Living cells and biological mechanisms as prototypes for developing chemical artificial intelligence.

Artificial Intelligence (AI) is having a revolutionary impact on our societies. It is helping humans in facing the global challenges of this century. Traditionally, AI is developed in software or through neuromorphic engineering in hardware. More recently, a brand-new strategy has been proposed. It is the so-called Chemical AI (CAI), which exploits molecular, supramolecular, and systems chemistry in wetware to mimic human intelligence. In this work, two promising approaches for boosting CAI are described. One regards designing and implementing neural surrogates that can communicate through optical or chemical signals and give rise to networks for computational purposes and to develop micro/nanorobotics. The other approach concerns "bottom-up synthetic cells" that can be exploited for applications in various scenarios, including future nano-medicine. Both topics are presented at a basic level, mainly to inform the broader audience of non-specialists, and so favour the rise of interest in these frontier subjects.

Chromatophores efficiently promote light-driven ATP synthesis and DNA transcription inside hybrid multicompartment artificial cells.

The construction of energetically autonomous artificial protocells is one of the most ambitious goals in bottom-up synthetic biology. Here, we show an efficient manner to build adenosine 5'-triphosphate (ATP) synthesizing hybrid multicompartment protocells. Bacterial chromatophores from Rhodobacter sphaeroides accomplish the photophosphorylation of adenosine 5'-diphosphate (ADP) to ATP, functioning as nanosized photosynthetic organellae when encapsulated inside artificial giant phospholipid vesicles (ATP production rate up to ∼100 ATP∙s-1 per ATP synthase). The chromatophore morphology and the orientation of the photophosphorylation proteins were characterized by cryo-electron microscopy (cryo-EM) and time-resolved spectroscopy. The freshly synthesized ATP has been employed for sustaining the transcription of a DNA gene, following the RNA biosynthesis inside individual vesicles by confocal microscopy. The hybrid multicompartment approach here proposed is very promising for the construction of full-fledged artificial protocells because it relies on easy-to-obtain and ready-to-use chromatophores, paving the way for artificial simplified-autotroph protocells (ASAPs).

Explorative Synthetic Biology in AI: Criteria of Relevance and a Taxonomy for Synthetic Models of Living and Cognitive Processes.

This article tackles the topic of the special issue "Biology in AI: New Frontiers in Hardware, Software and Wetware Modeling of Cognition" in two ways. It addresses the problem of the relevance of hardware, software, and wetware models for the scientific understanding of biological cognition, and it clarifies the contributions that synthetic biology, construed as the synthetic exploration of cognition, can offer to artificial intelligence (AI). The research work proposed in this article is based on the idea that the relevance of hardware, software, and wetware models of biological and cognitive processes-that is, the concrete contribution that these models can make to the scientific understanding of life and cognition-is still unclear, mainly because of the lack of explicit criteria to assess in what ways synthetic models can support the experimental exploration of biological and cognitive phenomena. Our article draws on elements from cybernetic and autopoietic epistemology to define a framework of reference, for the synthetic study of life and cognition, capable of generating a set of assessment criteria and a classification of forms of relevance, for synthetic models, able to overcome the sterile, traditional polarization of their evaluation between mere imitation and full reproduction of the target processes. On the basis of these tools, we tentatively map the forms of relevance characterizing wetware models of living and cognitive processes that synthetic biology can produce and outline a programmatic direction for the development of "organizationally relevant approaches" applying synthetic biology techniques to the investigative field of (embodied) AI.

Chemical Systems for Wetware Artificial Life: Selected Perspectives in Synthetic Cell Research.

The recent and important advances in bottom-up synthetic biology (SB), in particular in the field of the so-called "synthetic cells" (SCs) (or "artificial cells", or "protocells"), lead us to consider the role of wetware technologies in the "Sciences of Artificial", where they constitute the third pillar, alongside the more well-known pillars hardware (robotics) and software (Artificial Intelligence, AI). In this article, it will be highlighted how wetware approaches can help to model life and cognition from a unique perspective, complementary to robotics and AI. It is suggested that, through SB, it is possible to explore novel forms of bio-inspired technologies and systems, in particular chemical AI. Furthermore, attention is paid to the concept of semantic information and its quantification, following the strategy recently introduced by Kolchinsky and Wolpert. Semantic information, in turn, is linked to the processes of generation of "meaning", interpreted here through the lens of autonomy and cognition in artificial systems, emphasizing its role in chemical ones.

A Role for Bottom-Up Synthetic Cells in the Internet of Bio-Nano Things?

