobels have some serious closeted skeletons from the early days -- prefrontal lobotomies for all!), but it's too bad so many miss out who would deserve it.

The curious case of the proteasome inhibitor Argyrin A

A burning set of questions in my old shop when I was there, and I have every reason to think is still aflame, is why does Velcade work in some tumors but not others and how could you predict which tumors it will work in. Does the sensitivity of myelomas & certain lymphomas generally (and a seemingly random scatter of solid tumor examples) to proteasome inhibition follow a pattern? And is this pattern a reflection of the inner workings of these cells or more how the drug is distributed throughout the body?

An even broader burning question is whether any other proteasome inhibitor would behave differently at either level. Would a more potent inhibitor of the proteasome have a different spectrum of tumors which it hit?

Now, while Velcade (bortezomib, fka PS) is the only proteasome inhibitor on the market, it will probably not always be that. Indeed, since Velcade has proven the therapeutic utility of proteasome inhibition, other companies and academics have been exploring proteasome inhibitors. The most advanced that I am aware of is a natural product being developed by Nereus Pharmaceuticals, which I will freely confess to not really following.

The featured (and therefore free!) article in July's Cancer Cell describes a new proteasome inhibitor, another natural product. Argyrin A was identified in a screen for compounds which stabilize p27Kip1, an important negative regulator of the cell cycle. Kip1 is one of the a host of proteins reported to be an important protein stabilized by proteasome inhibition (one of duties back on Landsdowne Street was to catalog the literature on such candidates). While there are probably many ways to stabilize p27Kip1, what they reported on is this novel proteasome inhibitor.

By straightforward proteasome assays Argyrin A shows a very similar profile to Velcade. That is, the proteasome has multiple protease activities which can be chemically distinguished, and the pattern of inhibition by the two compounds is very similar. However, by a number of approaches they make the case that there are significant biological differences in the response to Velcade & Argyrin A.

Now there is a whole lot of data in this paper & I won't go into detail on most of it. But I will point out something a bit curious -- very curious. They performed transcriptional profiling (using Affymetrix chips) on samples treated with Velcade, Argyrin A, and siRNA vs an ensemble of proteasome subunits, each at different timepoints. In their analysis they saw lots of genes perturbed by Velcade but a very small set perturbed by Argyrin A and the siRNA. Specifically, they claim 10,500(!) "genes" (probably probesets) for Velcade vs 500 for Argyrin A. That's a huge fraction of the array moving!

Now, I'll confess things are a bit murky. Back at MLNM I would have had the right tools at my disposal & could quickly verify things; now I have to rely on my visual cortex & decaying memory. But when I browse through their lists of genes for Argyrin A in the supplementary data, I don't see a bunch of genes which are a distinct part of the proteasome inhibition signature. At MLNM, huge numbers of proteasome inhibition experiments were done & profiled on arrays, using a number of structurally unrelated proteasome inhibitors in many different cell lines. Not only does a consistent signal emerge, but when an independent group published a signature for proteasome inhibition in Drosophila there was a lot of overlap in their signature & our signature once you mapped the orthologs.

What's the explanation? Well, it could be that I'm not recognizing what is there due to poor memory, though I'm pretty sure. One thing that is worrisome is that the Argyrin A group's data is based on a single profile per drug x timepoint; there are no biological replicates. That's not uncommon due to the expense and challenge of microarray studies, but good experiments are easy. Nor was there any follow-up by another technology (e.g. RT-PCR) to show the effects across biological replicates or other cell lines. Given that these are in tissue culture cells, which can behave screwy if you stare at them the wrong way, that's very unfortunate. Even small differences in the culturing of the cells -- such as edge effects on plates or humidity differences, can lead to huge artifacts.

Another possible explanation is that the Bortezomib cells were watched too late; the first Velcade timepoint is at 14 hours. After 14 hours, the cells are decidedly unhealthy and heading for death. The right times to sample were always a point of contention, but one suggestion that there is an issue is the lack of correlation between the different timepoints for Velcade vs the strong correlation for the other treatments (Figure 7). That works (in my head at least) in reverse too -- it's downright odd that their other treatments are so auto-correlated between 14 and 48 hours with Argyrin A -- if cells are not yet dead at 14 hours but committed to die, one would expect there to be some sort of movement away from the original profile.

One other curiosity. They do report looking for the Unfolded Protein Response (UPR) and report seeing it in the Velcade treated cells but not Argyrin A treated ones. The UPR is the cell's response to misfolded proteins -- and since disposal of misfolded proteins is a role of the proteasome, it has never surprised anyone that the UPR is induced by proteasome inhibitors. Can you really have a proteasome inhibitor that doesn't induce the UPR? If this is truly the case, it is very striking and deserves its own study.

Is the paper wrong? Obviously I can't say, but I really wonder about it. I also wonder if the referees brought up the same questions. Hopefully we'll see some more papers in the future which explore this compound in a wider range of cell lines and with more biological replicates

Nickeleit et al
Argyrin a reveals a critical role for the tumor suppressor protein p27(kip1) in mediating antitumor activities in response to proteasome inhibition.
Cancer Cell. 2008 Jul 8;14(1):23-35.

Forging into the gap

Gaps are important. There is a major brand by that name. Controversy over a perceived "missle gap" was a major issue in the Nixon-Kennedy election of 1960. Budget gaps cause governments to trim services. About a half an hour's drive west of where I grew up is the town of Gap, and a bunch of generations ago my ancestors probably passed through the Cumberland Gap.

Gaps occupy a special place in computational biology, specifically in the alignment of sequences and structures. As sequences evolve, they can acquire new residues (insertions) or lose residues (deletions), and so if we wish to align a pair of sequences we must put a gap in. Pairwise algorithms such as Needleman-Wunsch-Sellers and Smith-Waterman insert the optimal gaps -- given certain assumptions which include, but are not limited to, the match, mismatch, gap insertion and gap deletion penalties. Some pairwise alignment problems have been addressed by even more complicated gapping schemes. For example, if I am aligning a cDNA to a genomic sequence I may wish to have separate consideration of introns (a special case of gaps), gaps that would insert or remove multiples of three (codons) or gaps which don't all in either of those categories.

Multiple sequence alignment gets even harder. There are no exact algorithms to compute a guaranteed best alignment, so all methods have some degree of heuristics to them. Many algorithms are progressive, first aligning two sequences and then aligning another to that alignment and then another and so on, or perhaps aligning pairs of sequences and then aligning the aligned pairs and so on. Placement of gaps becomes especially tricky, as their placement in early alignments greatly influences the placement in later alignments, which could well be a bad thing.

Protein alignments in particular have the problem of trying to serve three masters, who are often but not always in agreement. An alignment can be a hypothesis of which parts of a protein serve the same role, a hypothesis as to which amino acids occupy similar positions in space, or a hypothesis as to which amino acids derive from codons with a shared ancestry. Particularly in the strongly conserved core of proteins these three are likely to be in agreement, but in the hinterlands of structural loops in proteins or disordered regions it's not so clear. There is also a bit of aesthetics that comes in; alignments just look neater and simpler when there are fewer gaps. Perhaps not quite Occam's Razor in action, but simplicity is appealing.

The June 20th issue of Science (yep, Science & Nature have been piling up) has a paper that addresses this issue and builds an algorithm unapologetically aligned to just the one goal: find the most plausible evolutionary history. They point out that while insertions and deletions are treated symmetrically by pairwise programs, they are quite asymmetric for progressive multiple alignment. The alignment gets to pay once for deleting something, but insertions (like overdue credit cards) incur a penalty with each successive alignment. It seems unlikely that nature works the same way, so this is undesirable.

One solution to this has been to have site-specific insertion penalties. Loytnoja & Goldman point out that this compensation often doesn't work and causes insertions to be aligned which are not homologous, in the sense that they each arose from a different event (indeed, these insertions should not be aligned with anything from an evolutionary point-of-view, though structurally or functionally an alignment is reasonable).

As an alternative, their method flags insertions made in early alignments so that they are treated specially in later alignments. The flagging scheme even allows insertions at the same position to be treated as independent -- they neither help nor penalize the alignment and are reported as separate entities.

