The bottom-up branch of synthetic biology includes—among others—innovative studies that combine cell-free protein synthesis with liposome technology to generate cell-like systems of minimal complexity, often referred to as synthetic cells. The functions of this type of synthetic cell derive from gene expression, hence they can be programmed in a modular, progressive and customizable manner by means of ad hoc designed genetic circuits. This experimental scenario is rapidly expanding and synthetic cell research already counts numerous successes. Here, we present a review focused on the exchange of chemical signals between liposome-based synthetic cells (operating by gene expression) and biological cells, as well as between two populations of synthetic cells. The review includes a short presentation of the “molecular communication technologies,” briefly discussing their promises and challenges.

The long-term use of antibiotics has led to the emergence of multidrug-resistant bacteria. A promising strategy to combat bacterial infections aims at hampering their adaptability to the host environment without affecting growth. In this context, the intercellular communication system quorum sensing (QS), which controls virulence factor production and biofilm formation in diverse human pathogens, is considered an ideal target. Here, we describe the identification of new inhibitors of the QS system of the human pathogen by screening a library of 1,600 U.S. Food and Drug Administration-approved drugs. Phenotypic characterization of engineered strains and molecular docking demonstrated that the antifungal drugs clotrimazole and miconazole, as well as an antibacterial compound active against Gram-positive pathogens, clofoctol, inhibit the system, probably by targeting the transcriptional regulator PqsR. The most active inhibitor, clofoctol, specifically inhibited the expression of-controlled virulence traits in , such as pyocyanin production, swarming motility, biofilm formation, and expression of genes involved in siderophore production. Moreover, clofoctol protected larvae from infection and inhibited the QS system in isolates from cystic fibrosis patients. Notably, clofoctol is already approved for clinical treatment of pulmonary infections caused by Gram-positive bacterial pathogens; hence, this drug has considerable clinical potential as an antivirulence agent for the treatment of lung infections.

Recent developments in bottom-up synthetic biology (e.g., lipid vesicle technology integrated with cell-free protein expression systems) allow the generation of semi-synthetic minimal cells (in short, synthetic cells, SCs) endowed with some distinctive capacities of natural cells. In particular, such approaches provide technological tools and conceptual frameworks for the design and engineering of programmable SCs capable of communicating with natural cells by exchanging chemical signals. Here we describe the generation of giant vesicle-based SCs which, via gene expression, synthesize in their aqueous lumen an enzyme that in turn produces a chemical signal. The latter is a small molecule, which is passively released in the medium and then perceived by the bacterium Pseudomonas aeruginosa, demonstrating that SCs and bacteria can communicate chemically. The results pave the way to a novel basic and applied research area where synthetic cells can communicate with natural cells, for example for exploring minimal cognition, developing chemical information technologies, and producing smart and programmable drug-producing/drug-delivery systems.

The aim of the present study was to determine the role of milk endogenous proteolytic enzymes in sheep milk cheesemaking ability during lactation. Plasmin, plasminogen, and plasminogen activator in ewe bulk milk were not significantly affected by stage of lactation, probably because of the good health of the ewe udders throughout lactation as indicated by somatic cell count, which never exceeded 600,000 cells/mL. Elastase content increased significantly during lactation, whereas cathepsin showed the greatest content in mid lactation. Early and mid lactation milk showed impaired renneting parameter compared with late lactation milk, probably because of greater alpha-casein degradation, brought about by cathepsin, and lesser fat and casein (CN) milk contents. Changes in macrophage and neutrophil levels in ewe bulk milk during lactation were also investigated. Macrophages minimally contributed to leukocyte cell count in milk and had the greatest levels at the beginning of lactation. An opposite trend was recorded for polymorphonuclear neutrophilic leucocytes (PMNL) that increased throughout lactation, showing the greatest value in late lactation. Urea-PAGE of sodium caseinate (NaCN) incubated with isolated and concentrated PMNL at 37 degrees C after 48 h at pH 8 showed massive casein degradation that could be ascribed to proteases yielded by PMNL. The increase of PMNL percentage and elastase content in milk, despite the relatively low SCC, suggests that PMNL and elastase underwent a physiological increase associated to the remodeling of mammary gland in late lactation.