The potential role of bottom-up Synthetic Cells (SCs) in the Internet of Bio-Nano Things (IoBNT) is discussed. In particular, this perspective paper focuses on the growing interest in networks of biological and/or artificial objects at the micro- and nanoscale (cells and subcellular parts, microelectrodes, microvessels, etc.), whereby communication takes place in an unconventional manner, i.e., via chemical signaling. The resulting "molecular communication" (MC) scenario paves the way to the development of innovative technologies that have the potential to impact biotechnology, nanomedicine, and related fields. The scenario that relies on the interconnection of natural and artificial entities is briefly introduced, highlighting how Synthetic Biology (SB) plays a central role. SB allows the construction of various types of SCs that can be designed, tailored, and programmed according to specific predefined requirements. In particular, "bottom-up" SCs are briefly described by commenting on the principles of their design and fabrication and their features (in particular, the capacity to exchange chemicals with other SCs or with natural biological cells). Although bottom-up SCs still have low complexity and thus basic functionalities, here, we introduce their potential role in the IoBNT. This perspective paper aims to stimulate interest in and discussion on the presented topics. The article also includes commentaries on MC, semantic information, minimal cognition, wetware neuromorphic engineering, and chemical social robotics, with the specific potential they can bring to the IoBNT.

Exploiting the photoactivity of bacterial reaction center to investigate liposome dynamics.

Charge recombination kinetics of bacterial photosynthetic protein Reaction Center displays an exquisite sensitivity to the actual occupancy of ubiquinone-10 in its QB-binding site. Here, we have exploited such phenomenon for assessing the growth and the aggregation/fusion of phosphocholine vesicles embedding RC in their membrane, when treated with sodium oleate.

Highly oriented photosynthetic reaction centers generate a proton gradient in synthetic protocells.

Photosynthesis is responsible for the photochemical conversion of light into the chemical energy that fuels the planet Earth. The photochemical core of this process in all photosynthetic organisms is a transmembrane protein called the reaction center. In purple photosynthetic bacteria a simple version of this photoenzyme catalyzes the reduction of a quinone molecule, accompanied by the uptake of two protons from the cytoplasm. This results in the establishment of a proton concentration gradient across the lipid membrane, which can be ultimately harnessed to synthesize ATP. Herein we show that synthetic protocells, based on giant lipid vesicles embedding an oriented population of reaction centers, are capable of generating a photoinduced proton gradient across the membrane. Under continuous illumination, the protocells generate a gradient of 0.061 pH units per min, equivalent to a proton motive force of 3.6 mV⋅min-1 Remarkably, the facile reconstitution of the photosynthetic reaction center in the artificial lipid membrane, obtained by the droplet transfer method, paves the way for the construction of novel and more functional protocells for synthetic biology.

Gene Expression Inside Liposomes: From Early Studies to Current Protocols.

Synthesizing proteins inside liposomes and other microcompartments is a well-established practice. However, the origin of this research is not from the distant past, dating back to 1999-2004, when the first successful attempts were published. Protein synthesis inside artificial compartments is now under strong expansion in the context of synthetic biology (in bottom-up approaches), and, in particular, it strongly contributes to the construction of artificial cell-like systems. These systems, often called "synthetic cells", can be used to model cellular processes, including membrane-centered ones. They are very innovative models that complement traditional studies and promise future applications. This review does not discuss all current directions in synthetic cell research; in particular, it does not include all kinds of artificial compartments. Instead, it is uniquely dedicated to the analysis of historical and technical developments of protein synthesis inside liposomes, highlighting a selected list of open questions. One of the goals is to note the importance of mastering liposome technology together with cell-free systems for the successful realization of this specific type of synthetic cell. With this aim, four currently employed protocols are compared and discussed, with a major emphasis on the droplet transfer method, which is attractive due to its simplicity and encapsulation efficiency.

Human Serum Albumin Is an Essential Component of the Host Defense Mechanism Against Clostridium difficile Intoxication.

