Virulence and your UTI

Nobody wants to belong to the party of losers. One of the best strategies in such a case is evidently an interpretation of the change as a gradual accumulation of knowledge while their work has always been at the cutting edge. — Kalevi Kull


Take on the role of a virus competing to infect a host cell and replicate your viral components!

If you have played this game, you probably know more about virulence than the researchers and journalists who report on it.

Bacterial virulence phenotypes of Escherichia coli and host susceptibility determine risk for urinary tract infections

…our findings suggest that UTI risk and outcome may be determined by complex interactions between host susceptibility and the urovirulence potential of diverse bacterial strains.

Reported as: Urinary tract infections reveal the importance of interactions between host susceptibility and bacterial gene expression

…infection depends on both the host environment and gene expression levels in the bacteria.

The epigenetically-effected host environment biophysically constrains viral latency in all living genera. Viral latency exemplifies how ecological variation must be linked to nutrient energy-dependent pheromone-controlled ecological adaptations and gene expression in E. coli, which prevent virus-driven genomic entropy in all living genera.

They reveal the importance of energy-dependent pheromone-controlled quorum sensing, which links virus-driven energy theft to messenger RNA degradation and all pathology via what is known about bacteria in the light organ of the bobtail squid and the microRNAome of all mammals. 

  …sperm have been reported to carry both RNA and microRNA to the fertilized zygote [17], [18]. MicroRNA (miRNA) are important regulators in translation, and their altered expression often leads to disease or cancer.

Telling us about UTIs does nothing to reveal the importance of biophysically constrained energy-dependent viral latency to the understanding of all pathology.

For example, if you were taught that antibiotic resistance is due to mutations in bacteria such as E.coli, which causes most UTIs, you may never learn that bacteriophages drive the nutrient-dependent pheromone-controlled ecological adaptations in the bacteria and viral latency is the key to healthy longevity in all living genera. UTI risk is predicted by differences in the microRNA/messenger RNA balance that link amino acid substitutions in the host to protection from amino acid substitutions in viruses that cause virulence.

Ask yourself where the terms virus and virulence came from — if not from what was suspected or what is known about virus-driven energy theft.


Energy-dependent pheromone-controlled entropy (2)

See also: Energy-dependent pheromone-controlled entropy (1)
Let’s make peer review scientific

…peer review is often biased and inefficient. It is occasionally corrupt, sometimes a charade, an open temptation to plagiarists. Even with the best of intentions, how and whether peer review identifies high-quality science is unknown. It is, in short, unscientific.

The androgynous baby-faced boy or girl pictured among those wearing pink “crotch” hats appears to representing others at the March for Women. The sign may also be representing the intent of one of the organizers whose support for terrorists became better known after this March.
Next comes the March for Science, where anyone whose photo appears can be asked what area of scientific expertise most interested them. Attempts will be made to learn more about what they did to link energy-dependent changes in angstroms from sunlight to ecosystems in all living genera via hydrogen-atom transfer in DNA base pairs in solution.

See for example: Quantifying Intracellular Rates of Glycolytic and Oxidative ATP Production and Consumption Using Extracellular Flux Measurements

Abstract excerpt:

Measurement of ATP use revealed no significant preference for glycolytic or oxidative ATP by specific ATP consumers. Overall, we demonstrate how extracellular fluxes quantitatively reflect intracellular ATP turnover and cellular bioenergetics.

The energy-dependent extracellular fluxes link pheromone-controlled entropy from cellular bioenergetics to the physiology of reproduction. Virus-driven energy theft compromises the conserved molecular mechanisms of cellular bioenergetics.
See for example: Endothelial cell tropism is a determinant of H5N1 pathogenesis in mammalian species

…our study demonstrates that endothelial cell tropism is a determinant of the high virulence associated with HPAI H5N1 infection in mammalian hosts. By utilizing endogenous miRNA mediated restriction of viral tropism, we demonstrate that H5N1 infection of endothelial cells results in increased cytokine production in the lungs and loss of endothelial barrier function, which culminates in vascular leakage in the lungs. In addition, extrapulmonary spread of H5N1 virus likely occurs via the hematogenous route by infection of endothelial cells.

