(I)n another paper by Axe and Ann Gauger (Model and Laboratory Demonstrations That Evolutionary Optimization Works Well Only If Preceded by Invention—Selection Itself Is Not Inventive” BIO-Complexity 2015 (2):1–13. doi:10.5048/BIO-C.2015.2), the authors asked if two variants of a canonical TEM beta-lactamase - one inactive due to targeted deletion of the active site, and the other the low-activity, temperature-sensitive variant that was used in Axe’s 2004 JMB paper - could be optimized by repeated rounds of amplification and selection. In Tables 1 and 2 of this report, they state that the lowest concentrations of ampicillin that completely inhibited growth of cells expressing the respective constructs had overlapping ranges (5-10 µg/mL and 10-45 µg/ml for the inactive and low-activity variants, respectively). Moreover, the ranges of highest concentrations of ampicillin that permitted appropriately-transfected E. coli to grow (5-7 and 5-40 µg/mL, respectively) also overlapped. These concentration ranges include the concentration (10 µg/mL) Axe used in his 2004 paper. The ramification of this is that the assay Axe used in the 2004 study cannot accurately distinguish between constructs (plasmids) encoding enzymes with very low activity and plasmids that encode enzymes devoid of any activity. He does not know if variants scored as nonfunctional may actually have low (say, 2 or 5-fold lower than the starting enzyme) activity, or for that matter if variants scored as active are actually inactive. In my opinion, since the purpose of the 2004 study was to enumerate functional variants (even those with much lower activity than the initial enzyme) as well as non-functional variants, the fact that the assay cannot reliably discern between the “starting point” and controls with no activity (let along variants with slightly lower activity) renders the entire study meaningless.
“So, to assess the minimal constraints for proper enzyme function, the approach should be first to obtain an extensively degraded reference sequence that just passes a low selection threshold (Figure 2b, left) and then to subject locally randomized variants of that sequence to selection at the same threshold…”
This describes how he evaluated the variants.
Why, then, are you scientists here busy debating a rube like me? And you won’t step up to debating Axe, by a note in a journal. I was in academia for a while, in computer science, and this happens all the time. People have these dialogs in notes, in notes to the editor, and the back-and-forth can go on for some time.
I will just add (and be curious to see what @Art thinks) that Art’s demolition of the experimental design of the “study” applies to Axe’s laughable barnase data but even if those experiments were better designed, even if they actually assayed enzyme activity, etc., they would be experiments on a SINGLE protein that were then used in extrapolations and speculations that were dubious even in 2004. Re-reading the meandering and tedious paper is a jarring reminder of how weak the whole thing was, even then, and how irrelevant it always was, but especially: how utterly unimportant it is today.
Actually, I came here to discuss it, and see what people had to say to refute the paper.
Actually, I asked a question, and your reply does not actually answer the question. Yes, scientists are human, but it seems the reviewers should have been able to spot a seriously flawed setup, along with all the other problems you all are pointing to. That’s what reviewers set out to do, that’s their job. You all are claiming a complete failure by the reviewers, by implication. I find that shocking. I deem that unlikely.
I protest that you all should present your objections to the journal, and find it also shocking that you all are completely unwilling to do this.
Actually, I noticed one paper, and stopped there. I should have checked the other papers, my mistake.
A quick Google search however, turned up Douglas Axe’s reply:
“the final two paragraphs of the discussion focus on what this value means for the evolution of new protein folds, concluding with this critique of the evolutionary model: “generating new folds from parts of old ones may be much less feasible than has been supposed.” [1] Keep in mind that three experts in enzyme function and evolution approved of the paper in order for it to be published, so my opinion that the work is relevant to protein evolution is shared by others who know the subject very well.”
“And as you’ve probably guessed, meaningful 42-character combinations are far more rare than 1 in 1000, which explains why the islands of meaning are always isolated—mere dots in an enormously vast ocean of nonsense. Billions of sensible things can be said in 42 characters, and almost all of them can be said in many different ways, but none of that amounts to anything compared to the quadrillion quadrillion quadrillion quadrillion possible character combinations of that length (27^42 = 10^60).
That is the sense in which functional protein sequences appear to be rare, and it has everything to do with their isolation.”
“Objection 3: Because Axe measured mutational sensitivity from a weakly functional starting sequence rather than the fully functional natural enzyme, the mutants he generated were inappropriately disadvantaged, and this is why he arrived at such a low value for the prevalence of functional sequences.
