High-yield recombinant bacterial expression of 13 C-, 15 N-labeled, serine-16 phosphorylated, murine amelogenin using a modified third generation genetic code expansion protocol.

Amelogenin constitutes ~90% of the enamel matrix in the secretory stage of amelogenesis, a still poorly understood process that results in the formation of the hardest and most mineralized tissue in vertebrates-enamel. Most biophysical research with amelogenin uses recombinant protein expressed in Escherichia coli. In addition to providing copious amounts of protein, recombinant expression allows 13 C- and 15 N-labeling for detailed structural studies using NMR spectroscopy. However, native amelogenin is phosphorylated at one position, Ser-16 in murine amelogenin, and there is mounting evidence that Ser-16 phosphorylation is important. Using a modified genetic code expansion protocol we have expressed and purified uniformly 13 C-, 15 N-labeled murine amelogenin (pS16M179) with ~95% of the protein being correctly phosphorylated. Homogeneous phosphorylation was achieved using commercially available, enriched, 13 C-, 15 N-labeled media, and protein expression was induced with isopropyl ß-D-1-thiogalactopyranoside at 310 K. Phosphoserine incorporation was verified from one-dimensional 31 P NMR spectra, comparison of 1 H-15 N HSQC spectra, Phos-tag SDS PAGE, and mass spectrometry. Phosphorus-31 NMR spectra for pS16M179 under conditions known to trigger amelogenin self-assembly into nanospheres confirm nanosphere models with buried N-termini. Lambda phosphatase treatment of these nanospheres results in the dephosphorylation of pS16M179, confirming that smaller oligomers and monomers with exposed N-termini are in equilibrium with nanospheres. Such 13 C-, 15 N-labeling of amelogenin with accurately encoded phosphoserine incorporation will accelerate biomineralization research to understand amelogenesis and stimulate the expanded use of genetic code expansion protocols to introduce phosphorylated amino acids into proteins.

The phylogenetic distribution of the cell division system would not imply a cellular LUCA but a progenotic LUCA.

The stage reached by the evolution of cellularity in the Last Universal Common Ancestor (LUCA) has not yet been identified. In actual fact, it has not been clarified whether the LUCA was a cell (genote) or a protocell (progenote). Recently, Pende et al. (2021) analysed the phylogenetic distribution of the cell division system present in bacteria and archaea reaching the conclusion that LUCA was a cell and not a progenote. I find this conclusion unreasonable with respect to the observations they presented. One of the points is that the presence in the domains of life of many genes - some paralogs - which would define the membrane-remodeling superfamily would seem to imply a tempo and a mode of evolution for the LUCA more typical of the progenote than the genote. Indeed, the simultaneous presence of different genes - in a given evolutionary stage and with functions that are also partially correlated - would seem to define a heterogeneity that would appear to be the expression of a rapid and progressive evolution precisely because this evolution would have taken place in the diversification of all these genes. Furthermore, the presence of different genes coding for the function of cell division and related functions could reflect a progenotic status in LUCA, precisely because these functions might have originated from a single ancestral gene instead coding for a protein (or proteins) with multiple functions, and therefore an expression of a rapid and progressive evolution typical of the progenote. I also criticize other aspects of considerations made by Pende at al. (2021). The arguments presented here together with those existing in the literature make the hypothesis of a cellular LUCA favoured by Pende et al. (2021) unlikely.

Archetypes and code biology.