They link virus-driven energy theft from microRNAs in bacteria to messenger RNA degradation in archaea and L-forms (cells without walls). They fail to link energy theft to the substitution of nutrient energy-dependent amino acid substitutions in viruses, which links viruses to pathology in all living genera.
See for example: Identification of amino acid substitutions supporting antigenic change of influenza A(H1N1)pdm09 viruses

In conclusion, substitutions in or near the RBS can influence the antigenic properties of A(H1N1)pdm09 viruses. Based on the current and previous studies of antigenic change of influenza A viruses (11, 12), it is probable that emerging antigenic variants of A(H1N1)pdm09 viruses will escape from population immunity because of substitutions in or near the RBS. However, our results also suggest that the presence of antibodies directed to epitopes on seasonal A(H1N1) and A(H1N1)pdm09 viruses in much of the population limits the number of antigenic variants that can emerge to cause new epidemics.

Antigenic variants that are biophysically constrained cannot “emerge.” Nutrient stress or social stress must first break the biophysical constraints or there would be no new epidemics. That fact means that nutrient-dependent pheromone-controlled neurogenesis links the physiology of reproduction to ecological adaptations in all cell types of all individuals of all living genera, including organisms with no brain. If you start from neurogenesis in the human brain without realizing that it is pheromone-controlled, you may never learn how food odors and pheromones link the physiology of reproduction to the transgenerational epigenetic inheritance of all morphological and behavioral diversity via conserved molecular mechanisms of endogenous RNA interference.
See also from 6 years ago: Reproduction: A New Venue for Studying Function of Adult Neurogenesis?

Recent studies disclose that SVZ neurogenesis is under regulation of reproductive cues like pheromones.

Some recent debate about this fact was published to the Discover Magazine blog site in the context of accurate claims that Ben Carson made about learning and memory. A anonymous character with the screen name “Neuroskeptic” caused me to voice some concerns about the intelligence of people who accused Ben Carson, a pediatric neurosurgeon, of being wrong about anything.
See: Ben Carson and the Power of the Hippocampus 3/10/17

See also: Social phobia: Indication of a genetic cause — reported on “medicalxpress”
Excerpt: The cause of genetic illnesses often lies in the SNPs.
My comment: The energy-dependent differences in the SNPs is RNA-mediated. In this case, the differences link RNA methylation from endogenous RNA interference to learning and memory via experience-dependent cell type differentiation during life history transitions.
For example, one amino acid substitution (COMT Val158Met) was already linked to differences in the behavior of adolescents and adults. When will Neuroskeptic and others admit that they need to learn more about biologically-based cause and effect before they attack people like Ben Carson.

My comment from 3/11/17
See also:Reproduction: A New Venue for Studying Function of Adult Neurogenesis?” (2011) Cell Transplant

Excerpt: …the number of dividing neurons in the hippocampus of female sheep increased robustly (43), which is accompanied with a sharp increase in circulating luteinizing hormone. Additionally, luteinizing hormone level and hippocampal neurogenesis were also upregulated by the soiled bedding. With the use of prolactin and leutinizing hormone receptor knock-out mice, it was confirmed that the hippocampal neurogenesis is due to the increase of the luteinizing hormone but not prolactin; in contrast, prolactin is the regulatory factor of SVZ neurogenesis (69).

Neuroskeptic displayed the ignorance of all theorists and left them with no excuse to say anything more about Ben Carson, or anyone else who intends to help the President of the United States “Make America Great Again.” Only biologically uniformed liberals will continue their attempts to stop President Trump. The anti-entropic effect of pheromones on GnRH and luteinizing hormone links food odors and pheromones from feedback loops to the physiology of reproduction in all vertebrates via the substitution of the achiral amino acid glycine in position 6 of the GnRH decapeptide.

With co-authors, Donald Pfaff did this in the context of autism. For example, substitution of the achiral amino acid glycine in position 6 of the GnRH decapeptide also links food odors and pheromones from stress-linked changes and feedback loops to sex-specific gene–environment interactions via the hypothalamic-pituitary-gonadal axis.
See: Sex-specific gene–environment interactions underlying ASD-like behaviors

…we found a significant three-way interaction on corticotropin-releasing hormone receptor-1 (Crhr1) gene expression, in the left hippocampus specifically, which co-occurred with epigenetic alterations in histone H3 N-terminal lysine 4 trimethylation (H3K4me3) over the Crhr1 promoter. Although it is highly likely that multiple (synergistic) interactions may be at work, change in the expression of genes in the hypothalamic–pituitary–adrenal/stress system (e.g., Crhr1) is one of them. The data provide proof-of-principle that genetic and environmental factors interact to cause sex-specific effects that may help explain the male bias in ASD incidence.