Using synthetic data they tested their program against a number of other popular multiple aligners and found (surprise!) it did a better job of created the correct alignment. They also simulated what getting additional, intermediate data does for the alignments -- and scarily for the older alignment programs gap placement got worse (less reflective of the actual insertion/deletion history of the synthetic data).

The article closes with an interesting question: has our view of sequence evolution been shaped by incorrect algorithms? Is the dominant driver of sequence change in protein loops point mutants or small insertions/deletions.

Phylogeny-Aware Gap Placement Prevents Errors in Sequence Alignment and Evolutionary Analysis
Ari Löytynoja and Nick Goldman
http://www.sciencemag.org/cgi/content/abstract/320/5883/1632
p. 1632

If life begins at conception, when does life start & when does it end?

Yesterday's Globe carried an item that Colorado is considering adopting a measure which would define a legal human life as beginning at conception. Questions around reproductive ethics and law raise strong emotions, and I won't attempt to argue either one of them. However, law & ethics should be decided in the context of the correct scientific framework, and that is what I think is too often insufficiently explored.

Defining when life "begins" is often presented as a simple matter by those who are proponents of "life begins at conception" definition. However, to a biologist the definition of conception is not so simple. Conception involves a series of events -- at one end of these events are two haploid cells and at the other is a mitotic division of a diploid cell. In between a number of steps occur.

The question is not mere semantics. Many observers have commented that a number of contraceptive measures, such as IUDs and the "morning after" pill would clearly be illegal under such a statute, as they work at least in part by preventing the implantation of a fertilized egg into the uterine wall. Anyone attempting to develop new female contraceptives might view the molecular events surrounding conception as opportunities for new pharmaceutical contraceptives. For example, a compound might prevent the sperm from homing with the egg, binding to the surface, entering the egg, discharging its chromosomes, locking out other sperm from binding, or prevent the pairing of the paternal chromosomes with maternal ones (there's probably more events; it's been a while since I read an overview). Which are no longer legal approaches under the Colorado proposal?

At the other end, if we define human life by a particular pairing of chromosomes and metabolic activity, then when does life end? Most current definitions are typically based on brain or heart activity -- neither of which is present in a fertilized zygote.

Again, the question is not academic. One question to resolve is when it is permissible to terminate a pregnancy which is clearly stillborn. Rarer, but even more of a challenge for such a definition, are events such as hydatiform moles and "absorbed twins".

In a hydatiform mole an conception results in abnormal development; the chromosome complement (karyotype) of these tissues is often grossly abnormal. Such tissues are often largely amorphous, but sometimes recognizable bits of tissue (such as hair or even teeth) can be found. Absorbed twins are the unusual, but real, phenomenon of one individual carrying a remnant of a twin within their body. Both of these conditions are rare (though according to Wikipedia in some parts of the world 1% of pregnancies are hydatiform moles!) but can be serious medical issues for the individual carrying the mole or absorbed twin.

Are any these questions easy to answer? No, of course not. But they need to be considered.

Do-it-yourself genomics: bad advice is bad advice

GenomeWeb's frequently entertaining Daily Scan notes that Wired magazine has a wiki which gives instructions on how to explore your own genome, including how to do your own genetic testing by home-PCRing your DNA and sending it to a contract lab for sequencing.

It isn't a very good idea, but that doesn't mean people won't try it. Doing a simple PCR really is pretty easy; I've done it in a hotel ballroom (proctoring a high school science fair sponsored by Invitrogen). Instructions for homebrew thermocyclers are surely out there; a number were published in the early days of PCR. But that doesn't mean getting good results is easy. Sticking to a purely technical level, are Wired's instructions very good?

I'd say no. I suppose I should even register to edit the wiki, but at the moment I'll limit myself to pointing out some of the technical issues that are ignored or glossed over (the material I quote below may well change, since it is a wiki).

The first obvious area is primer design. Wired's instructions are pretty simple

Designing them may be the hardest step. Look up the DNA sequence flanking your genetic marker of interest in a database like dbSNP. Pick a segment that is about 20 bases long and slightly ahead of the marker. That is your forward primer. Pick another 20ish base sequence that is behind the region of DNA that you want to study. Use a web app of your choice to find its reverse complement.

Alas, this will frequently be a recipe for disaster. As for my own qualifications for making that claim I will state that (a) I regularly design PCR amplicons in my professional life and (b) I have a much greater appreciation for my ignorance about how PCR can go awry than the average biologist. Leading the list of pitfalls is designing a primer with too low a Tm -- if those 20 nucleotides are mostly A & T, it won't work well. Second would be if the two primers will anneal to each other; you'll get lots of primer-dimer and little else. Equally bad would be a primer that can prime off itself. Third would be if the primers aren't specific to your targeted region of the genome. Prime off a conserved Alu piece and you are in real trouble.

The really silly part about this advice is that there are free primer design programs all over the internet, and some of the sites will perform nearly all of the checks mentioned above.

The rules for placement are much trickier than suggested. If you are going to sequence (and you might be sequencing heterozygous DNA; see below), then you really need the primers to be at least 50 nucleotides away from what you care about -- there is a front of unincorporated dye which often drops the quality any closer than this.

Even more of a concern is the sequence data itself. Wired makes it sound easy

Once that's done, you can buy sequencing equipment and do it yourself, or send the sample off to any one of many sequencing companies and they will do it for about five dollars.

If you are sequencing uncloned PCR products, then you are sequencing a population. If you are heterozygous for a single nucleotide, that means that nucleotide will read out as a mix -- two overlapping peaks of perhaps half height. A deletion or insertion ("indel") will make the trace "double peaked" from that spot on.

Those are the best case scenarios. If you had poor quality amplification (due to badly designed primers or just a miserable to amplify region), all those truncated PCR products will be in the sequencing mix as well -- further degrading your signal. If your SNP is in a region expanded due to copy number variation, then life is even harder.

Which gets to another point: Wired seems to be ignorant of copy number variants. Their testing recipe certainly won't work there.

The idea of untrained, emotionally involved individuals trying to interpret good genetic data is scary enough (Wired's example of celiac disease, as pointed out over at DNA and You, is a particularly problematic one); scarier is to overlay lots of ambiguity and error due to sloppy amateur technique. Hopefully, few will have the energy & funds to try it.

History Forget: How not to explain the impact of Prozac

Having escaped the usual abode for the weekend, there were a pile of the accumulated newspapers to digest on the train this morning. The Sunday Globe Ideas section caught my eye with an item by Jonah Lehrer titled "Head Fake: How Prozac sent the science of depression in the wrong direction". It's not an awful article -- once you get past that subtitle. But, it isn't a great article either.

The article puts forth the thesis that Prozac led to a chemical theory of depression, which recent literature has seriously upended. Alas, that greatly distorts the history.

Prozac was not the first successful drug nor the real antecedent to a chemical theory of depression. Early antidepressives such as the tricyclics and monoamine oxidase inhibitors opened the path to thinking that depression was due to imbalances in specific neurotransmitters. Prozac itself, as a Selective Serotonin Reuptake Inhibitor (SSRI), was an outgrowth of that work -- given the previous success with psychoactive drugs which seemed to affect many neurotransmitters and evidence that specific neurotransmitters might be more important for specific psychological diseases, it was natural to try to zoom in on one neurotransmitter. Prozac then is not a paradigm shifter (ala Kuhn) but was an extension of the existing paradigm. The success of SSRIs, partly due to a significantly attenuated side effect profile and partly due to a lot of popular press and partly due to marketing, merely pushed an existing theory up the ranks, particularly in the popular zeitgeist.

Lehrer does do a nice job of summarizing some recent work suggesting how antidepressants may really work, which is that they may help neurons heal (a new paradigm of depression as a neurodegenerative disease). In a recent conversation a clinician acquaintance noted to me some of the same key points (I'll confess to having not read the literature myself), so there's nothing wrong here. He also notes that it was the investigation of inconsistencies of observation with the predictions of the chemical imbalance theory, such as the frequently observed time lag between beginning antidepressant therapy and seeing results, which led to the new theory.