As a clinical psychologist, I observe stereotyped formulas of behavior in action every day in the consulting room, despite differences in age, race, or culture; they present themselves as codified rules or typical modes of behavior in archetypical situations. Such circumstances coincide with what C.G. Jung defended: the existence of archetypes stored in an inherited/phylogenetic repository, which he called the collective unconscious - somewhat similar to the notion of an ethogram, as shown by ethology. Psychologists can use a perspective to facilitate understanding the phenomenon: the code biology perspective (Barbieri 2014). This approach can help us recognize how these phenomenological events have an ontological reality based not only on the existence of organic information but also on the existence of organic meaning. We are not a tabula rasa (Wilson 2000): despite the explosive diversification of the brain and the emergence of conscience and intentionality, we observe the conservation of basic instincts and emotions (Ekman 2004; Damasio 2010) not only in humans but in all mammals and other living beings; we refer to the neural activity on which the discrimination behavior is based, i.e., the neural codes. The conservation of these fundamental set-of-rules or conventions suggests that one or more neural codes have been highly conserved and serves as an interpretive basis for what happens to the living being who owns them (Barbieri 2003). Thus, archetypes' phenomenological reality can be understood not as something metaphorical but as an ontological (phylogenetic) fact (Goodwyn 2019). Furthermore, epigenetic regulation theories present the possibility that the biomolecular process incorporates elements of the context where it takes place; something fundamental to understand our concept - the archetype presents itself as the mnesic remnant of the behavioral history of individuals who preceded us on the evolutionary scale. In short: brains are optimized for processing ethologically relevant sensory signals (Clemens et al., 2015). From the perspective of the corporeal mind (Searle 2002), in this paper, we will show the parallels between code biology and the concept of the archetype, as Jung defended it and as it appears in clinical practice.

The lower threshold as a unifying principle between Code Biology and Biosemiotics.

Whether we emphasize the notion of 'sign' or the notion of 'code', either way the main interest of biosemiotics and Code Biology is the same, and we argue that the idea of the lower threshold is what still unifies these two groups. Code Biology concentrates on the notion of code: living organisms are defined as code-users or code-makers, and so it may be called the 'lower coding threshold' in this case. The semiotic threshold on the other hand is a concept without a specific definition. There are many possible ways of understanding this term. In order to maintain the lower threshold as the unifying concept between Code Biology and biosemiotics, it is important to be very clear about where this threshold is located and how it is defined. We focus on establishing the lower semiotic threshold at protein biosynthesis, and we propose basing the semiotic understanding of the lowest life forms on the following criteria: arbitrariness, representation, repetition, historicity and self-replication. We also offer that this definition of the lower threshold need not include the notion of interpretation, in the hope that this newly specified common principle of the lower threshold be accepted as a way forward in the conversation between Code Biology and biosemiotics.

Metacode: One code to rule them all.

The code of codes or metacode is a microcosm where biological layers, as well as their codes, interact together allowing the continuity of information flow in organisms by increasing biological entities' complexity. Through this novel organic code, biological systems scale towards niches with higher informatic freedom building structures that increase the entropy in the universe. Code biology has developed a novel informational framework where biological entities strive themselves through the information flow carried out through organic codes consisting of two molecular or functional landscapes intertwined through arbitrary linkages via an adaptor whose nature is autonomous from molecular determinism. Here we will integrate genomic and epigenomic codes according to the evidence released in ENCODE (phase 3), psychENCODE and GTEx project, outlining the principles of the metacode, to address the continuous nature of biological systems and their inter-layered information flow. This novel complex metacode maps from very constrained sets of elements (i.e., regulation sites modulating gene expression) to new ones with greater freedom of decoding (i.e., a continuous cell phenotypic space). This leads to a new domain in code biology where biological systems are informatic attractors that navigate an energy metaspace through a complexity-noise balance, stalling in emergent niches where organic codes take meaning.

Code biology and the problem of emergence.

It should now be recognized that codes are central to life and to understanding its more complex forms, including human culture. Recognizing the 'conventional' nature of codes provides solid grounds for rejecting efforts to reduce life to biochemistry and justifies according a place to semantics in life. The question I want to consider is whether this is enough. Focussing on Eigen's paradox of how a complex code could originate, I will argue that along with Barbieri's efforts to account for the origins of life based on the ribosome and then to account for the refined codes through a process of ambiguity reduction, something more is required. Barbieri has not provided an adequate account of emergence, or the basis for providing such an account. I will argue that Stanley Salthe has clarified to some extent the nature of emergence by conceptualizing it as the interpolation of new enabling constraints. Clearly, codes can be seen as enabling constraints. How this actually happens, though, is still not explained. Stuart Kauffman has grappled with this issue and shown that it radically challenges the assumptions of mainstream science going back to Newton. He has attempted to reintroduce real possibilities or potentialities into his ontology, and argued that radically new developments in nature are associated with realizing adjacent possibles. This is still not adequate. What is also involved, I will suggest, utilizing concepts developed by the French natural philosopher Gilbert Simondon, is 'transduction' as part of 'ontogenesis' of individuals in a process of 'individuation', that is, the emergence of 'individuals' from preindividual fields or milieux.