According to Hunt, I “molded a variant that would be exquisitely sensitive to mutation.” [2] Venema expressed the same concern, that the starting sequence I used was “intentionally ‘hamstrung’ with multiple mutations to render it far less functional than its natural counterpart.” [7]
Both Hunt and Venema seem to think the outcome would have been more favorable (i.e., functional sequences would have been more prevalent) had I used the highly proficient natural enzyme as a starting point rather than the handicapped version. Actually, as a demonstration will show, the opposite is true.”
This is, by the way, relevant to Mercer and others’ comments along these lines, here. Now please address Axe’s comments in response to your article…
Yeah what seems far fetched to me isn’t that Axe did shoddy work and had a falling out with his supervisor or putative co-authors. It’s that he was running around basically admitting openly that it was all a grift and that he deliberately botched his experiment to get the result that he wanted.
I can even believe he flunked the experiment to get the result that he wanted (or at the very least was deluded enough that he could convince himself that he did legitimate work), it’s the part where he purportedly admits this to people present and admits he did it to secure a future as a creationist propagandist. That seems rather fantastic to me.
To entertain that possibility I have to tell myself a story about how he’s actually a sociopath and took pleasure in trolling his co-workers with stuff he knew they could do nothing about, or something to that effect. That’s just too self-serving for me to believe. Once we allow ourselves this degree of ad-hoc storytelling we’ve basically become creationists.
That’s what he writes. Is it true, Lee? Is that how we should assess the minimal constraints for proper enzyme function?
Assuming that is what you want to find, I’d think, and I’d be curious if you have a different opinion(?), that what we should do is find the lowest concentration of antibiotic that measurably affects fitness, then find what the lowest enzymatic activity that is necessary to produce a measurable fitness advantage is. That means, I think, that we have to experiment with increasingly diluted concetrations of antibiotic and measure doubling times (usually by OD) of the bacteria in question, until we can no longer distinguis the effect of antibiotic on growth rate from just experimental noise. Once we’ve done that, we’ve found the lowest threshold where we can at least measurably show that an enzyme should have a fitness effect. Then we can proceed to measure the effects on doubling time with bacteria encoding enzymes of different activities (themselves established first by assays of enzyme kinetics).
I think that’s what we’d want to do if we truly were trying to establish “the minimal constraints for proper enzyme function.”
Are you going to engage your own brain and think about this, or are you just going to feed us this bs line about taking it up with the journal because you yourself aren’t smart enough to work this stuff out and all you ever do is blindly parrot what the clever academics on your side say to you?
Thanks for digging this ups, Lee. unfortunately, Axe does not (in the reply you cite or anywhere else as far as I know) respond to the chief criticism that I discussed in my PT essay many moons ago.
More to the point of this thread, the excerpt you provide does not at all address the fact that Axe never, ever assayed any of the variants for enzymatic activity. Nor does it address the point I raised a few posts back in this thread, that his assay cannot reliably distinguish between inactive and active TEM beta-lactamase variants. Not that I would expect him to, at least in the essay you pointed to. Axe’s response was written well before the fundamental flaws in his study - no biochemical data, and the unreliability of the plating assay - came to light in this forum.
Maybe someone can point you to Ann Gauger’s fumbling response to these issues in this forum. As I recall, she rather choked on these points, and never responded in any way to these facts. I mention this because, as a matter of fact, the crew here has directly engaged with Axe’s coauthors, and it is they who have failed utterly and completely to rescue Axe’s work. This is probably why Axe has declined invites to join us, and will likely never, ever join a discussion with any of us, or with people who are informed about these matters.
That is because of your ignorance. It clearly happened in this instance, and it is not really all that uncommon.
My, but you are easily shocked. Please be sure you are never too far from a fainting couch and a vial of smelling salts.
It would be a total waste of time to point out the flaws in an obscure, 20 year article that has had zero impact in the field. If someone was to write to the editors, they would probably just think “WTF?” and file it in the circular filing cabinet.
I will repeat the question that you have, so far, ignored: How many articles on evolution has the DI claimed were flawed over the 20 years the organization has been in existence? How many times has the DI written to the editors expressing their criticisms? How often have the articles been retracted (or “refuted”, as you put it)?
Or, if answering that is too much work, you can save time and simply admit that you are a shameless hypocrite. Either will do.