But getting back to that irksome subtitle, did Prozac steer "the science of depression in the wrong direction" or simply on a winding path? Yes, the chemical imbalance theory looks like it may be down for the count. However, it was that very same theory, via its shortcomings, that led to the new theory. This is how science works -- it's often indirect & messy. That's an important message that's lost (or nearly so) in the piece. SSRIs were perhaps a blunt tool, but they are the tool which has unlocked a new understanding of the topic.

Could we have gotten to the current understanding of depression without SSRIs and other chemical antidepressants? That's an exercise in alternative history best left to experts in the field, if anyone. Perhaps we might have, but perhaps not -- or would have via an even more tortuous path. It is important to get out the story of how pharmaceutical antidepressants do and do not work, but it is equally important to get out the story of how science really works.

Myeloma unified?

Multiple myeloma is a complex disease. Perhaps one metaphor is that of the mythical Hydra -- each time a new molecular tool is thrown at it the number of vicious heads increases. For example, there are different chromosomal translocations which lead to myeloma. If you look at myeloma samples by transcriptional profiling, then one can find distinct expression signatures for each translocation -- and just as easily find ways to split those signatures into further subtypes. For example, some translocations activate one gene disrupted by the translocation whereas other instances of the same translocation will activate both deranged genes.

Another possible metaphor is the old fable of blind men examining an elephant -- each reports that the object is different, based on examining a different portion of the beast. In the case of myeloma, one examiner might focus on the subset with large portions of the genome amplified, others on specific deletions on chromosome 13, another on those cases where bone destruction is rampant. My own experience with palpitating the pachyderm looked at the response to a specific drug.

Now the Staudt lab has come out with a paper in Nature which proposes lumping everything back together again. Initially using a retroviral RNAi screen they identified the transcription factor IRF4 as a unifying theme of myeloma. IRF4 is activated in one characteristic translocation and plays an important role in B-cell development, so it's not a total shock. But linking it across multiple types is surprising.

The screen achieved 2-8 fold knockdown of IRF4 in 3 different myeloma cell lines, each possessing a different hallmark translocation (one of which was an IRF4 translocation). This was later extended to additional myeloma lines with similar lethality, but the knockdown of IRF4 in lymphoma lines had little effect, save one line possessing a translocation of IRF4.

One interesting surprise is that with the exception of the known IRF4 translocation bearing line, none of the lines have amplifications or other obvious derangements of IRF4. Only one showed point mutations upon resequencing. Hence, somehow IRF4 is being activated but not via a painfully obvious mechanism.

RNAi approaches can suffer from off-targets, genes not meant to be hit which cause the phenotype being studied rather than the believed target. The paper provides strong evidence that the effects really are driven by IRF4 knockdown -- not only were multiple shRNAs targeting IRF4 found to kill myeloma cells, but one of these targets the 3' untranslated region of IRF4 -- and the phenotype could be rescued by expressing IRF4 lacking the 3' UTR.

Transcriptional profiling of the knockdown lines in comparison with parental lines revealed a number of candidate IRF4 targets, and a large number of these were also identified by chromatin immunoprecipitation-chip (ChIP-chip) studies, confirming them as direct IRF4 targets. As noted, some direct targets may have been missed by ChIP-chip due to limitations with the arrays used. One other interesting aspect: the IRF4 target list in myeloma lines somewhat resembles a union of that in plasma cells (the normal cell myelomas are most kin to) with that of antigen-stimulated B-cells.

A particularly interesting direct IRF4 target identified in this study is the notorious oncogene MYC. A number of identified IRF4 targets are also known MYC targets, suggesting synergistic activation. They also found that both IRF4 and MYC bind upstream of IRF4 -- suggesting a complex web of positive feedback loops.

An interesting further bit of work targeted various identified IRF4 targets and showed these knockdowns to be lethal to myeloma cell lines. Hence it is suggested that IRF4 ablation in myeloma would lead to tumor cell death by many routes. Mice heterozygous for IRF4 deletion are viable, suggesting that IRF4 could be targeted safely.

The catch would be targeting IRF4 -- transcription factors are on nobody's list of favorite targets. The authors cite as points of optimism approaches targeting p53 & BCL6. However, the p53 targeting route is by inhibiting an enzyme which destabilizes p53, so an analogous approach to IRF4 would require first identifying key determinants of its stability. The BCL6 example they cite uses a peptide mimic, not something the medicinal chemists love much.

Other approaches to targeting IRF4 might focus on "druggable" (if any) genes in the IRF4 target lists, or perhaps something else. I'll try to put together a post next week on one of those candidate elses.

Now that Staudt's group has brought things together, it is tempting to contemplate slicing off some more Hydra heads. How do IRF4 target gene profiles differ across the chromosomal abberation subtypes of myleoma? Do IRF4 targets have any predictive value for determining the appropriate medication or show differential response to different medications?

Laying the groundwork for the one ton tomato

Somewhere in life I've heard a children's/novelty song about a one ton tomato; eventually (if I remember correctly) it ends up as a similar quantity of ketchup.

Nearly half-ton pumpkins show up pretty regularly at the big agricultural fairs every fall, but tomatoes aren't in that league. But, the difference between an ancestral tomato (small berries) and a multi-pound beefsteak is nothing to sneeze at. Domestication has made great strides.

A paper last month in Nature Genetics laid out part of this process. Interestingly, there are two different developmental processes that have been utilized to enlarge tomatoes. A tomato fruit is composed of multiple subunits, the carpels. One change has increased the number of cells per carpel by tinkering with the cell cycle -- a much more delicious change than what a similar process will yield in a person. The new work details the genetic change which increased the number of carpels.

Of course, of interest is how universal these mechanisms are. Most domestic fruits are greatly enlarged over their wild counterparts -- though perhaps raspberries show very little enlargement & blueberries it is a small multiple. On the other end are those monster curcurbits at the fair and their watermelon cousins.

But getting back to the title. Now the question is whether these mechanisms have reached their biological maximum or simply what a few mutations can do (there are also practical considerations, such as the stem strength required to support larger tomatoes). Or, can we use this new knowledge to bring up the laggards -- or figure out why there are no fist-sized raspberries or basketball-like blueberries? A strawberry the size of my dog? Of course, purely economic forces might lead to the fruits commanding the most money per unit weight -- perhaps pomegranates will have an order of magnitude more seeds! Healthy for you -- so long as you watch where you eat them.

Don't do it Josh!

The Globe this week had a number of articles on the passing of the $1B biotech bill in Massachusetts and the proxy fight for Biogen Idec. But a third item really raised my eyebrows.

Vertex's CEO Joshua Boger announced that Vertex is contemplating moving out of the state. The apparent driver of this is a concern that Vertex might outgrow the Boston area and that now might be the time to move, before the company grows even larger. Previous discussion of moving had produced a striking plan to relocate to the Boston waterfront.

Now, I'll confess a certain personal interest. I'm probably going to be in this area for most of my employment life, so I don't want to see employers leave (I can see Vertex headquarters from my office). Furthermore, I believe big companies like Vertex, BiogenIdec and such have a beneficial effect on their overall corporate neighborhood -- they tend to grow more talent than they need and those persons tend to start new ventures near the old ones.

Which is the point -- people don't really like to move. Yes, some folks will follow their job to the ends of the earth, but a lot of folks won't. So atop the disruption & distraction of moving, a lot of good people will leave in a short timespan. My general prejudice is that planners recognize such costs but then grossly underestimate them.

Why might Vertex be contemplating such a move? The most cynical explanation is to try to extract tax incentives from either Massachusetts or wherever they move to. Such incentives have driven previous moves or new sites, with mixed success. Rhode Island trumpeted extracting Alpha-Beta from Massachusetts, until Alpha-Beta failed in the clinic and disappeared into the dust.