This was reported as: Study tests the ‘three-hit’ theory of autism

…the researchers looked for molecular changes within these rodents’ brains that might help to explain the differences in behavior. They found an increase in the expression of a gene that helps to kick off stress responses, in a brain region called the left hippocampus. With help from C. David Allis’s lab, they looked for chemical alterations in the packaging of DNA that might explain this uptick in gene activity. This effort revealed one particular chemical change in the nerve cell nucleus that encourages the expression of this stress-relevant gene.

The chemical alterations are energy-dependent and RNA-mediated via methylation, which alters gene activation to help ensure that all organisms of all living genera have the best opportunity to ecologically adapt. If they fail to adapt, they die. They do not mutate and become another species.
See: Every amino acid matters: essential contributions of histone variants to mammalian development and disease  (2014) by senior author C. David Allis.

…numerous histone variants seem to be restricted to specific cell lineages or tissue types, yet it remains unclear how such expression patterns are maintained and what the consequences are of increasing or reducing combinatorial variant deposition across cell types. Aberrations in these processes result in detrimental phenotypic outcomes across numerous mammalian systems, including humans. Although we are clearly still in the infancy of this ever-expanding and diverse field, we imagine that future endeavours related to histone variant biology will hold great promise for human health and disease.

The tag-team of Pfaff and Allis will continue to prevent others from what is known to all serious scientists about epigenetically-effected gene-environment interactions among all living genera. The interactions are nutrient-energy-dependent and pheromone controlled by the physiology of reproduction.
I posted this question to the CRISPR Cas 9 FB group and to the miRNA & siRNA FB group

Does any experimental evidence of biologically-based cause and effect suggest that microRNA-mediated host-induced gene silencing is not linked from biophysically constrained viral latency to energy-dependent RNA-mediated cell type differentiation via amino acid substitutions in the cell types of all living genera?

For example, could host-induced gene silencing occur outside the context of natural selection for energy-dependent codon optimality and endogenous RNA interference and the physiology of reproduction?

See also: What’s Next for Diagnostic Patents After Ariosa v. Sequenom

…we should use language that highlights the inventive piece of the invention rather than the natural law underlying it.
The natural law underlying all links from ecological variation to ecological adaptation could be applied to the patent for RNA-Guided Human Genome Engineering. If so, the examiners will find that host-induced gene silencing is the obvious link from nutrient energy-dependent RNA methylation to the pheromone-controlled physiology of reproduction in all living genera via Kohl’s Laws of Biology.
Simply put, Kohl’s Laws includes two facts:
1) all organisms must eat or they die.
2) all species must reproduce or they become extinct.

Can you imagine what the value of future patents on drugs will be if researchers continue to deny what is known about those two natural laws?


Happy Darwin Day (2017)

My series of blog posts about the refutation of theistic evolution by George Church led him to contact me via email.

  1. He asked why he would get credit for or against the refutations
  2. He claimed he was trained in quantum physics.
  3. He claimed that he has authored peer reviewed papers on protein folding, biodiversity, supercoiling, etc.
  4. He wanted to know more but did not know enough about my target audience to realize why I included information about the viral hecatomb.
  5. He also claimed to have written more on what is known about endogenous RNA interference than on exogenous RNA interference.

I invited him to discuss this further on my FB group, or on this domain. He declined. That was a great end to my 2017 Darwin Day.
See Evolution-guided optimization of biosynthetic pathways, which was co-authored by George Church and published December 1, 2014.
There is no such thing as evolution-guided optimization. Natural selection for energy dependent codon optimality is the only link from ecological variation to ecological adaptation in all living genera. That means we can move forward without George Church and still place his comments into the context of “Trust me, I’m a biologist.
I think that most serious scientists agree that you can’t trust evolutionary theorists For comparison, you can trust Darwin’s “conditions of life.”
Darwin’s “conditions of life” link the anti-entropic virucidal energy of sunlight to the physiology of reproduction in all living genera. Can you trust anyone who claims evolution did that?
See: RNA-Guided Human Genome Engineering 

Repetitive elements or endogenous viral elements can be targeted with engineered Cas+gRNA systems in microbes, plants, animals, or human cells to reduce deleterious transposition or to aid in sequencing or other analytic genomic/transcriptomic/proteomic/diagnostic tools (in which nearly identical copies can be problematic).