Karyotype coding: The creation and maintenance of system information for complexity and biodiversity.

The mechanism of biological information flow is of vital importance. However, traditional research surrounding the genetic code that follows the central dogma to a phenotype faces challengers, including missing heritability and two-phased evolution. Here, we propose the karyotype code, which by organizing genes along chromosomes at once preserves species genome information and provides a platform for other genetic and nongenetic information to develop and accumulate. This specific genome-level code, which exists in all living systems, is compared to the genetic code and other organic codes in the context of information management, leading to the concept of hierarchical biological codes and an 'extended' definition of adaptor where the adaptors of a code can be not only molecular structures but also, more commonly, biological processes. Notably, different levels of a biosystem have their own mechanisms of information management, and gene-coded parts inheritance preserves "parts information" while karyotype-coded system inheritance preserves the "system information" which organizes parts information. The karyotype code prompts many questions regarding the flow of biological information, including the distinction between information creation, maintenance, modification, and usage, along with differences between living and non-living systems. How do biological systems exist, reproduce, and self-evolve for increased complexity and diversity? Inheritance is mediated by organic codes which function as informational tools to organize chemical reactions, create new information, and preserve frozen accidents, transforming historical miracles into biological routines.

The drawbridge of nature: Evolutionary complexification as a generation and novel interpretation of coding systems.

The phenomenon of evolutionary complexification corresponds to the generation of new coding systems (defined as а codepoiesis by Marcello Barbieri). The whole process of generating novel coding statements that substantiate organizational complexification leads to an expansion of the system that incorporates externality to support newly generated complex structures. During complexifying evolution, the values are assigned to the previously unproven statements via their encoding by using new codes or rearranging the old ones. In this perspective, living systems during evolution continuously realize the proof of Gödel's theorem. In the real physical world, this realization is grounded in the irreversible reduction of the fundamental uncertainty appearing in the self-referential process of internal measurement performed by living systems. It leads to the formation of reflexive loops that establish novel interrelations between the biosystem and the external world and provide a possibility of active anticipatory transformation of externality. We propose a metamathematical framework that can account for the underlying logic of codepoiesis, outline the basic principles of the generation of new coding systems, and describe main codepoietic events in the course of progressive biological evolution. The evolutionary complexification is viewed as a metasystem transition that results in the increase of external work by the system based on the division of labor between its components.

Life and living beings under the perspective of organic macrocodes.

A powerful and concise concept of life is crucial for studies aiming to understand the characteristics that emerged from an inorganic world. Among biologists, the most accepted argument define life under a top-down strategy by looking into the shared characteristics observed in all cellular organisms. This is often made highlighting (i) autonomy and (ii) evolutionary capacity as fundamental characteristics observed in all cellular organisms. Along the present work, we assume the framework of code biology considering that biology started with the emergence of the first organic code by self-organization. We reinforces that the conceptual structure of life should be reallocated from the ontology class of Matter to its sister class of Process. Along the emergence and early evolution of biological systems, biological codes changed from open systems of "naked" molecules (at the progenote era), to close, encapsulated systems (at the organismic era). Living beings appeared at the very moment when nucleic acids with coding properties became encapsulated. This led to the origin of viruses and, then, to the origin of cells. In this context, we propose that the single character that makes a clear distinction between the abiotic and the biotic world is the capacity to process organic codes. Thus, life appears with the self-assembly of a genetic code and evolves by the emergence of other overlapping codes. Once life has been clearly conceptualized, we go further to conceptualize organisms, parents, lineages, and species in terms of code biology.

Genes on the circular code alphabet.

