A simple software, to be used as an aid in the identification of non-tryptic peptides based on low resolution (3D-ion trap) tandem (MS/MS) and sequential (MS3) mass spectrometry data, is presented. The program, named INSPIRE (Identification of Non-tryptic peptide Sequences based on Product Ions m/z ratios and RElative abundances), provides alternative rankings for the several candidate sequences usually arising from protein database searches when non-tryptic peptides are involved and only low resolution MS/MS data are available. The rankings, based on parameters related to m/z ratios and relative abundances of experimental product ions matching with predicted ones, can be exploited to reduce the number of candidates to be included in subsequent data processing based on MS3 measurements. The latter usually represents a mandatory step towards a reliable peptide identification when high resolution MS/MS data are not accessible. Sets of peptide sequences arising from MS/MS-based database searches for 63 previously identified non-tryptic peptides (all generated from milk proteins) were exploited to check the INSPIRE performance. It was found that, if retrieved among candidates after the database search, the correct sequence was always ranked among the first 10 ones when parameters calculated by INSPIRE were adopted for discrimination purposes. Under the same conditions the ranks provided by popular database search programs were significantly worse in a remarkable number of cases.

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

A theoretical study of 1,3-benzenedimethanethiol adsorption on Au(111) planar surface at low coverage is conducted. Several configurations were taken into consideration but all of them had both sulphur atoms deprived of the terminal H atom. Also, the case of gold surface reconstruction was examined and results are reported for a configuration analogous to one proposed for methylthiolate adsorption that has lately gained much consensus, one in which both sulphur atoms are coordinated to a single gold adatom.

This article deals with artificial vesicles and their membranes as reaction promoters and regulators. Among the various molecular assemblies which can form in an aqueous medium from amphiphilic molecules, vesicle systems are unique. Vesicles compartmentalize the aqueous solution in which they exist, independent on whether the vesicles are biological vesicles (existing in living systems) or whether they are artificial vesicles (formed in vitro from natural or synthetic amphiphiles). After the formation of artificial vesicles, their aqueous interior (the endovesicular volume) may become – or may be made – chemically different from the external medium (the exovesicular solution), depending on how the vesicles are prepared. The existence of differences between endo- and exovesicular composition is one of the features on the basis of which biological vesicles contribute to the complex functioning of living organisms. Furthermore, artificial vesicles can be formed from mixtures of amphiphiles in such a way that the vesicle membranes become molecularly, compositionally and organizationally highly complex, similarly to the lipidic matrix of biological membranes. All the various properties of artificial vesicles as membranous compartment systems emerge from molecular assembly as these properties are not present in the individual molecules the system is composed of. One particular emergent property of vesicle membranes is their possible functioning as promoters and regulators of chemical reactions caused by the localization of reaction components, and possibly catalysts, within or on the surface of the membranes. This specific feature is reviewed and highlighted with a few selected examples which range from the promotion of decarboxylation reactions, the selective binding of DNA or RNA to suitable vesicle membranes, and the reactivation of fragmented enzymes to the regulation of the enzymatic synthesis of polymers. Such type of emergent properties of vesicle membranes may have been important for the prebiological evolution of protocells, the hypothetical compartment systems preceding the first cells in those chemical and physico-chemical processes that led to the origin of life.

‘ENVIRONMENT’ is a computational platform that has been developed in the last few years with the aim to simulate stochastically the dynamics and stability of chemically reacting protocellular systems. Here we present and describe some of its main features, showing how the stochastic kinetics approach can be applied to study the time evolution of reaction networks in heterogeneous conditions, particularly when supramolecular lipid structures (micelles, vesicles, etc) coexist with aqueous domains. These conditions are of special relevance to understand the origins of cellular, self-reproducing compartments, in the context of prebiotic chemistry and evolution. We contrast our simulation results with real lab experiments, with the aim to bring together theoretical and experimental research on protocell and minimal artificial cell systems.