Nutrient energy-dependent microRNAs link natural selection for energy-dependent codon optimality to viral latency and protection of organized genomes from endogenous viral elements. Targeting endogenous viral elements with RNA-mediated amino acid substitutions is the key to biophysically constrained cell type differentiation. Fixation of amino acid substitutions prevents problematic nearly identical copies. There is no need for uncontrolled copies if there is no need to find another energy source. Uncontrolled copies link virus-driven energy theft from mutations to all pathology in all genera.
For example, energy theft from bacteria links messenger RNA degradation to morphological and behavioral phenotypes of archaea. Similarly, messenger RNA degradation in humans links the transgenerational epigenetic inheritance of Zika virus-damaged DNA to craniofacial morphology and brain development in infants.
Taken together with what is known about differences in energy-dependent endogenous RNA interference in nematodes, all ridiculous misrepresentations of Darwin’s works must be reversed to show the truth about what virus-driven energy theft does. It links RNA-mediated cell type differentiation from the energy-dependent creation of bacteria to the energy-dependent creation of humans and it links virus-driven energy theft to all pathology.
Riding the Evolution Paradigm Shift With Eugene Koonin

The entire evolution of the microbial world and the virus world, and the interaction between microbes and viruses and other life forms have been left out of the Modern Synthesis… 

Is Eugene Koonin joking about what was left out? If not, big bang cosmologists and neo-Darwinian theorists have never tried to support their ridiculous theories with any experimental evidence of biologically based cause and effect. They never examined the role of energy-dependent microRNAs or virus-driven energy theft. Instead, they invented gene-centric theories of mutation-driven evolution.

See for comparison: Membrane Patterns Carry Ontogenetic Information that is Specified Independently of DNA
Reported as: Peer-Reviewed Paper: Development Needs Ontogenetic Information that Cannot Arise from Neo-Darwinian Mechanisms

With over 400 citations to the technical literature, this well-researched and well-documented article shows that embryogenesis depends on crucial sources of information that exist outside of the DNA.

A 16 page monograph with 12 pages of citations is an unparalleled achievement for anyone who is not a polymath or someone who has not already linked physics and chemistry from molecular epigenetics to all biophysically constrained cell type differentiation in all living genera via fixation of nutrient energy-dependent RNA-mediated amino acid substitutions in supercoiled DNA.
None of the cited works appear to link what is known about virus-driven energy theft to all pathology. Again, it is time to move forward.
See: Charles Darwin’s Ocean Upwelling

It’s hard to overstate how vital Darwin’s coral reef theory was in developing his career and thinking. It paved the way, conceptually and methodologically, for everything to come — particularly his transmutation theory [natural selection]. The likenesses startle. Like the transmutation theory, the coral reef theory described how small, virtually unnoticeable changes could create differences of essential type in seemingly immutable forms — and in doing so, account for broad patterns of development and difference.

Changes in coral reefs are nutrient energy-dependent and controlled by the physiology of reproduction. De vries defined “mutation” in 1902, which means that Darwin could not have had a transmutation theory. Also, Darwin repeatedly asserted the claim that his “conditions of life” must come before any claims about natural selection.
Darwin Day 2017 may become known as the day George Church refused to publicly discuss evolution or to admit there is no such thing as evolution outside the context of virus-driven energy theft and the evolution of  pathology. On the same day, The Smithsonian National Museum of Natural History blog site published a post that misrepresented everything known to serious scientists about biophysically constrained energy-dependent RNA-mediated cell type differentiation and healthy longevity.  Who could ask for a better Darwin Day than one during which Darwin’s “conditions of life” clearly triumphed over the ridiculous claims made by neo-Darwinian theorists and others who refuse to admit to the facts that link energy-dependent changes in chirality from autophagy to endogenous RNA interference and to supercoiled DNA, which prevents virus-driven energy theft from causing the mutations that all serious scientists have linked to all pathology?