More practically Boston does have its drawbacks & tradeoffs. Traffic is awful; but that's true of a lot of America. Housing prices are insane. Neither of these encourages new workers. On the other hand, the academic & hospital environment is huge and Boston has a decent transit system, which somewhat offsets the traffic issue. It is striking that so many large biotech & pharma have been trying to move in to Cambridge/Boston over the last decade or so (Merck, Novartis, Schering, Astra, Amgen, Sanofi-Aventis, etc).

But in any case, I return to my main argument. I'm sure Vertex could thrive in many places -- Boston is not Mecca, and if they moved they would recover and thrive again -- but after paying a steep price of disruption & lost talent.

Are there other options? One of course is to stick it out in Boston. Another is to have multiple locations, which incurs its own inefficiencies. No solution is perfect. But please leave migrations for the birds!

Visiting a time capsule

The Next Generation & I went to the Boston Museum of Science today (we're members this year) and one of the exhibits where he lingered was the one of biotechnology.

I was a bit surprised to find that it dated to 1993; I didn't remember it always being in the spot it's in, so either my memory is flaky (not an unreasonable idea) or it was moved or in storage at some time. But it has been out for a while.

Simply looking at the list of sponsors is a bit of a memory jogger. While some are unchanged (BASF, Genencor), some simply went bust (Alpha-Beta), some were absorbed in corporate actions (Genetics Institute, Perseptive Biosystems) while others remain but under somewhat different names (lawyers Hale & Dorr have several more '&' in the name now; Biogen is now Biogen Idec).

Reading the text is interesting too. For example, we can learn that the human genome maybe, possibly might be sequenced one day.

One of the displays proposes that the dye indigo might one day be synthesized by bacteria (which had been demonstrated) instead of synthesized from petroleum (which had supplanted the original natural source about a century ago); that process has apparently not (yet?) become commercially feasible.

One of the games involves performing gene therapy for cystic fibrosis using a cold virus. That's certainly still a dream, but not for lack of trying.

Another game has you adding an antifreeze gene to tomatoes to prevent their freezing; this was once an active pursuit, but I haven't heard anything lately. Certainly the no-soften tomato was a commercial flop; I'm still eagerly awaiting some tomasil seeds.

This isn't meant to ridicule the display; in general I think it was well done & carefully thought out (Aspirin has been misspelled on the display all these years, but oh well!). Making interesting, interactive exhibits on molecular biology themes remains challenging.

Perhaps what has aged the least on the displays was the addressing of ethical concerns -- when does gene therapy go too far, what privacy rights do we have to our genes, etc.

Isn't The Great Filter something in the Whatman catalog?

Twice in the last week the Globe has run pieces on a concept called 'The Great Filter', once on the Op-Ed page and now in the Star Watch astronomy column. I've read both, and the pseudo-statistical thinking in them just irks me.

The headline on the star watch column suggests the hubris that is perhaps what is goading me: "Why a microbe on Mars would change humanity's future". I'd completely agree that discovering microbial life on Mars would be exciting, but where it goes from there is bizarre.

The gist of the argument can be found in this quote

If life arose independently twice in just one solar system, it would mean that the life formation process is easy and common. Life would be abundant everywhere. Most starts have planets, os the entire universe would be teeming with living things.
Good news? No. The chance for humanity's long-term survival would immediately look worse.
Follow carefully now. Whether or not simple life is common, we know that intelligen, technological life -- like us -- is probably rare. Otherwise, goes the arugment, it would have noticed such a good planet as Earth and come here to colonize as early as hundreds of millions of years ago

.
Given that we haven't yet found signs of other advanced life (or any life) elsewhere

If life is common, something apparently stops it from developing to the point of gaining interstellar travel and settling the galaxy...Apparently, some kine of "Great Filter" preveents life from evolving to the point of getting starships. If the Great Filter lies early in evolution -- such as if the origin of life itself is a rare fluke -- then we, humanity, have already gotten through it. If the Great Filter lies ahead of us -- such as, for instance, if technological civilizations always destroy themselves as soon as they get to power -- then we have no more chance of making it than all the others who have failed and left the cosmos silent.
The more advanced the fossils of living things that Mars may hold, the greater the chance that the Great Filter lies not behind us but ahead.

Okay, just where to start. First, the current Mars mission finding life on Mars is a far cry from finding that life arose independently on Mars. We know that rocks make the transit occasionally, and while we think we sterilized all the probes, the possibility that any life form found really shares a common heritage must first be ruled out. Gary Ruvkun has suggested an experiment for a future probe to look for & sequence ribosomal RNA (if I remember correctly); that would be an appropriate follow-up.

There's also the problem of an N of one: Mars is one planet. Maybe you count an N of 2 with Earth as the second case, though since you're trying to predict on it that's a case of training on your test set. Mars is hardly an independent sample; the same solar system, which may or may not have some unusual properties.

But perhaps more irksome is conflating the reasonable idea that there are difficult barriers against spacefaring species to arise with the rather silly one that there is a single "Great Filter". Mars is a particularly poor example, as we would have a good guess what the filter is there: the planet quit being a nice place to live.

How improbable is life? How often do planets get life but it stays unicellular? How often multicellular but never ambulatory, sentient beings? How often do those sentient beings come up with some way to prevent travel to the stars -- a religion that forbids it, self-extermination (which our species has toyed with). Perhaps some inhabited planets have a super Van Allen belt which dissuaded their residents from becoming star travelers. Perhaps there are intelligent cultures far away -- but with a timing such that their signals can't yet reach us.

The fact is, any estimates of the probability of any one of these (or anything else you can imagine) are nothing but personal priors, wild guesses without much basis in fact. Feel free to make them, but spare us the headlines about predicting doom and gloom.

Cuddle up to a phage!

While searching Amazon for a book, I came across a very funny (in a geeky way) line of plush toys: all sorts of microbes! GiantMicrobes.com has quite a taxonomy of them. I think my visual favorite is the T4 phage , but there's lots of other fun stuff here.

You can get a whole range of common (E.coli) and nasty (a whole line of venereal disease agents. Human pathogens are not monopolized: to terrorize Miss Amanda (or make voodoo chew toys) there's mange, rabies & heartworm.

The E.coli are a flagellated strain. You can buy one or a trio (Petri dish) . Surprisingly, there isn't a package deal on T4+E.coli, nor do they (yet?) have a pBR322 to accessorize your E.coli. Perhaps a future product line extension will include GFP-expressing glow-in-the-dark variants, or perhaps some scent-enhanced ones.

House ATG.GAC.

I don't watch a lot of network television, but there are a handful of programs that have latched onto me. At the end of this season, there were just two and by accident rather than design (or perhaps it is the current plethora of such) they are both hospital-based. Last week I viewed the last of the new episodes off my PVR – so in place of a new episode this week, I’ll try to sketch out my own

House M.D. is an hourlong drama focusing on Dr. Gregory House, a brilliant diagnostician who is also an extremely difficult human being. He terrorizes his three junior colleagues, who are trapped in his orbit like the inner moons of Jupiter -- and subject to similar violent (though only psychologically) tidal forces. Three previous assistants have attained somewhat more distant orbits, though one has spiraled back in. His boss & a colleague attempt to be friends, but get much grief for their efforts.

As with most series TV, there is a basic formula, a framework which the writers decorate or modify each week, rarely breaking it entirely. The scheme here generally starts with a patient arriving with some strange, dramatic set of symptoms (usually exposited prior to the opening credits). House is either intrigued or blackmailed by his boss into taking the case Lots of diagnostic dead ends follow (and new symptoms appear), accompanied by exorbitant amounts of testing. House's assistants provide the union of all high tech medicine & are capable of running any diagnostic under the sun (somehow, the hospital lacks lab techs!). By the end, the case is solved -- and more often than not the patient survives (a few lose the lottery).

One thing you actually DON'T see much of is DNA testing -- once in a while, but it hardly shows up as much as on a CSI/Law & Order type police procedural. DNA testing just doesn't televise well; the best you can do is show someone drawing their own blood (what, no buccal swabs?). In contrast, the MRI room has lots of fun angles -- private conversations behind the console, bouts of claustrophobia, or dramatic races to reach the suddenly stricken patient. Sequencers just aren't very dramatic.