The X motifs, motifs from the circular code X, are enriched in the (protein coding) genes of bacteria, archaea, eukaryotes, plasmids and viruses, moreover, in the minimal gene set belonging to the three domains of life, as well as in tRNA and rRNA sequences. They allow to retrieve, maintain and synchronize the reading frame in genes, and contribute to the regulation of gene expression. These results lead here to a theoretical study of genes based on the circular code alphabet. A new occurrence relation of the circular code X under the hypothesis of an equiprobable (balanced) strand pairing is given. Surprisingly, a statistical analysis of a large set of bacterial genes retrieves this relation on the circular code alphabet, but not on the DNA alphabet. Furthermore, the circular code X has the strongest balanced circular code pairing among 216 maximal C3 self-complementary trinucleotide circular codes, a new property of this circular code X. As an application of this theory, different tRNAs studied on the circular code alphabet reveal an unexpected stem structure. Thus, the circular code X would have constructed a coding stem in tRNAs as an outline of the future gene structure and the future DNA double helix.

Determining amino acid scores of the genetic code table: Complementarity, structure, function and evolution.

The Standard Genetic Code (SGC) table was investigated with respect to the three-dimensional codon arrangement, and all possible 24 hierarchical base partitions (4! = 24). This was done by determining the amino acid scores for each codon hierarchy in relation to the 1st horizontal, 2nd vertical and 3rd horizontal sub-tables. Marked differences were observed for the hydrophobicity and lipophilicity parameters encoded by the second base of the SGC table. The nucleotide hierarchy U < C < G < A and its complement A < G < C < U at the second base correlated best with the amino acid hydrophobicity and polarity. By contrast, the hierarchy C < G < U < A and its backwards transcript A < U < G < C at the second base were associated with the amino acid parameters of lipophilicity and accessible surface area. No association was observed between 24 base hierarchies of the codons at the 1st and 3rd positions with respect to the hydropathy, polarity, lipophilicity and accessible surface area. The results imply that the second base possesses the majority of information content with respect to the physicochemical properties observed. It is shown that amino acid information obtained by determining the scores of the bases and codon weightings in digital form coincides with physicochemical properties, and the temperature range between 25 °C and 100 °C does not affect the hydrophobicity, the related prediction of α- and ß-protein structure, codon scores, or the complementarity code for sense and antisense peptide interactions. The amino acid scores determined for the SGC table enable the construction of rules and algorithms for the analysis of the structure, function and evolution of proteins. It has been demonstrated that IUPAC-based encoding of nucleobase and amino acid sequences could be used for the representation of the bases with the Semiotic (Greimas) Square and probabilistic square of opposition. It is concluded that the structural, functional and evolutionary patterns of the protein sequences may be modeled using codon based amino acid information, instead of using the information based on amino acid physicochemical properties only.

Sperm RNA code programmes the metabolic health of offspring.

Mammalian sperm RNA is increasingly recognized as an additional source of paternal hereditary information beyond DNA. Environmental inputs, including an unhealthy diet, mental stresses and toxin exposure, can reshape the sperm RNA signature and induce offspring phenotypes that relate to paternal environmental stressors. Our understanding of the categories of sperm RNAs (such as tRNA-derived small RNAs, microRNAs, ribosomal RNA-derived small RNAs and long non-coding RNAs) and associated RNA modifications is expanding and has begun to reveal the functional diversity and information capacity of these molecules. However, the coding mechanism endowed by sperm RNA structures and by RNA interactions with DNA and other epigenetic factors remains unknown. How sperm RNA-encoded information is decoded in early embryos to control offspring phenotypes also remains unclear. Complete deciphering of the 'sperm RNA code' with regard to metabolic control could move the field towards translational applications and precision medicine, and this may lead to prevention of intergenerational transmission of obesity and type 2 diabetes mellitus susceptibility.

[DNA for information storage?] / L'ADN comme mémoire informatique ? - Chroniques génomiques.

The very high information density of DNA has prompted speculations on its use for information storage. The high costs of DNA synthesis and sequencing made this highly unpractical; however recent progress (notably array oligonucleotide synthesis) is changing the situation. A recent paper shows encoding and decoding of significant amounts of data (200 MB) with random access to individual files and faithful retrieval of content, at a cost that is still high but not extreme. Much progress remains to be achieved, but this use of DNA in now technically achievable and may eventually become practical.

Hierarchical groove discrimination by Class I and II aminoacyl-tRNA synthetases reveals a palimpsest of the operational RNA code in the tRNA acceptor-stem bases.