Synthetic or semi-synthetic minimal cells are those cell-like artificial compartments that are based on the encapsulation of molecules inside lipid vesicles (liposomes). Synthetic cells are currently used as primitive cell models and are very promising tools for future biotechnology. Despite the recent experimental advancements and sophistication reached in this field, the complete elucidation of many fundamental physical aspects still poses experimental and theoretical challenges. The interplay between solute capture and vesicle formation is one of the most intriguing ones. In a series of studies, we have reported that when vesicles spontaneously form in a dilute solution of proteins, ribosomes, or ribo-peptidic complexes, then, contrary to statistical predictions, it is possible to find a small fraction of liposomes (<1%) that contain a very large number of solutes, so that their local (intravesicular) concentrations largely exceed the expected value. More recently, we have demonstrated that this effect (spontaneous crowding) operates also on multimolecular mixtures, and can drive the synthesis of proteins inside vesicles, whereas the same reaction does not proceed at a measurable rate in the external bulk phase. Here we firstly introduce and discuss these already published observations. Then, we present a computational investigation of the encapsulation of transcription-translation (TX-TL) machinery inside vesicles, based on a minimal protein synthesis model and on different solute partition functions. Results show that experimental data are compatible with an entrapment model that follows a power law rather than a Gaussian distribution. The results are discussed from the viewpoint of origin of life, highlighting open questions and possible future research directions.

Synthetic cells can be constructed by encapsulating an appropriate set of molecules inside lipid vesicles (liposomes). They can be used as primitive cell model or for biotechnological applications. At these aims, it is important to develop experimental methods for liposome formation, solute encapsulation, and solute exchange across the lipid membrane. Here we focus on (i) the encapsulation of a polymer-enzyme (peroxidase) conjugate inside giant vesicles (GVs) prepared from phospholipids, and its reaction with Amplex Red reagent; and (ii) on the formation of amphotericin B pores allowing the entrance of cobalt ions inside calcein-containing vesicles, with the aim of developing experimental methods for constructing cell-like systems capable of processing chemicals. The presented approach is also shortly discussed from the viewpoint of chemical computing.

Two kinetic models describing the emergence of autopoietic chemical units are presented and discussed: the single reagent autopoietic mechanism (SRAM) and a reduced version (rSRAM). The proposed schemes are inspired to the autopoietic vesicles studied by Zepik et al (2001 Angew. Chem., Int. Ed. Engl. 40 199–202). Deterministic and stochastic analyses are then performed in order to obtain conditions for growth, homeostasis and decay time behaviours of the overall amphiphiles concentration. Only the reduced SRAM is able to exhibit all the three regimes as experimentally observed and in order to obtain details on the time evolution of the aggregates’ size distribution, stochastic simulations are carried out. What emerges from the rSRAM simulation outcomes is that random fluctuations can act as selection rules for the size of the autopoietic units in the homeostatic regime suggesting how, in a prebiotic scenario, stochastic fluctuations can select the more robust, in this case larger, as the fittest ‘organisms’.

Recent experimental work in the field of synthetic protocell biology has shown that prebiotic vesicles are able to ‘steal’ lipids from each other. This phenomenon is driven purely by asymmetries in the physical state or composition of the vesicle membranes, and, when lipid resource is limited, translates directly into competition amongst the vesicles. Such a scenario is interesting from an origins of life perspective because a rudimentary form of cell-level selection emerges. To sharpen intuition about possible mechanisms underlying this behaviour, experimental work must be complemented with theoretical modelling. The aim of this paper is to provide a coarse-grain mathematical model of protocell lipid competition. Our model is capable of reproducing, often quantitatively, results from core experimental papers that reported distinct types vesicle competition. Additionally, we make some predictions untested in the lab, and develop a general numerical method for quickly solving the equilibrium point of a model vesicle population.

Recent advancements in synthetic biology pave the way to the design and construction of synthetic cells of increasing complexity, capable of performing specific functions in programmable manner. One of the most exciting goal is the development of a molecular communication technology based on the exchange of chemical signals between synthetic and natural cells. We are currently involved in such a research program. Following our previous contributions to WIVACE workshops (2012–2013), here we present the project, and discuss some general considerations on the use of synthetic cells for developing novel bio-chemical Information and Communication Technologies (bio-chem-ICTs). Moreover, by analysing in detail a mathematical model of synthetic cell/natural cell communication process, we provide some hints that can be valuable for the next experimental steps.