Well, he does, actually: “So, to assess the minimal constraints for proper enzyme function, the approach should be first to obtain an extensively degraded reference sequence that just passes a low selection threshold (Figure 2b, left) and then to subject locally randomized variants of that sequence to selection at the same threshold…”
Wait, now he did an assay? It seems you just claimed he didn’t. And I expect the reviewers of his paper saw what he did, and approved it. As Axe wrote in his reply to you, “Keep in mind that three experts in enzyme function and evolution approved of the paper in order for it to be published, so my opinion that the work is relevant to protein evolution is shared by others who know the subject very well.”
Um, Axe did respond to your article, in the link I found. I picked some excerpts just for illustration, it would now be your job to respond to his points he made about your article on Panda’s Thumb.
Axe, in attempting to defend his 2004 paper against this objection (the objection that new proteins can evolve from parts of old ones), cites [1] which is … his 2004 paper.
I’d hate to play chess with Axe. “When your queen threatens my king, he just shoots a laser at her! Checkmate evolutionists!”
Well let me cite this post I wrote on a 2019 dissertation exploring this exact question to see if Axe 2004 somehow shows that generating new functional proteins from parts of other already functional proteins is “much less feasible than has been supposed”:
Here’s a small snippet:
Rather, given that Axe’s work is once again being cited and is doing the rounds in ID-creationist circles, I thought this would be as good a time as any to visit some work in experimental molecular biology that massively contradicts Axe’s number. And to not beat around the bush too much, we’re talking a 72 order-of-magnitude discrepancy between Axe’s frequency of 1 in 1077 and what other experiments in probing protein sequence space reveal.
I will give an account of experiments done to probe a particular process of novel protein evolution, called gene fusion, which turned out to be able to find functional proteins at a rate of 1 in 3.3×105. To put the magnitude of the discrepancy into context, it’s 1 followed by 72 zeroes, which is a number that seems to fit in the arena concerning things like the number of atoms in the observable universe (estimated at ~1080). Quite a margin of error!
So we go from Axe estimating that new functional proteins require searching 1077 on average before we find a single new one. And then an experiment using randomly fusing fragments of already existing proteins finds new functional ones at roughly 1 in ~300 000.
This one is ridiculously false. Axe uses the example of a readable sentence to try to demonstrate this, which is so confused it’s difficult to resist the interpretation that he wrote it to obfuscate.
So let’s just correct his laughably inept example right here in a way that should be immediately comprehensible to any rational person that wants to understand.
So first of all, to calculate his final number, Axe calculates the “pass rate” (fraction of substitutions tolerated at a typical position) of his temperature sensitive enzyme mutants, raised to the 153th power (length of protein), multiplied by some hydropathic signature factor (which he estimates at 10-13), to get to his 10-77 number.
In his experiment, using the temperature sensitive enzyme, he averages the pass rates for each segment and calculates the fraction of substitutions tolerated at a typical position to be 0.38.
So it’s 0.38153 × 10-13 = 10-77
So the key number is 0.38. That’s the one his temperature sensitivity directly affects. By making it more sensitive to temperature, he has already mutated it, and made it less tolerant of further mutation. With this as the basis for his experiment he finds that 38% of substitutions at a typical position can still be tolerated and gives a functional enzyme.
He took the wild type sequence, which we can like Axe demonstrate with a sentence.
The “wild-type” sentence: Did Douglas Axe Disprove Evolution? Spoiler: No
Then he created a highly degraded one that can barely even be read: Dxd DoNglfs Ake Dispvuge EVolTtijn? SGoilah: No
I now pose the question: Which sentence can tolerate the most mutations before it entirely stops working - the wild-type, or the already mutated one?
This is like asking: What would be the passe rate for the wild-type enzyme? Would it be higher than 0.38, or lower than 0.38?
Had he not used the already degraded version, what per-position fraction of random mutants of the wild-type sentence would have retained readability?
Any thinking person can immediately see that the wild-type sequence should have a higher pass rate. More than 0.38. The already degraded, already mutated one can barely take any more before readability disappears entirely.
For forks sake, just think! Already mutating the wild-type sequence is how he got the temperature sensitive variant to begin with. He has already thrown mutations at the enzyme he uses in his experiment but doesn’t factor that into his calculation.
Even if we cannot by reason alone know how much higher than .38 that number would be if we used the wild-type sequence, we can see immediately just by looking at the sequences that obviously the wild-type sentence can tolerate more substitutions and retain readability.
Axe lies about what the result would be if he had used the wild-type protein in his experiment. It would NOT have made the result worse. That’s a BOLD FACED LIE and his explanation that purports to show this is deliberate obfuscation.