Background: The pathogenic effects of Clostridium difficile are primarily attributable to the production of the large protein toxins (C difficile toxins [Tcd]) A (TcdA) and B (TcdB). These toxins monoglucosylate Rho GTPases in the cytosol of host cells, causing destruction of the actin cytoskeleton with cytotoxic effects. Low human serum albumin (HSA) levels indicate a higher risk of acquiring and developing a severe C difficile infection (CDI) and are associated with recurrent and fatal disease. Methods: We used a combined approach based on docking simulation and biochemical analyses that were performed in vitro on purified proteins and in human epithelial colorectal adenocarcinoma cells (Caco-2), and in vivo on stem cell-derived human intestinal organoids and zebrafish embryos. Results: Our results show that HSA specifically binds via its domain II to TcdA and TcdB and thereby induces their autoproteolytic cleavage at physiological concentrations. This process impairs toxin internalization into the host cells and reduces the toxin-dependent glucosylation of Rho proteins. Conclusions: Our data provide evidence for a specific HSA-dependent self-defense mechanism against C difficile toxins and provide an explanation for the clinical correlation between CDI severity and hypoalbuminemia.

Antioxidant activity of hydroxytyrosyl esters studied in liposome models.

The properties and the antioxidant activity of a series of hydroxytyrosyl esters having different carbon chain lengths (C4, C8, C12 and C18) have been measured in phosphatidylcholine model membrane (liposomes) using specific probes for the bilayer and liposome lumen microenvironment, i.e., 1,6-diphenyl-1,3,5-hexatriene (DPH) and 2',7'-dichlorodihydrofluorescein (H2DCF), respectively. Antioxidants self-assembly and their interaction with liposomes has been evaluated by light scattering, fluorescence, turbidimetry, gel filtration chromatography and microfiltration measurements, allowing the determination of critical aggregation concentration, bound fraction, capacity of crossing the lipid bilayer. The distribution of hydroxytyrosyl long chain esters has been proved to depend quite specifically on their lipophilic chain length, and this turns to have deep effects on their antioxidant behaviour. Shedding new light on the cut off effect and antioxidant behaviour of phenolipids, this study also put forward the relevance of cell-free liposome-based cellular models, like giant liposomes, for further characterization of analogous systems.

Spectroscopic and calorimetric characterization of spermine oxidase and its association forms.

Spermine oxidase (SMOX) is a flavin-containing enzyme that oxidizes spermine to produce spermidine, 3-aminopropanaldehyde, and hydrogen peroxide. SMOX has been shown to play key roles in inflammation and carcinogenesis; indeed, it is differentially expressed in several human cancer types. Our previous investigation has revealed that SMOX purified after heterologous expression in Escherichia coli actually consists of monomers, covalent homodimers, and other higher-order forms. All association forms oxidize spermine and, after treatment with dithiothreitol, revert to SMOX monomer. Here, we report a detailed investigation on the thermal denaturation of SMOX and its association forms in native and reducing conditions. By combining spectroscopic methods (circular dichroism, fluorescence) and thermal methods (differential scanning calorimetry), we provide new insights into the structure, the transformation, and the stability of SMOX. While the crystal structure of this protein is not available yet, experimental results are interpreted also on the basis of a novel SMOX structural model, obtained in silico exploiting the recently solved acetylspermine oxidase crystal structure. We conclude that while at least one specific intermolecular disulfide bond links two SMOX molecules to form the homodimer, the thermal denaturation profiles can be justified by the presence of at least one intramolecular disulfide bond, which also plays a critical role in the stabilization of the overall three-dimensional SMOX structure, and in particular of its flavin adenine dinucleotide-containing active site.

The Arabidopsis polyamine oxidase/dehydrogenase 5 interferes with cytokinin and auxin signaling pathways to control xylem differentiation.

In plants, the polyamines putrescine, spermidine, spermine (Spm), and thermospermine (Therm-Spm) participate in several physiological processes. In particular, Therm-Spm is involved in the control of xylem differentiation, having an auxin antagonizing effect. Polyamine oxidases (PAOs) are FAD-dependent enzymes involved in polyamine catabolism. In Arabidopsis, five PAOs are present, among which AtPAO5 catalyzes the back-conversion of Spm, Therm-Spm, and N1-acetyl-Spm to spermidine. In the present study, it is shown that two loss-of-function atpao5 mutants and a 35S::AtPAO5 Arabidopsis transgenic line present phenotypical differences from the wild-type plants with regard to stem and root elongation, differences that are accompanied by changes in polyamine levels and the number of xylem vessels. It is additionally shown that cytokinin treatment, which up-regulates AtPAO5 expression in roots, differentially affects protoxylem differentiation in 35S::AtPAO5, atpao5, and wild-type roots. Together with these findings, Therm-Spm biosynthetic genes, as well as auxin-, xylem-, and cytokinin-related genes (such as ACL5, SAMDC4, PIN1, PIN6, VND6, VND7, ATHB8, PHB, CNA, PXY, XTH3, XCP1, and AHP6) are shown to be differentially expressed in the various genotypes. These data suggest that AtPAO5, being involved in the control of Therm-Spm homeostasis, participates in the tightly controlled interplay between auxin and cytokinins that is necessary for proper xylem differentiation.