Superparamagnetic iron oxide nanoparticles are used in a rapidly expanding number of research and practical applications in biotechnology and biomedicine. Recent developments in iron oxide nanoparticle design and understanding of nanoparticle membrane interactions have led to applications in magnetically triggered, liposome delivery vehicles with controlled structure. Here we study the effect of external physical stimuli-such as millimeter wave radiation-on the induced movement of giant lipid vesicles in suspension containing or not containing iron oxide maghemite (γ-Fe2O3) nanoparticles (MNPs). To increase our understanding of this phenomenon, we used a new microscope image-based analysis to reveal millimeter wave (MMW)-induced effects on the movement of the vesicles. We found that in the lipid vesicles not containing MNPs, an exposure to MMW induced collective reorientation of vesicle motion occurring at the onset of MMW switch "on." Instead, no marked changes in the movements of lipid vesicles containing MNPs were observed at the onset of first MMW switch on, but, importantly, by examining the course followed; once the vesicles are already irradiated, a directional motion of vesicles was induced. The latter vesicles were characterized by a planar motion, absence of gravitational effects, and having trajectories spanning a range of deflection angles narrower than vesicles not containing MNPs. An explanation for this observed delayed response could be attributed to the possible interaction of MNPs with components of lipid membrane that, influencing, e.g., phospholipids density and membrane stiffening, ultimately leads to change vesicle movement.

A model is here presented to analyse how vesicles may turn into protocells that synthesize their own lipid components and the consequences that this may have on the properties of the resulting membrane (in particular, on its permeability), as well as on the overall stability of the system

How did primitive living cells originate? The formation of early cells, which were probably solute-filled vesicles capable of performing a rudimentary metabolism (and possibly self-reproduction), is still one of the big unsolved questions in origin of life. We have recently used lipid vesicles (liposomes) as primitive cell models, aiming at the study of the physical mechanisms for macromolecules encapsulation. We have reported that proteins and ribosomes can be encapsulated very efficiently, against statistical expectations, inside a small number of liposomes. Moreover the transcription-translation mixture, which realistically mimics a sort of minimal metabolic network, can be functionally reconstituted in liposomes owing to a self-concentration mechanism. Here we firstly summarize the recent advancements in this research line, highlighting how these results open a new vista on the phenomena that could have been important for the formation of functional primitive cells. Then, we present new evidences on the non-random entrapment of macromolecules (proteins, dextrans) in phospholipid vesicle, and in particular we show how enzymatic reactions can be accelerated because of the enhancement of their concentration inside liposomes.

Background: The wet-lab synthesis of the simplest forms of life (minimal cells) is a challenging aspect in modern synthetic biology. Quasi-cellular systems able to produce proteins directly from DNA can be obtained by encapsulating the cell-free transcription/translation system PURESYSTEM™(PS) in liposomes. It is possible to detect the intra-vesicle protein production using DNA encoding for GFP and monitoring the fluorescence emission over time. The entrapment of solutes in small-volume liposomes is a fundamental open problem. Stochastic simulation is a valuable tool in the study of biochemical reaction at nanoscale range. QDC (Quick Direct-Method Controlled), a stochastic simulation software based on the well-known Gillespie’s SSA algorithm, was used. A suitable model formally describing the PS reactions network was developed, to predict, from inner species concentrations (very difficult to measure in small-volumes), the resulting fluorescence signal (experimentally observable). Results: Thanks to suitable features specific of QDC, we successfully formalized the dynamical coupling between the transcription and translation processes that occurs in the real PS, thus bypassing the concurrent-only environment of Gillespie’s algorithm. Simulations were firstly performed for large liposomes (2.67μm of diameter) entrapping the PS to synthetize GFP. By varying the initial concentrations of the three main classes of molecules involved in the PS (DNA, enzymes, consumables), we were able to stochastically simulate the time-course of GFPproduction. The sigmoid fit of the GFP-production curves allowed us to extract three quantitative parameters which are significantly dependent on the various initial states. Then we extended this study for small-volume liposomes (575 nm of diameter), where it is more complex to infer the intra-vesicle composition, due to the expected anomalous entrapment phenomena. We identified almost two extreme states that are forecasted to give rise to significantly different experimental observables. Conclusions: The present work is the first one describing in the detail the stochastic behavior of the PS. Thanks to our results, an experimental approach is now possible, aimed at recording the GFP production kinetics in very small microemulsion droplets or liposomes, and inferring, by using the simulation as a reverse-engineering procedure, the internal solutes distribution, and shed light on the still unknown forces driving the entrapment phenomenon.