So, I'm going to suggest an episode. Perhaps this qualifies as a "treatment" in Hollywood-speak. I have no desire for a career there, but if the writers take the idea I'd hardly turn down a walk-on.

A patient arrives at Princeton-Plainsboro seeking House due to a mysterious set of symptoms which has afflicted her for years. As usual with such, House is disdainful -- until the patient tries to hand him a DVD but dramatically collapses instead with some interesting symptom along the way. When the patient regains conciousness in a hospital bed, they start asking about the DVD again -- and then deliver the trump card: the DVD has her genome sequence on it.

House has no great interest in the DVD, and argues how useless it is. He's patently annoyed by it. One of the assistants makes the mistake of rising to the bait and proposing that perhaps a critical clue lies within -- and thereby gets assigned the task of cross-referencing EVERY polymorphism against the patient's symptoms. Several dead ends come from the DNA data, but nothing useful -- or in reality, just too many hypotheses which are too tenuous to do anything with. That doesn't stop the young assistants from batting some around and debating the now and future utility of such scans.

Now, as an aside, the story really (in my opinion) needs a complete genome scan. However, if there is a desire to garner some product placement that would narrow the candidates to one (Knome) at this stage. SNP scans are quite as dramatic!

At the end, the patient's puzzle is solved & they get to proceed in life knowing what they have & able to manage it. But, the kicker is that the assistant now cross-references the now known disease against the polymorphisms and comes up with an answer -- but it was buried deep within hundreds of other equally supported hypotheses. Finish the episode with some more back-and-forth amongst the characters about how this might play out the next time. How their careers might change. How well (or not so well) their training has prepared them for this.

Starting to add up to some real money

Last week's Globe carried an item that a real estate firm is planning a 5-year, $1 billion dollar, 1.5M square foot biotech complex in Cambridge. Given all the recent news about Gov. Patrick's $1 billion biotech initiative, perhaps Sen Dirksen was right. Predictably, one letter to the editor proposed that the private money obviates the need for the public mone.

Of course, they're addressing two different things, well, mostly. The original biotech proposal was going to be heavily research oriented, but now there is the earmarks for education & earmarks for local infrastructure. The real estate development is going to provide space for future growth, space that the company is hoping will exist.

Real estate in general & biotech specifically are a boom-and-bust phenomenon in Cambridge, though the trend is clearly weighted a bit towards boom. Even before the genomics boom there was a shortage of space & all sorts of old factory space was converted -- one MLNM site was known as the "Box Factory", as it had previously manufactured heart-shaped candy boxes for Valentine's Day. New buildings went up, such as the cluster of current & former MLNM buildings and the Cambridge beachhead for Partners Healthcare's research empire. The really big daddy's were the conversion of a candy factory to the Novartis site & Genzyme's beautiful building. When the tech boom crashed, space intended for companies such as Akamai was hastily converted.

Then the genomics era came crashing down, and suddenly MLNM wasn't gobbling up space but instead dumping it. Sites such as 640 Memorial Drive sat largely vacant, along with many smaller ones. Signs for 'Biotech Space Available'.

The pendulum is apparently closer to boom again, and several biotechs are heading to the suburbs for cheaper rents or more space. Cambridge will never be cheap, that's for sure.

A billion dollars is no pocket change. One unintentionally humorous element in the story was that no clients had been lined up yet -- like anybody in this business can plan 5 years ahead! MLNM got burnt multiple times on shorter term planning -- stuck in a long lease at 640, buildings configured for the wrong mix of chemistry & biology labs, etc.

Biotech buildings have all sorts of additional requirements, many of which I've only recently become aware of. Heavy-duty floors are needed to support equipment. Complicated ventilation infrastructure. Systems to pH neutralize waste water. Some companies have systems to move waste solvents downstairs; Cambridge's fire department has strict limits which grow tighter the higher the floor. Trying to get leeway there is a non-starter; a year and a half ago a non-biotech solvent explosion blew apart a neighborhood in a town north of Boston.

The location is very good; close to a lot of existing biotech, major road routes, and two mass transit lines -- one of which will probably be extended by the middle of the next decade. The area is already congested, but where isn't?

In the image, the Charles River is the dark slash in the lower right corner, and the Genzyme building anchors the lower left corner.
View Larger Map The big parking lot in the center would be a key site, and has begged for redevelopment for a while. The parking lot above it and to the right would also be included -- but also the low rise buildings going diagonally up to the upper left. These are apparently currently low-rent startup space, a useful commodity, but the new buildings will be much taller -- critical in the increasingly crowded biotech zone. A little bit of the space will be restaurant/retail, but with Kendall Square & the Cambridge Galleria nearby, it won't be lacking for eating & errands.

Misadventures in social networking

My one previous foray into social networking sites was LinkedIn. During one of MLNM's scythe-to-the-workforce exercises folks started setting up the sites, and it seemed like a good idea. Growth was slow at first -- which was fine by me as I've set a personal rule only to link to people I can actually remember interacting with. I also tended to only link to those already possessing accounts; little proselytizing for me. At one point though, I invited one person in just so I'd have one link with nothing to do with MLNM. However, Miss Amanda has shown no interest -- I suppose she won't until digital scent technology picks up)

Over time my network has grown & I have found the tool useful. For one, it's a way to keep connected to folks even as email addresses go dead due to job moves or internet provider changes. However, it's hardly foolproof there -- too often someone's LinkedIn account still points at the old email address. There's the related problem of someone having multiple accounts, having lost their access to one because it points to a defunct email. At least the last time I looked, LinkedIn's interface made it hard to distinguish them when you're trying to delete one -- you just see the person's name.

I also found it useful during my post-MLNM job search to scout out a company -- who do I know at company X?

Purely social sites such as MySpace don't have much appeal to me, but particularly in the past year I've gotten exposed to other sites -- usually by someone inviting me in. For example, SciLink is run out of Boston and the founder is a friend-of-a-friend, so we've actually met. This site starts building your network off your publications, a clever trick (though it does make me glad I'm not a John Smith).

For whatever reason, this morning I decided to check out some of those other invites that have been enjoying benign neglect in my email box. One thought was to get some minor fodder for this page. I uncovered the invite for Spock and also one for Doostang and thought about polishing up my SciLink entry.

For whatever reason, I picked Doostang first. Why I don't know. I sure don't understand the name. My quick associations to it are Durmstrang, boomslang & doofus. -- not good associations (I'm more of a Hogwarts partisan & I care not to meet a boomslang up close). But, what's the harm?

So I followed the link someone sent me & set up an account. Wrote up some skeleton information about my days of being blue & crimson and my job experience.

Now most of the sites have some feature to mine your email addresses for possible links. In keeping with past practice, I thought I'd use this to find others already on the site and try to link to them. Yep, just invite them, that's the plan.

The first inkling of disaster came when I got an email. From myself. Inviting me to Doostang. Actually, it wasn't addressed to me -- but to a Boston area informatics mailing list. OOOPS! Major social faux pas.

Then another email. From myself. This time to a company mailing list. Luckily, my colleagues had a sense of humor about it.

Then I check my email box: people are responding fast-and-furious. One person asks if my invite is spam -- hmmm, not quite sure how to answer that. Several are long lost colleagues, relatives & friends -- okay, that's a good thing. Many had nice things to say such as "thank's for thinking of me" -- I'm getting some social credit I probably didn't really earn.

Another is someone who's email address is a simple mutation away from my own -- people misaddress mail to me there. Nice to meet my not-quite-doppelganger, but pretty strange. Wierder is my inadvertant attempt to cozy up with by good buddies at subscriptions (at) nature.com -- yeah, they'll love me there!

Luckily, I've gotten out of the habit of debating science with crypto-creationists and have restrained myself from e-arguing with the staff right-winger at the Glob. Who knows how many Nigerian finance whizzes & providers of lists of MDs I'm now linked to! I probably should check my account to see what unsavory types I'm now e-collegial with. Please, no emails inviting me to join a network at www.MadeMan.com!

Well, the damage is done. I'll go easy on Spock & probably just return my invite to Meri Jeevan Kahani linger -- at least until I clear out all those messages from Doostang! I've always been aware I can be a bit socially awkward in real settings; now I get to bring that talent to the instant world of the Internet!