Vesicle aggregates as a model for primitive cellular assemblies.

Primitive cell models help to understand the role that compartmentalization plays in origin of life scenarios. Here we present a combined experimental and modeling approach towards the construction of simple model systems for primitive cellular assemblies. Charged lipid vesicles aggregate in the presence of oppositely charged biopolymers, such as nucleic acids or polypeptides. Based on zeta potential measurements, dynamic light scattering and cryo-transmission electron-microscopy, we have characterized the behavior of empty and ferritin-filled large unilamellar POPC vesicles, doped with different amounts of cationic (DDAB, CTAB) and anionic (sodium oleate) surfactants, and their aggregation upon the addition of anionic (tRNA, poly-l-glutamic acid) and cationic (poly-l-arginine) biopolymers, respectively. The experimental results are rationalized by a phenomenological modeling approach that predicts the average size of the vesicle aggregates as function of the amount of added biopolymers. In addition, we discuss the mechanism of vesicle aggregation induced by oppositely charged biopolymers. Our study complements previous reports about the formation of giant vesicle clusters and thus provides a general vista on primitive cell systems, based on the association of vesicles into compartmentalized aggregates.

Stability of spermine oxidase to thermal and chemical denaturation: comparison with bovine serum amine oxidase.

Spermine oxidase (SMOX) is a flavin-containing enzyme that specifically oxidizes spermine to produce spermidine, 3-aminopropanaldehyde and hydrogen peroxide. While no crystal structure is available for any mammalian SMOX, X-ray crystallography showed that the yeast Fms1 polyamine oxidase has a dimeric structure. Based on this scenario, we have investigated the quaternary structure of the SMOX protein by native gel electrophoresis, which revealed a composite gel band pattern, suggesting the formation of protein complexes. All high-order protein complexes are sensitive to reducing conditions, showing that disulfide bonds were responsible for protein complexes formation. The major gel band other than the SMOX monomer is the covalent SMOX homodimer, which was disassembled by increasing the reducing conditions, while being resistant to other denaturing conditions. Homodimeric and monomeric SMOXs are catalytically active, as revealed after gel staining for enzymatic activity. An engineered SMOX mutant deprived of all but two cysteine residues was prepared and characterized experimentally, resulting in a monomeric species. High-sensitivity differential scanning calorimetry of SMOX was compared with that of bovine serum amine oxidase, to analyse their thermal stability. Furthermore, enzymatic activity assays and fluorescence spectroscopy were used to gain insight into the unfolding process.

Identification of an estrogen receptor α non covalent ubiquitin-binding surface: role in 17ß-estradiol-induced transcriptional activity.

Ubiquitin (Ub)-binding domains (UBDs) located in Ub receptors decode the ubiquitination signal by non-covalently engaging the Ub modification on their binding partners and transduce the Ub signalling through Ub-based molecular interactions. In this way, inducible protein ubiquitination regulates diverse biological processes. The estrogen receptor alpha (ERα) is a ligand-activated transcription factor that mediates the pleiotropic effects of the sex hormone 17ß-estradiol (E2). Fine regulation of E2 pleiotropic actions depends on E2-dependent ERα association with a plethora of binding partners and/or on the E2 modulation of receptor ubiquitination. Indeed, E2-induced ERα polyubiquitination triggers receptor degradation and transcriptional activity, and E2-dependent reduction in ERα monoubiquitination is crucial for E2 signalling. Monoubiquitinated proteins often contain UBDs, but whether non-covalent Ub-ERα binding could occur and play a role in E2-ERα signalling is unknown. Here, we report an Ub-binding surface within the ERα ligand binding domain that directs in vitro the receptor interaction with both ubiquitinated proteins and recombinant Ub chains. Mutational analysis reveals that ERα residues leucine 429 and alanine 430 are involved in Ub binding. Moreover, impairment of ERα association to ubiquitinated species strongly affects E2-induced ERα transcriptional activity. Considering the importance of UBDs in the Ub-based signalling network and the central role of different ERα binding partners in the modulation of E2-dependent effects, our discoveries provide novel insights into ERα activity that could also be relevant for ERα-dependent diseases.

Protein Synthesis in Sub-Micrometer Water-in-Oil Droplets.