Class I and II aaRS recognition of opposite grooves was likely among the earliest determinants fixed in the tRNA acceptor stem bases. A new regression model identifies those determinants in bacterial tRNAs. Integral coefficients relate digital dependent to independent variables with perfect agreement between observed and calculated grooves for all twenty isoaccepting tRNAs. Recognition is mediated by the Discriminator base 73, the first base pair, and base 2 of the acceptor stem. Subsets of these coefficients also identically compute grooves recognized by smaller numbers of aaRS. Thus, the model is hierarchical, suggesting that new rules were added to pre-existing ones as new amino acids joined the coding alphabet. A thermodynamic rationale for the simplest model implies that Class-dependent aaRS secondary structures exploited differential tendencies of the acceptor stem to form the hairpin observed in Class I aaRS•tRNA complexes, enabling the earliest groove discrimination. Curiously, groove recognition also depends explicitly on the identity of base 2 in a manner consistent with the middle bases of the codon table, confirming a hidden ancestry of codon-anticodon pairing in the acceptor stem. That, and the lack of correlation with anticodon bases support prior productive coding interaction of tRNA minihelices with proto-mRNA.

Causation, constructors and codes.

Relational biology relies heavily on the enriched understanding of causal entailment that Robert Rosen's formalisation of Aristotle's four causes has made possible, although to date efficient causes and the rehabilitation of final cause have been its main focus. Formal cause has been paid rather scant attention, but, as this paper demonstrates, is crucial to our understanding of many types of processes, not necessarily biological. The graph-theoretic relational diagram of a mapping has played a key role in relational biology, and the first part of the paper is devoted to developing an explicit representation of formal cause in the diagram and how it acts in combination with efficient cause to form a mapping. I then use these representations to show how Von Neumann's universal constructor can be cast into a relational diagram in a way that avoids the logical paradox that Rosen detected in his own representation of the constructor in terms of sets and mappings. One aspect that was absent from both Von Neumann's and Rosen's treatments was the necessity of a code to translate the description (the formal cause) of the automaton to be constructed into the construction process itself. A formal definition of codes in general, and organic codes in particular, allows the relational diagram to be extended so as to capture this translation of formal cause into process. The extended relational diagram is used to exemplify causal entailment in a diverse range of processes, such as enzyme action, construction of automata, communication through the Morse code, and ribosomal polypeptide synthesis through the genetic code.

Mathematical fundamentals for the noise immunity of the genetic code.

Symmetry is one of the essential and most visible patterns that can be seen in nature. Starting from the left-right symmetry of the human body, all types of symmetry can be found in crystals, plants, animals and nature as a whole. Similarly, principals of symmetry are also some of the fundamental and most useful tools in modern mathematical natural science that play a major role in theory and applications. As a consequence, it is not surprising that the desire to understand the origin of life, based on the genetic code, forces us to involve symmetry as a mathematical concept. The genetic code can be seen as a key to biological self-organisation. All living organisms have the same molecular bases - an alphabet consisting of four letters (nitrogenous bases): adenine, cytosine, guanine, and thymine. Linearly ordered sequences of these bases contain the genetic information for synthesis of proteins in all forms of life. Thus, one of the most fascinating riddles of nature is to explain why the genetic code is as it is. Genetic coding possesses noise immunity which is the fundamental feature that allows to pass on the genetic information from parents to their descendants. Hence, since the time of the discovery of the genetic code, scientists have tried to explain the noise immunity of the genetic information. In this chapter we will discuss recent results in mathematical modelling of the genetic code with respect to noise immunity, in particular error-detection and error-correction. We will focus on two central properties: Degeneracy and frameshift correction. DEGENERACY: Different amino acids are encoded by different quantities of codons and a connection between this degeneracy and the noise immunity of genetic information is a long standing hypothesis. Biological implications of the degeneracy have been intensively studied and whether the natural code is a frozen accident or a highly optimised product of evolution is still controversially discussed. Symmetries in the structure of degeneracy of the genetic code are essential and give evidence of substantial advantages of the natural code over other possible ones. In the present chapter we will present a recent approach to explain the degeneracy of the genetic code by algorithmic methods from bioinformatics, and discuss its biological consequences. FRAMESHIFT CORRECTION: The biologists recognised this problem immediately after the detection of the non-overlapping structure of the genetic code, i.e., coding sequences are to be read in a unique way determined by their reading frame. But how does the reading head of the ribosome recognises an error in the grouping of codons, caused by e.g. insertion or deletion of a base, that can be fatal during the translation process and may result in nonfunctional proteins? In this chapter we will discuss possible solutions to the frameshift problem with a focus on the theory of so-called circular codes that were discovered in large gene populations of prokaryotes and eukaryotes in the early 90s. Circular codes allow to detect a frameshift of one or two positions and recently a beautiful theory of such codes has been developed using statistics, group theory and graph theory.