When biotech pork doesn't mean GFP spare ribs

Tuesday morning's Globe carries a front page item, with the headline above the fold, outlining the wayward course which Governor Patrick's biotech initiative has taken. Originally outlined as a broad sweep to nurture biotech growth in the Commonwealth with an emphasis on academia, the project has morphed in the Legislature into a set of earmarks.

None of the earmarks are completely devoid of biotech relevance, but they certainly aren't going for broad strokes. $13M for an interchange (near where I live) to relieve commuter congestion around a big Wyeth biopharmaceutical production facility & $13M for a water treatment plant in Framingham which Genzyme needs to support an expanded production plant there. Both will help retain existing biotech facilities which are important employers, but neither of these is likely to drive any growth outside the specific plant targeted (should the Wyeth environs sprout a plethora of omics companies, I will happily figure out a way to eat crow during my exponentially shortened commute!).

Other funds are targeting state university favorites of legislators. U Mass was always going to get a new stem cell repository (which it was pointed out originally was a clever hand to Harvard, which wouldn't mind getting a graceful exit from that business), but the tab is now up to $195M. Nearly $50M will go to build a life science center at a Western Mass school not known for life sciences education; indeed, it doesn't even have a graduate program in the field -- but does have a powerful pol as an alumnus.

University professors & at least one biotech CEO (Genzyme's) are already crying foul, but this is unlikely to have much effect. Massachusetts is effectively a one party state with little involuntary turnover in the Legislature (or the U.S. Congress seats come to think of it). The pols already have retreated to "It's the public money & we have the perogative to spend it!" -- true, but not exactly a justification for how they're spending it.

Who is to blame for the mess? Governor Patrick need look no farther than his mirror. First he made insane estimates of the job creation it would drive -- something in excess of 4X the current employment in the entire existing life sciences sector. Then he burned all his political capital trying to get casino gambling legalized in the state, and then at the moment of the key vote was off to New York signing a book deal rather than corraling a few last votes. With no real leverage, he's at the mercy of the Legislature. The quote in the article suggests that he's ready to sign whatever comes his way, a hollow victory preferable to an honorable defeat.

One thing Patrick clearly underestimated, perhaps because he really is even newer to the state than I am (not quite to the 2 decade mark) is that there is an enormous geographical divide (not that I anticipated it when I initially reacted to it just over a year ago either!). The conditions that favor biotech tend to be in Boston, Cambridge and some surrounding areas -- with Worcester (about 1 hour away) the one other large outpost. Everyone feels that anyone closer to Boston is getting a better deal than they are. So a biotech bill likely to favor the apparently favored was going to have a hard time without a bit of bacon fat to grease the skids -- but once you wave some pancetta before the pols, it's hard to get them to stop.

When biotech pork doesn'

Sherlock Holmes, Omicist

A nice item in GenomeWeb about a new NIH initiative that's just brilliant -- using omics to try to solve rare disease mysteries. I've blogged on this topic before, and it's an obvious way to go -- particularly since the price of these genome studies is dropping so precipitiously.

As noted by the patient named in the report, finding a cause is not (alas!) the same as finding a treatment. But if many patients with mystery diseases are screened, there will almost certainly be some clues that do lead to useful remedies. It is also important to remember that very rare syndromes often shed important light on very common disorders. For example, a large number of rare tumor syndromes have illuminated key cellular mechanisms broadly relevant to tumorigenesis -- von Hippel-Lindau, neurofibromatosis, and many others. Having some molecular clue to the disease is infinitely better than a baffling list of symptoms.

When you care enough to send the very best DNA

Yesterday was Mother's Day, and while searching for a card I spied what looked like a double helix on the front of one card. Finding this odd, I checked the card in detail -- and indeed it was DNA!

DNA is clearly in the public consciousness -- years of Law & Order and CSI have ensured that, but I found it striking that the image of a double helix is deemed recognizable by as mainstream & middlebrow a company as Hallmark.

A nice twist is the card actually bore a message along the lines of 'even though you didn't give me any DNA...' -- a card for mother figures, not birth mothers. So this isn't a sign of rampant DNA deterministic thinking, but rather the imprint of DNA on the public (or at least corporate) mind

A missed creative science opportunity?

Either Science or Nature (I can't find the item now) had a blurb noting that a Chilean observatory will play a prominent role in an upcoming James Bond movie -- the hideout of the villain (original press release here). A bit later in the item it is mentioned that the observatory will basically be simply compensated for its costs.

Given the state of public science funding, it's too bad they didn't extort something more. This isn't somebody's production-costs-charged-to-my-personal-Visa indie film, but 007 himself. Budget never seems to matter much in those films, so why not extract a bit of cash?

The movie is at least titled 'Quantum of Solace', so maybe that's some science there. If the observatory could have held out a bit longer, perhaps they could have gotten something better. Imagine, for instance, the effect on interesting young males in science if Bond's love interest was an astronomer, with a requisite seduction scene taking place around a telescope! Imagine the classic Bondian double entendre opportunities!

Ah well, perhaps it's just jealousy. Nobody builds funky buildings for biologists in stunningly scenic locations (the Salk Institute perhaps excepted). Q's gadgets haven't yet involved synthetic biology (I suppose it doesn't film well) -- alas, no devices made from codons.

Space, the final bio-frontier?

In case it hasn't been obvious from the occasional post, I am a spaceflight aficionado. As a very young child I watched some of the last moon landings. Many hours of play were spent imagining riding a rocket, playing with toy rockets, and building Lego spaceships.

At some point I realized I really didn't quite have the Right Stuff. Clearly I was never going to cut it as a pilot (I carry scale models of Hubble's corrective lenses on my nose daily), and in the end my scientific interests weren't really going to support traveling to space. So it became purely an observational hobby, though the dream has been rekindled a bit by the notion of buying a rocket ticket (alas, 2001 has come-and-gone without the vision of 2001). When Millennium changed travel agents a few years back & we needed to fill out new travel preference forms, I put Virgin Galactic as my preferred carrier.

A more inner struggle, one reflected in much of the space community, is the appropriate role of humans in space, or perhaps more pointedly, of government funding of humans in space. It is one thing for some gazillionaire to pay multi-millions to take a joy ride (anyone want to spot me $50M for a week PLUS a spacewalk?); it's another for governments to continue to spend billions to put people up there. Manned flight is thrilling, but robots tend to get more data.

An item in The Scientist (free registration may be required) points to this debate again, and close to my scientific home. Lobbying is firing up again for biology research in orbit, and given that the company (Spacehab) lobbying for it builds manned research gear, they're pushing the manned angle.

Space research has yielded many earthly benefits, but they're mostly in areas such as communications & remote sensing. It is difficult to really prove a case for very many other areas. Spaceflight remains rare, unpredicable & expensive, three qualities that few like to associate with their research programs.

Two biotech claims are advanced to support space research. One is the long-standing issue of crystallography -- the claim is that crystals for X-ray diffraction studies can be grown in space that are either higher quality than ground samples or which simply can't be made on the ground. The other is a very new claim of vaccine research.

If anyone knows of a good, balanced (not in the Fox News sense!) review of space-based crystallography, I'd love to have a pointer. I'm not in that community, but my general impression is that while useful data has been collected on space-grown crystals, it really hasn't taken that community by storm. Perhaps if flights were cheap & frequent it could, but other approaches such as high-throughput condition screening have had a bigger impact.

The vaccine claims are based on a paper published last year in PNAS (also covered in The Scientist) which found that spaceflight changed some key gene expression programs in Salmonella and that the space-flown bugs were more virulent. A quick scan suggests that the paper is reasonably well done on the transcriptional profiling side (both biological & technical replicates). But, it also points to the challenge of space research -- when is the next flight opportunity to determine how general the effect is?