Iron-sulfur (Fe-S) clusters are ubiquitous co-factors essential for life. It is largely thought that the emergence of oxygenic photosynthesis and progressive oxygenation of the atmosphere led to the origin of multiprotein machineries (ISC, NIF, and SUF) assisting Fe-S cluster synthesis in the presence of oxidative stress and shortage of bioavailable iron. However, previous analyses have left unclear the origin and evolution of these systems. Here, we combine exhaustive homology searches with genomic context analysis and phylogeny to precisely identify Fe-S cluster biogenesis systems in over 10,000 archaeal and bacterial genomes. We highlight the existence of two additional and clearly distinct "minimal" Fe-S cluster assembly machineries, MIS and SMS, which we infer in the Last Universal Common Ancestor (LUCA), and we experimentally validate SMS as a bona fide Fe-S cluster biogenesis system. These ancestral systems were kept in Archaea whereas they went through stepwise complexification in Bacteria to incorporate additional functions for higher Fe-S cluster synthesis efficiency leading to SUF, ISC, and NIF. Horizontal gene transfers and losses then shaped the current distribution of these systems, driving ecological adaptations such as the emergence of aerobic lifestyles in archaea. Our results show that dedicated machineries were in place early in evolution to assist Fe-S cluster biogenesis, and that their origin is not directly linked to Earth oxygenation.

The encapsulation of transcription–translation (TX–TL) machinery inside lipid vesicles and water-in-oil droplets leads to the construction of cytomimetic systems (often called ‘synthetic cells’) for synthetic biology and origins-of-life research. A number of recent reports have shown that protein synthesis inside these microcompartments is highly diverse in terms of rate and amount of synthesized protein. Here, we discuss the role of extrinsic stochastic effects (i.e. solute partition phenomena) as relevant factors contributing to this pattern. We evidence and discuss cases where between-compartment diversity seems to exceed the expected theoretical values. The need of accurate determination of solute content inside individual vesicles or droplets is emphasized, aiming at validating or rejecting the predictions calculated from the standard fluctuations theory. At the same time, we promote the integration of experiments and stochastic modeling to reveal the details of solute encapsulation and intra-compartment reactions.

The emergence of antibiotic resistant bacterial pathogens is increasing at an unprecedented pace, calling for the development of new therapeutic options. Small molecules interfering with virulence processes rather than growth hold promise as an alternative to conventional antibiotics. Anti-virulence agents are expected to decrease bacterial virulence and to pose reduced selective pressure for the emergence of resistance. In the opportunistic pathogen Pseudomonas aeruginosa the expression of key virulence traits is controlled by quorum sensing (QS), an intercellular communication process that coordinates gene expression at the population level. Hence, QS inhibitors represent promising anti-virulence agents against P. aeruginosa. Virtual screenings allow fast and cost-effective selection of target ligands among vast libraries of molecules, thus accelerating the time and limiting the cost of conventional drug-discovery processes, while the drug-repurposing approach is based on the identification of off-target activity of FDA-approved drugs, likely endowed with low cytotoxicity and favorable pharmacological properties. This study aims at combining the advantages of virtual screening and drug-repurposing approaches to identify new QS inhibitors targeting the pqs QS system of P. aeruginosa. An in silico library of 1,467 FDA-approved drugs has been screened by molecular docking, and 5 hits showing the highest predicted binding affinity for the pqs QS receptor PqsR (also known as MvfR) have been selected. In vitro experiments have been performed by engineering ad hoc biosensor strains, which were used to verify the ability of hit compounds to decrease PqsR activity in P. aeruginosa. Phenotypic analyses confirmed the impact of the most promising hit, the antipsychotic drug pimozide, on the expression of P. aeruginosa PqsR-controlled virulence traits. Overall, this study highlights the potential of virtual screening campaigns of FDA-approved drugs to rapidly select new inhibitors of important bacterial functions.

In this article we present novel aspects of the\ud impact that synthetic biology (SB) can express in a field\ud traditionally based on computer science: information and\ud communication technologies (ICTs), an area that we will\ud consider taking into account also possible implications for\ud artificial intelligence (AI) research. In the first part of this\ud article we will shortly introduce some recent theoretical\ud and experimental issues related to our approach in SB,\ud discussing their relevance and potentiality in the field.\ud Next, we define an original SB research programme that\ud aims at contributing to the development of bio-chem-ICTs\ud and AI based on chemical communication between natural\ud and synthetic cells. In particular we present (i) a mathematical\ud model that allows us to simulate the main features\ud of the system under construction; and (ii) preliminary wetlab\ud experiments showing the feasibility of our research\ud programme. Based on the bottom-up construction of synthetic\ud cells, the traits of this novel approach and their\ud connections with recent conceptual and technological\ud trends are finally discusse