Towards measuring the semantic capacity of a physical medium demonstrated with elementary cellular automata.

The organic code concept and its operationalization by molecular codes have been introduced to study the semiotic nature of living systems. This contribution develops further the idea that the semantic capacity of a physical medium can be measured by assessing its ability to implement a code as a contingent mapping. For demonstration and evaluation, the approach is applied to a formal medium: elementary cellular automata (ECA). The semantic capacity is measured by counting the number of ways codes can be implemented. Additionally, a link to information theory is established by taking multivariate mutual information for quantifying contingency. It is shown how ECAs differ in their semantic capacities, how this is related to various ECA classifications, and how this depends on how a meaning is defined. Interestingly, if the meaning should persist for a certain while, the highest semantic capacity is found in CAs with apparently simple behavior, i.e., the fixed-point and two-cycle class. Synergy as a predictor for a CA's ability to implement codes can only be used if context implementing codes are common. For large context spaces with sparse coding contexts synergy is a weak predictor. Concluding, the approach presented here can distinguish CA-like systems with respect to their ability to implement contingent mappings. Applying this to physical systems appears straight forward and might lead to a novel physical property indicating how suitable a physical medium is to implement a semiotic system.

Fundamental principles of the olfactory code.

Sensory coding represents a basic principle of all phyla in nature: species attempt to perceive their natural surroundings and to make sense of them. Ultimately, sensory coding is the only way to allow a species to make the kinds of crucial decisions that lead to a behavioral response. In this manner, animals are able to detect numerous parameters, ranging from temperature and humidity to light and sound to volatile or non-volatile chemicals. Most of these environmental cues represent a clearly defined stimulus array that can be described along a single physical parameter, such as wavelength or frequency; odorants, in contrast, cannot. The odor space encompasses an enormous and nearly infinite number of diverse stimuli that cannot be classified according to their positions along a single dimension. Hence, the olfactory system has to encode and translate the vast odor array into an accurate neural map in the brain. In this review, we will outline the relevant steps of the olfactory code and describe its progress along the olfactory pathway, i.e., from the peripheral olfactory organs to the first olfactory center in the brain and then to the higher processing areas where the odor perception takes place, enabling an organism to make odor-guided decisions. We will focus mainly on studies from the vinegar fly Drosophila melanogaster, but we will also indicate similarities to and differences from the olfactory system of other invertebrate species as well as of the vertebrate world.

Cyclic Concatenated Genetic Encoder: A mathematical proposal for biological inferences.

The organization of the genetic information and its ability to be conserved and translated to proteins with low error rates have been the subject of study by scientists from different disciplines. Recently, it has been proposed that living organisms display an intra-cellular transmission system of genetic information, similar to a model of digital communication system, in which there is the ability to detect and correct errors. In this work, the concept of Concatenated Genetic Encoder is introduced and applied to the analysis of protein sequences as a tool for exploring evolutionary relationships. For such purposes Error Correcting Codes (ECCs) are used to represent proteins. A methodology for representing or identifying proteins by use of BCH codes over ℤ20 and F4×ℤ5 is proposed and cytochrome b6-f complex subunit 6-OS sequences, corresponding to different plants species, are analyzed according to the proposed methodology and results are contrasted to phylogenetic and taxonomic analyses. Through the analyses, it was observed that using BCH codes only some sequences are identified, all of which differ in one amino acid from the original sequence. In addition, mathematical relationships among identified sequences are established by considering minimal polynomials, where such sequences showed a close relationship as revealed in the phylogenetic reconstruction. Results, here shown, point out that communication theory may provide biology of interesting and useful tools to identify biological relationships among proteins, however the proposed methodology needs to be improved and rigorously tested in order to become into an applicable tool for biological analysis.