Well, the DI has taken a different approach, they have published articles like Douglas Axe did, but mostly they have written books describing the problems with NeoDarwinism. Notably, Gunter Bechly set out some years ago to create a display, with a scale holding books on evolution on one side, and Intelligent Design books on the other. Of course, the evolution side was set up to outweigh the ID side! Then Bechly decided he would read some of these books, and was convinced by their arguments. He became a proponent of ID, and consequently lost his post at the State Museum of Natural History in Stuttgart, Germany. He recently, before his tragic death, issued a challenge to his former colleagues, concerning species pairs: “Considering the fact that windows of time of only 5-10 million years account for most of the abrupt appearances of new body plans in the fossil record (Bechly & Meyer 2017, Bechly 2021), the Bayesian likelihood of not finding a single example of similar morphological disparity having originated on a similar time frame among the millions of living species is basically close to zero. I consider this simple argument as a final nail in the coffin of Darwinian unguided evolution.”
So here he is addressing, in effect, the varied papers and arguments for evolution, and challenging them.
I know. It doesn’t explain why he evaluated a continuous variable (enzymatic activity, cheaply assayed) as a binary one. Does that make any sense to you?
We’re trying to explain. If your goal is debate, why are you also claiming that you came here to discuss? In what way do you think that parroting Axe constitutes debate or discussion?
Most importantly, why isn’t Axe doing more science?
Why don’t you read that journal and point us to the debates that have been published there?
All the time? So why haven’t you cited any in the Journal of Molecular Biology that would make your point?
I completely agree. Doug Axe probably can’t recover any scientific credibility after feeding (and perhaps creating) a wholly fictional mythology around his paper. I struggle to understand his conduct. I just can’t get my head around the shameless dishonesty that he has to haul around.
But a sociopath bragging about their own lawlessness? No I don’t picture Axe that way. If he worked in the White House I might reconsider…
I’m not sure why this is needed, though, shouldn’t you just use a standard antibiotic dose, and see if bacteria survive? Why make it easier for bacteria, isn’t that just going to take longer to see if there is any effect?
It seems that instead we’ve just made it more difficult to determine if an enzyme has fitness, though.
I like Axe’s approach better, where he starts with a minimally fit enzyme, and varies it, testing with the same threshold, for fitness.
I will also note that you offer no citation for your Bechly quote, unless the link “challenge” is intended to be; but that doesn’t go anywhere. And the citations internal to the quote go not to the peer-reviewed literature but to edited apologetics volumes, one of which at least is out of print. Not helpful.
What are some of these new body plans that Bechly refers to?
Several problems here. Most importantly, he assumes that the probability of evolving a new body plan has not changed over time. Note that you could equally falsify creation (or, in Bechly’s notion, saltation) if you assume that the probability of creating (possibly by zapping an existing species, not ex nihilo) a new body plan doesn’t change over time. What’s the difference?
Second, given that the fossil record is incomplete, how can you assume that this 5-10my window is not just an artifact of preservation?
Third, “body plan” is ill-defined. How do you recognize a new one from a modified old one?
@lee_merrill, I apologize for being loose with my terminology. I should be more careful.
When I state that “Axe never, ever assayed any of the variants for enzymatic activity”, I mean that he never directly assayed any the purified variants for beta-lactamase activity, say using the methods John refers to in this thread. Rather, he used a proxy - ability of E. coli carrying a plasmid with genes encoding variants to grow on minimal concentrations of ampicillin. Most of us who have worked with E. coli know that many, many factors other than the presence of beta-lactamase activity can promote growth on low concentrations of ampicillin and related antibiotics. I refer to this as the plating assay. This assay is not a direct measure of enzyme activity. And, as Axe and Gauger themselves have shown, it is not a reliable emasure of low amounts of activity.
With all due respect, I have no problem claiming that I am a better judge of this study than these anonymous reviewers. Also, as many other have stated in this thread, peer review is not the end of the scientific process. So further considerations, such as you are learning here, that clearly reveal fatal flaws in a work take precedence.
Sorry, @lee_merrill, but you are just plain wrong. Axe has never responded to my specific criticisms, and his proxies (such as Ann Gauger) have failed to rescue his ideas when confronted with these facts.
Actually, I gave him a sterling example, that of the Hawaiian Silverswords. Bechly’s counterargument amounted to the ridiculous assertion that all angiosperms have the same body plan. This counterargument deserves a doubleface-palm.