I do believe there are a lot of fascinating fundamental questions to ask about biology in space. Many would be in the developmental & cellular world: to what degree does gravity influence various developmental processes. Some other research might be less about space & more about behavior: Skylab astronauts had spiders spin webs, and it took a number of trials for the spiders to learn to do it in Zero-G. It could be a fascinating way to study such behaviors & how an animal adapts to a changed environment. But, most space biology questions have an importance scaled to our commitment to manned presence in space. I'm a bit skeptical that the Salmonella experiments really help understand virulence on the ground (or more importantly, are going to be generally relevant -- but sometimes it doesn't hurt to be lucky!), but I'd sure want that line of work driven hard if I was going to spend months in space!

Bizarre inanity from the financial analysis world

In general I try to ignore the various bleatings of stock pickers. Given the mountain of evidence in favor of the efficient market hypothesis, claims of successful stock picking should be generally lumped in with schemes for perpetual motion machines.

However, sometimes something truly ludicrous crosses my eyes & keeps them crossed. I've previously http://omicsomics.blogspot.com/2007_06_01_archive.html, but now I get to pick on someone calling a stock a buy.

The stock is (surprise!) Millennium, which Zacks.com is diligent to inform us is still a buy in their opinion. When I saw the headline I did a double-take, and then had to read the article. It's just as bizarre as I expected. The author describes a complex analysis leading to a target price of $25, miraculously the same as what Takeda is offering. They note all sorts of good news which might occur to Millennium.

But that's irrelevant, as Takeda has set the price for MLNM: $25/share. Given that MLNM has accepted the offer, the price ain't going higher without another bidder -- and unlike eBay auctions bidders don't tend to swoop in at the last minute. Indeed, Zacks isn't saying "buy this because the price will go higher". Yes, MLNM is currently priced a bit south of $25, but that's because there is really a difference of getting $25 when the deal closes versus getting money today. The gap prices in the transactional costs, the time value of getting (or giving) money now, and the tiny risk the deal won't go through -- but Zacks doesn't comment on any of those. Nope, according to them you should buy because MLNM might have good news!

It should be noted that Zacks in Feb called MLNM a buy with a target of $18. Either they got lucky or they really can pick. However, nowhere do they explain how the calculations really changed between then and now -- supposedly they plugged new numbers in and got a new value. But which numbers changed & why? No talk there.

Scrambling E.coli

On a more scientific and interesting note, a new paper in Nature reports on what happens to E.coli if you mess with its regulatory network in a big way. Not only is the paper interesting, but fellow blogger Pedro Beltrao is one of the authors.

The paper takes various promoters and various transcriptional regulators and reassorts which are attached to which. Nearly 600 such combinations were constructed in wild-type E.coli, meaning that the same regulator was also present in its normal regulatory context. The regulators tested also had GFP downstream, so fluorescence could be used to get a rough guide to the level of transcription in the construct.

It is perhaps surprising that most such rewirings are viable; only a few couldn't be built. But, all sorts of perturbations in growth patterns were observed. Some were even more fit than wild type.

Bacteria generally appear to be genetic carpet sweepers, taking in all sorts of genes & trying them out. While most of those genes will be structural, some will be regulators which may bind to existing regulatory motifs (or random motifs in promoters) and activate those genes. Perhaps it is not surprising that E.coli can tolerate many rewirings, as such rewirings must frequently occur naturally -- and activators are often located near the potentially useful genes they activate. If you get the good stuff, you are likely to import an activator, so its useful to be able to adjust to it.

Death, Taxes & Shareholder Lawsuits

With depressing predictability, the Takeda purchase offer of Millennium has been followed by the filing of a shareholder lawsuit claiming that the MLNM Board of Directors has breached their fiduciary duty.

It is hard not to see this as anything other than a shakedown attempt, figuring that the companies would rather pay to see it go away. Or even more unscrupulous, somebody being conned into suing to provide a revenue stream & publicity for some shady lawyers. Or perhaps a bit of both.

Such a suit ignores the fact that Takeda is buying MLNM for a 50% premium over the previous days price. That price was lower than the recent peak, but not by much -- the Takeda offer represents about a 30% premium over the highest price in many years -- indeed, it was 6 years ago it was so high. So, MLNM was hardly sold cheap.

Of course, there are three other ways the suit could claim merit: either MLNM sat on some explosive positive information, the market was persistantly undervaluing MLNM by a lot, or MLNM directors colluded with Takeda. All highly unlikely.

Such suits are all too commonplace. They simultaneously demean & clog the legal system. Real corporate malfeasance does occur, but this ain't it.

The Day AFter

Okay, a day of reflection, buzz -- and two articles in the Boston Globe on the Millennium buyout. One leads from the front page & is a pretty neutral news item. But in the business section is a second piece that works on the theme that genomics was overhyped and has under-delivered. And who hyped it? "Nobody did more to raise those unrealistic expectations than Mark J. Levin"

Oh, really? Okay, I'll confess to not being a neutral bystander. I like Mark. He inspires you. He's also down-to-earth. He's genuine. And yes, he did tout genomics in general and Millennium in particular. But William Haseltine at HGS and Randy Scott at Incyte were hardly shrinking violets. J.C. Venter would never be confused with J.D. Salinger when it came to media access. Drs. Collins, Hood & Lander were hardly silent.

The article is actually a strange mix. It actually starts out with some balance, with an academic commenting how much genomics has forever altered basic biology. There are blurbs from Steve Holtzman (credited in the article as being a key architect of Millennium's business strategy) and Nick Galakatos (whose MLNM employment goes unmentioned), both sobered up but still convinced (as I am) that genomics continues to make an impact on medicine.

The article also repeats the canard about Millennium's drugs not being from genomics. Yes, the two marketed cancer drugs (Velcade & Campath) have very little to credit to genomics (not that we didn't try with Velcade!). On the other hand, unremarked is the pipeline of compounds that Takeda is presumably paying a lot for -- most if not all of those have some genomics heritage, though it is fair to say none of them can only trace back. That's the complexity ignored in articles such as this: genomics has perhaps failed to revolutionize drug discovery, but it has certain become many of the threads in the warp & woof of the drug discovery loom.

The other question that goes unasked is who exactly collaborated in hyping genomics? Hint: remove the silent 'e' & you get Glob. During Millennium's rise the Globe was remarkably charitable to Millennium, with many glowing pieces & routine coverage of every little deal on the front page of the business section. Only well after the genomics bubble burst did that cozy relationship noticeably cool, and indeed through stories such as the failed AnorMed acquisition attempt it seemed to be gone. Perhaps we in the genomics companies were hawking moonshine & snake oil, but the Globe certainly wasn't digging under then. Now, of course, they feel the need to bend over backwards the other way.

Sayonara Millennium?

Boy, if today's news can't break me out of my blogging neglect, then nothing can. Japanese pharma Takeda is buying my old shop for a 50% premium, putting MLNM's share price to a level it hasn't seen since before the Cor merger mistake & market cap at a level unseen since the genomics bubble.

Reports are still coming in, but apparently Takeda is really buying the company -- it is not a raid for the pipeline assets but an attempt to get more or less the whole enchilada. Retention plans are rumored to be in place & it's claimed Dunsire will be staying on. On the other hand, time will tell if Sidney Street will soon feel like a tepanaki table (at least I got the cuisine right this time!) with the chef twirling a large cleaver. A lot of the key folks from Cor were supposed to drive MLNM forward, but they pretty much all bailed after a while.

Management always wanted to get a Japanese deal going, but nothing ever seemed to go beyond secretive hints. Finally, it comes in and it is the ultimate deal.

Many thoughts spring to mind, and perhaps I'll try to cover some later. But in particular, was this the result of a deliberate selling attempt or just some talks that blossomed? Two years ago MLNM refused to sell to an unnamed suitor (though one friend of mine joked about it with a lawyer at a local biotech & decided he'd love to play poker with the lawyer, given the size of the 'tell'); this time Takeda was apparently welcomed with open arms. It will be interesting to see what the merger materials say about the timeline of the deal.