Water-in-oil (w/o) emulsions are used as a cellular model because of their unique cell-like architecture. Previous works showed the capability of eukaryotic-cell-sized w/o droplets (5-50 µm) to support protein synthesis efficiently; however data about smaller w/o compartments (<1 µm) are lacking. This work focuses on the biosynthesis of the enhanced green fluorescent protein (EGFP) inside sub-micrometric lecithin-based w/o droplets (0.8-1 µm) and on its dependence on the compartments' dynamic properties in terms of solute exchange mechanisms. We demonstrated that protein synthesis is strongly affected by the nature of the lipid interface. These findings could be of value and interest for both basic and applied research.

Pacific oyster polyamine oxidase: a protein missing link in invertebrate evolution.

Polyamine oxidases catalyse the oxidation of polyamines and acetylpolyamines and are responsible for the polyamine interconversion metabolism in animal cells. Polyamine oxidases from yeast can oxidize spermine, N(1)-acetylspermine, and N(1)-acetylspermidine, while in vertebrates two different enzymes, namely spermine oxidase and acetylpolyamine oxidase, specifically catalyse the oxidation of spermine, and N(1)-acetylspermine/N(1)-acetylspermidine, respectively. In this work we proved that the specialized vertebrate spermine and acetylpolyamine oxidases have arisen from an ancestor invertebrate polyamine oxidase with lower specificity for polyamine substrates, as demonstrated by the enzymatic activity of the mollusc polyamine oxidase characterized here. This is the first report of an invertebrate polyamine oxidase, the Pacific oyster Crassostrea gigas (CgiPAO), overexpressed as a recombinant protein. This enzyme was biochemically characterized and demonstrated to be able to oxidase both N(1)-acetylspermine and spermine, albeit with different efficiency. Circular dichroism analysis gave an estimation of the secondary structure content and modelling of the three-dimensional structure of this protein and docking studies highlighted active site features. The availability of this pluripotent enzyme can have applications in crystallographic studies and pharmaceutical biotechnologies, including anticancer therapy as a source of hydrogen peroxide able to induce cancer cell death.

A Simple Protein Synthesis Model for the PURE System Operation.

The encapsulation of transcription-translation (TX-TL) cell-free machinery inside lipid vesicles (liposomes) is a key element in synthetic cell technology. The PURE system is a TX-TL kit composed of well-characterized parts, whose concentrations are fine tunable, which works according to a modular architecture. For these reasons, the PURE system perfectly fulfils the requirements of synthetic biology and is widely used for constructing synthetic cells. In this work, we present a simplified mathematical model to simulate the PURE system operations. Based on Michaelis-Menten kinetics and differential equations, the model describes protein synthesis dynamics by using 9 chemical species, 6 reactions and 16 kinetic parameters. The model correctly predicts the time course for messenger RNA and protein production and allows quantitative predictions. By means of this model, it is possible to foresee how the PURE system species affect the mechanism of proteins synthesis and therefore help in understanding scenarios where the concentration of the PURE system components has been modified purposely or as a result of stochastic fluctuations (for example after random encapsulation inside vesicles). The model also makes the determination of response coefficients for all species involved in the TX-TL mechanism possible and allows for scrutiny on how chemical energy is consumed by the three PURE system modules (transcription, translation and aminoacylation).

The Glu²¹6/Ser²¹8 pocket is a major determinant of spermine oxidase substrate specificity.

SMO (spermine oxidase) and APAO (acetylpolyamine oxidase) are flavoenzymes that play a critical role in the catabolism of polyamines. Polyamines are basic regulators of cell growth and proliferation and their homoeostasis is crucial for cell life since dysregulation of polyamine metabolism has been linked with cancer. In vertebrates SMO specifically catalyses the oxidation of spermine, whereas APAO displays a wider specificity, being able to oxidize both N¹-acetylspermine and N¹-acetylspermidine, but not spermine. The molecular bases of the different substrate specificity of these two enzymes have remained so far elusive. However, previous molecular modelling, site-directed mutagenesis and biochemical characterization studies of the SMO enzyme-substrate complex have identified Glu²¹6-Ser²¹8 as a putative active site hot spot responsible for SMO substrate specificity. On the basis of these analyses, the SMO double mutants E216L/S218A and E216T/S218A have been produced and characterized by CD spectroscopy and steady-state and rapid kinetics experiments. The results obtained demonstrate that mutation E216L/S218A endows SMO with N¹-acetylspermine oxidase activity, uncovering one of the structural determinants that confer the exquisite and exclusive substrate specificity of SMO for spermine. These results provide the theoretical bases for the design of specific inhibitors either for SMO or APAO.