Biology to a Physicist

Natural selection for codon optimality and quantum viruses

Life is Quantum
Life is energy-dependent. Energy is information. Life is not quantum except in the context of hydrogen-atom energy transfer in DNA base pairs in solution. Claims that do not also address the effects of virus-driven energy theft fail to link what is know about quantum physics from the creation of the sun to quantum biology. That is why Matti Pitkanen and other plagiarists must take my model and use it as if they knew how to link ecological variation to ecological adaptation or to extinction before they learned from me how to do it with the same model.
MIT team genetically engineers a quantum virus for efficient energy transport

The wavelike nature of the particle provides a mechanism for it to simultaneously explore multiple pathways and ultimately resolve the optimal route. If the spacing of the chromophores, and the lifetimes of their excitons, are not “just so,” then the particle takes much longer to arrive at the reaction center. Much the same situation applies to electron tunneling through proteins in the mitochondrial respiratory chain. Lloyd whimsically describes these general phenomena as examples of the Quantum Goldilocks Effect: “Natural selection tends to drive quantum systems to the degree of quantum coherence that is ‘just right’ for attaining maximum efficiency.”

See for comparison: A rapidly acting glutamatergic ARC→PVH satiety circuit postsynaptically regulated by [alpha]-MSH

Here we report the discovery of this missing component: namely, glutamate-releasing neurons in the ARC that express the oxytocin receptor. We demonstrate that chemogenetic activation or inhibition of ARCVglut2/Oxtr neurons, in contrast to ARCPOMC neurons, rapidly and robustly decreases or increases hunger, respectively.

Reported as: Scientists catalogue ‘parts list’ of brain cell types in a major appetite center

The classic way of doing science is to ask questions and test hypotheses,” said Lowell, who is a professor of medicine in the Division of Endocrinology, Diabetes and Metabolism. “But the brain is so complex, we don’t even know how much we don’t know. This information fills in some of the unknowns so we can make new hypotheses. This work will lead to many discoveries that, without these data, people would never have even known to ask the question.
The established hypotheses were linked to biologically-based cause and effect by conserved molecular mechanisms of cell type differentiation. The mechanism link sunlight to energy-dependent changes in RNA-mediated amino acid substitutions via the nutrient energy-dependent pheromone-controlled physiology of reproduction in species from microbes to humans. In all mammals, Feedback loops link odor and pheromone signaling with reproduction. That fact became clear long before 2005.

All serious scientists learned that chemogenetic activation or inhibition of genes is the link from the nutrient-dependent pheromone-controlled physiology of reproduction to energy-dependent endogenous RNA interference and healthy longevity in all living genera. See also: Role of olfaction in Octopus vulgaris reproduction (2015)

Future work on O. vulgaris olfaction must also consider how animals acquire the odours detected by the olfactory organ and what kind of odour the olfactory organ perceives. The OL acting as control centre may be target organ for metabolic hormones such as leptin like and insulin like peptides, and olfactory organ could exert regulatory action on the OL via epigenetic effects of nutrients and pheromones on gene expression (Kohl, 2013; Elekonich and Robinson, 2000).

Virus-driven energy theft links mutations to all pathology via the failure of energy-dependent endogenous RNA interference, which typically protects organized genomes from the effects of messenger RNA degradation.

See for comparison:

Matti Pitkanen: Chemical qualia as number theoretical qualia? (link opens pdf)

…reactive and defensive attitudes interfere de-constructively with logical thinking. Mr X has been stubbornly claiming that I am touting pure nonsense and that all are laughing to me. Surprisingly, he also claims that I have stolen his ideas!

Yes, I am Mr. X and I approve these posts about Matti Pitkanen’s plagiarism. See: Physicists: Desperate Acts and Physicists: Desperate Acts (revisited)

See also: Mitochondria Help Cancers Grow

Jon Lieff has also been sneaking around and dismissing everything known to serious scientists about energy-dependent cell type differentiation, which links virus-driven energy theft to all pathology. He posted on changes in the mitochondria that he failed to link to energy-dependent healthy longevity. For example, mitochondria do not ‘produce’ the ‘cycles’ that link ‘food’ from one carbon metabolism to healthy longevity.

Mitochondria do not ‘adapt.’ The food enables thermodynamic cycles of nutrient energy-dependent protein biosynthesis and degradation. Your example of “doublespeak” obscures, disguises, distorts, or reverses the meaning of words. In that context Matti Pitkanen and Jon Lieff have done the same thing. They have distorted the works of serious scientists to make claims that link theories to biologically-based cause and effect. Theorists refuse to link experimental evidence from top-down causation to energy-dependent changes that link angstroms to ecosystems in all living genera via the physiology of reproduction.