Another key question is how tightly will Takeda attempt to integrate with Millennium? MLNM isn't the same loose place it was when the CEO dressed in drag every October (and just before I got there the high jinx bordered on Animal House), but it still had a soupcon of a laid back atmosphere. Last time I was in the lobby there was a display of each year's T-shirt; not your usual corporate display. I haven't had much dealing with Japanese companies, but this certainly doesn't fit the stereotype. Perhaps Takeda will see the wisdom in a largely hands-off approach, much like Warren Buffett does with his acquisitions -- the parent company funds the subsidiaries but otherwise just acts like a typical board member (though with Buffett, that's still a bit activist). Notable Buffett companies include a prominent local furniture store (where you can go to the movies or try to get free furniture if the Sox sweep the Series again) and the one insurance company unafraid to admit to a reptilian quality.

On the other hand, in theory the greatest value comes from integrating -- cross synergies, reduced duplicative effort, etc. My skepticism of such an approach scales with the distance both physical & cultural, so I doubt it would work. I've recently heard from a former colleague now in a large multi-national pharma how badly its integrated, and it's a company which has had years to do so & common language and nationality.

In any case, I'm sure the weekly sushi day in the cafe will be more popular than ever.

Do you prefer tomatoes or tomatomatoes?

My print version of Science showed up and the cover looks more like some foodie rag. I guessed wrong at first that they were peppers -- right family, wrong fruit. Nope, they are tomatoes.

I like growing tomatoes, though end-of-season output far outpaces my ability to consume them. It's fun growing different varieties, as there are so many different shapes, colors, sizes and flavors. Of course, all of the yard grown ones whip the store cardboard versions. On the other hand, it's hard to grow them around here this time of year -- though I once did have a rampant cherry tomato plant in the tearoom at Harvard (with bunsen burner supports & such staking it up!), though I didn't get any tomatoes until it dawned on me that I needed to play honeybee for the blossoms. When I interviewed at Millennium, someone had a tomato plant growing in the sequencing area (I'm sure the lab safety folks wouldn't let that happen any more!).

Anyway, the Science cover is for an interesting paper showing that a local gene duplication led to elongation of the fruit in one variety. Longer genes, longer fruit! The duplication is recent and was triggered by a retrotransposon, which altered the transcriptional environment around the gene. Cool!

History better learned late than never

I've always been interested in history, and the history of science is no exception. I thought I knew a bit about the history of DNA sequencing, so it was a bit of a rude surprise to read the obituaries on Wed for Dr. Ray Wu and discover that he had published one of the first DNA sequencing methods, a method that is credited with being the forerunner of Sanger sequencing. I was totally unaware of this history.
Sadly, the Wikipedia article on DNA sequencing doesn't cover this at all.

A bit of Medline hunting, aided by Dr. Wu's page at Cornell, found a few articles in PubMed, most sans abstracts and few with full text (Somebody PLEASE arrange legally to get classic J Mol Biol as free full text!). Luckily there are a few papers -- this NAR paper and an earlier PNAS one. If I'm reading it correctly, then it involved 2D analysis of digestion maps, which I had heard of so perhaps my historical knowledge isn't totally deficient.

The one question that occurs is why didn't Dr. Wu stay in the DNA sequencing business. I wonder if he left any thoughts -- was it just not interesting enough or did Sanger & Gilbert just jump ahead so he felt like it wasn't the right place to be. Whatever his reasons it can't really be criticized -- Wu had quite a publication record and appeared to be active essentially to the end of his life. It would just be interesting to understand why he took the direction he did.

Microsoft's irritating limits

Okay, it’s really time to face facts – there’s no avoiding the issue. I am a creature of habit, but the good ones tend to wax and wane with a rarely changing backdrop of the bad ones. Posting regularly to this forum was a good habit, but one which has been scarcely seen for a while.

For the explanations, you can round up the usual suspects. Work pressures. Holiday madness. Routines once disturbed being hard to reform. But in the end, those are just excuses which must be actively combated. Or perhaps it is a lack of passion? And how can passion be rekindled? What will put a new burr in the saddle – well (to badly mix metaphors), perhaps something to get the blood boiling: a good old-fashioned screed.

Now, there are whole websites devoted to griping about Microsoft. Perhaps everything I say has been said before. And, perhaps it is all obsolete carping – for reasons good and perhaps not-so-good I’m still using the previous version of Office. But, given that most of these problems have persisted through so many versions during a decade-and-a-half, I’m not optimistic.

Now, I’ve picked a very broad target. Easy to hit. Like an archer trying to hit the ocean with an arrow while standing in the foam. But I’ll try to focus.

I could gripe almost endlessly about Bill’s Army’s graphical choices. How poor contrast, cluttered charts are the defaults. I won’t (today, at least) go into that, but suffice to say it would be unhealthy for everyone concerned if I were to be trapped in an elevator with the Excel Charts programming team.

No, today I won’t focus on cluttered vision but on a limited vision, when Microsoft has a good idea but then shackles it bizarrely. When it gives the user choices, but insists on giving them very few.

Excel is a workhorse for myself and my colleagues as a data organization, filtering & delivery tool. This has been a habit acquired way back in graduate school and carried through many iterations. I often gravitate towards Excel for taking structured notes, with it’s relentless row-and-column layout forcing useful order. It’s also easy to take those tables and move them into other programs, and in my current post I’ve discovered Excel’s facility for slurping data out of relational databases. All great stuff.

Now, once you’ve got a mess of data in Excel you’d like to sort, filter & highlight it. Naturally, Excel can sort data, the handy Auto-Filter can filter it, and conditional formatting can highlight cells. But they are all broken in the same way: for reasons numerological or otherwise you can do any of these on only 3 criteria each. Sort on three columns. Have three different states to filter on format on.

Now, to pretend to be fair, with Auto-Filter that restriction is per-column, so in a many columned spreadsheet I can filter each column with three criteria -- except when it gives me only two - -I swear it happens, though I can’t remember when , as I’m usually too busy swearing. But, if you wish to see in your dining spreadsheet when you ate Thai, Italian, German or Fusion, you are out-of-luck.

A work-around, of course, is to compute columns with groupings. This can be useful, especially since you can’t save those filter settings for reuse later, but such columns quickly become a visual headache. A few can be useful, but in my hypothetical dining spreadsheet I really won’t want one column for every possible combination of cuisines!

The visual issue brings up another half-way measure: pane splitting. A useful way to deal with so many columns or rows is to split the panes horizontally or vertically (or often both) – but you can have only one split! So if I want to compare data in columns A,Q and BC, I’m out of luck – unless I copy columns or laboriously hide (and later unhide) the columns in between. One help is to make the top row or first column so it doesn’t scroll offscreen – except that blocks out pane splitting!

Now, somebody might be churlish enough to suggest that these limits are rationally chosen and have to do with tradeoffs. Hogwash! Spotfire has a spectacular interface for limiting a field all different ways, with different idioms for choosing (checkboxes, sliders, radio buttons). And if you are having problems with splitting too many ways not working well with various windowing components (such as scrollbars), it’s not like the company doesn’t have its paws in both areas.

A related class of shortcomings, but not quite in the same category, comes around Excel’s functions. If you want to add or count things conditional on some column, there are the COUNTIF and SUMIF functions. But what if I want a conditional AVERAGE? Well, that at least can be gotten from COUNTIF and SUMIF – but no such luck if I want QUARTILE or MEDIAN or STDEV such ways. Why isn’t there a function APPLYIF?

Similarly, I’ve recently gotten hooked on Pivot Tables, which are handy for summarizing data. Basically, a Pivot Table can give you summary statistics on some cross-reference of fields in rows – for example, I could easily see in my eating out spreadsheet the sums of cuisines versus the months of the year. Naturally, these statistics include sum, count, average, stdev, min, mean – but no MEDIAN or QUARTILE . Aargh!

Alas, for the moment I must let my blood boil. When in a company environment, there is an advantage to having a lingua franca, even if it is a flawed one. Plus, what alternative do I have? Perhaps some of this is better in OpenOffice, but I’m not optimistic – my previous experience (though at least a year old) with OO was that it generally aped Microsoft’s flaws & introduced some new ones. Yeah, with more time and energy I could fix that myself, given that it’s open source, but wasn’t the limits of personal time and energy how I opened this article?