The speed of light on contact with water is the obvious link to all energy-dependent biodiversity and virus-driven energy theft is the link to all pathology. Single-residue insertion switches the quaternary structure and exciton states of cryptophyte light-harvesting proteins. All serious scientists have links light harvesting proteins from plant sensors to RNA-mediated protein folding chemisty.

See: Multipurpose plant sensors startle scientists

 Evolutionary scientists did not predict such elaborate sensory integration in a single protein system.

Jon Lieff is one of the evolutionary theorists who has ignored that fact.

See also: Neuroimmune communication 

We present a special set of Review articles on neuroimmune communication that highlight how the immune system and nervous system are anatomically connected, mechanistically communicate and reciprocally influence the other’s function.

I did that last year and during the past 20 years of my publication history. See: RNA-mediated physics, chemistry, and molecular epigenetics

Published on 3 May 2016

Olfaction and the innate immune system link energy as information from the epigenetic landscape to the physical landscape of supercoiled DNA. The sun’s biological energy is the source of the information that links angstroms to ecosystems via physics, chemistry, and molecular epigenetics.

RNA-mediated protein folding chemistry and amino acid substitutions link the anti-entropic quantized energy of sunlight from the virucidal effects of ultraviolet (UV) light to healthy longevity via biophysically-constrained energy-dependent hydrogen-atom transfer in DNA base pairs in solution and cell type differentiation.

Biomarkers link energy-dependent differences in base pairs and amino acid substitutions to biosignatures across the healthy life span. RNA-mediated amino acid substitutions also reveal the increasing complexity of interactions among cell types as pathology progresses. For comparison, successful reproduction links energy from supercoiled DNA to protection of all organized genomes from virus-driven energy theft and pathology.

This model links the sun’s biological energy from top-down causation in microbes to the most recent model of bottom-up gene activation and cell type differentiation in vertebrates. Citations to extant literature provide examples of what is currently known about how ecological variation leads to biophysically constrained cell type differentiation in the context of nutritional epigenetics and Precision Medicine, which clearly link metabolic networks and genetic networks to pharmacogenomics.

See also:

2010 Synthesis, structure, and two-photon absorption studies of a phosphorus-based tris hydrazone ligand (S)P[N(Me)N=CH-C6H3-2-OH-4-N(CH2CH3)2]3 and its metal complexes.

2012 Evidence for two-photon absorption-induced ESIPT of chromophores containing hydroxyl and imino groups

The values of imino photons are linked to energy-dependent closed or open states via the Gibbs free energy difference. See: 2017 Base-pair opening dynamics of the microRNA precursor pri-miR156a affect temperature-responsive flowering in Arabidopsis

I am nearly convinced that no matter how much experimental evidence of biologically-based cause and effect links energy-dependent changes in base pairs from microRNAs to biophysically constrained protein folding chemistry, pseudoscientists will not recognize the need to include virus-driven energy theft when they invent new ridiculous theories.

The “tipping point” was reached in May, 2016, but there are now 7000 more indexed publications that link energy-dependent changes from the microRNA/messenger RNA balance to all biodiversity. microRNA = 57,721 on February 6, 2017

See also: Molecular basis for protection of ribosomal protein L4 from cellular degradation

The observed molecular recognition fundamentally differs from canonical promiscuous chaperone–substrate interactions. We demonstrate that the eukaryote-specific RpL4 extension harbours overlapping binding sites for Acl4 and the nuclear transport factor Kap104, facilitating its continuous protection from the cellular degradation machinery. Thus, Acl4 serves a dual function to facilitate nuclear import and simultaneously protect unassembled RpL4 from the cellular degradation machinery.

The nature of the energy-dependent interactions that link autophagy to endogenous RNA interference and healthy longevity is expressed without mention of RNA-mediated amino acid substitutions. The substitutions are reported in the context of residues.

The extensive nature of the interactions is best illustrated by the sheer number of residues directly involved in Acl4–RpL4LOOP binding: 42 out of 70 RpL4LOOP residues and 87 out of 333 Acl4 residues (Fig. 2a; Supplementary Fig. 2).