Here we report some recent biophysical issues on the preparation of solute-filled lipid vesicles and their relevance to the construction of “synthetic cells”. Firstly, we introduces the “semi-synthetic minimal cells” as the liposome-based cell-like systems which contain a minimal number of biomolecules required to display simple and complex biological functions. Next we focus on recent aspects related to the construction of synthetic cells. Emphasis is given to the interplay between the methods of synthetic cell preparations and the physics of solute encapsulation. We briefly introduce the notion of structural and compositional “diversity” in synthetic cell populations.

Minimal artificial cells (MACs) are self-assembled chemical systems able to mimic the behavior of living cells at a minimal level, i.e. to exhibit self-maintenance, self-reproduction and the capability of evolution. The bottom-up approach to the construction of MACs is mainly based on the encapsulation of chemical reacting systems inside lipid vesicles, i.e. chemical systems enclosed (compartmentalized) by a double-layered lipid membrane. Several researchers are currently interested in synthesizing such simple cellular models for biotechnological purposes or for investigating origin of life scenarios. Within this context, the properties of lipid vesicles (e.g., their stability, permeability, growth dynamics, potential to host reactions or undergo division processes…) play a central role, in combination with the dynamics of the encapsulated chemical or biochemical networks. Thus, from a theoretical standpoint, it is very important to develop kinetic equations in order to explore first—and specify later—the conditions that allow the robust implementation of these complex chemically reacting systems, as well as their controlled reproduction. Due to being compartmentalized in small volumes, the population of reacting molecules can be very low in terms of the number of molecules and therefore their behavior becomes highly affected by stochastic effects both in the time course of reactions and in occupancy distribution among the vesicle population. In this short review we report our mathematical approaches to model artificial cell systems in this complex scenario by giving a summary of three recent simulations studies on the topic of primitive cell (protocell) systems.

The computational platform ENVIRONMENT, developed to simulate stochastically reaction systems in varying compartmentalized conditions [Mavelli and Ruiz-Mirazo: Philos Trans R Soc Lond B Biol Sci 362:1789-1802, 2007; Physical Biology 7(3): 036002, 2010], is here applied to study the dynamic properties and stability of model protocells that start producing their own lipid molecules (e.g., phospholipids), which get inserted in previously self-assembled vesicles, made of precursor amphiphiles (e.g., fatty acids). Attention is mainly focused on the changes that this may provoke in the permeability of the compartment, as well as in its eventual osmotic robustness.

Background Over the last two decades, lipid compartments (liposomes, lipid-coated droplets) have been extensively used as in vitro “minimal” cell models. In particular, simple and complex biomolecular reactions have been carried out inside these self-assembled micro- and nano-sized compartments, leading to the synthesis of RNA and functional proteins inside liposomes. Despite this experimental progress, a detailed physical understanding of the underlying dynamics is missing. In particular, the combination of solute compartmentalization, reactivity and stochastic effects has not yet been clarified. A combination of experimental and computational approaches can reveal interesting mechanisms governing the behavior of micro compartmentalized systems, in particular by highlighting the intrinsic stochastic diversity within a population of “synthetic cells”. Methods In this context, we have developed a computational platform called ENVIRONMENT suitable for studying the stochastic time evolution of reacting lipid compartments. This software – which implements a Gillespie Algorithm – is an improvement over a previous program that simulated the stochastic time evolution of homogeneous, fixed-volume, chemically reacting systems, extending it to more general conditions in which a collection of similar such systems interact and change over the course of time. In particular, our approach is focused on elucidating the role of randomness in the time behavior of chemically reacting lipid compartments, such as micelles, vesicles or micro emulsions, in regimes where random fluctuations due to the stochastic nature of reacting events can lead an open system towards unexpected time evolutions. Results This paper analyses the so-called Ribocell (RNA-based cell) model. It consists in a hypothetical minimal cell based on a self-replicating minimum RNA genome coupled with a self-reproducing lipid vesicle compartment. This model assumes the existence of two ribozymes, one able to catalyze the conversion of molecular precursors into lipids and the second able to replicate RNA strands. The aim of this contribution is to explore the feasibility of this hypothetical minimal cell. By deterministic kinetic analysis, the best external conditions to observe synchronization between genome self-replication and vesicle membrane reproduction are determined, while its robustness to random fluctuations is investigated using stochastic simulations, and then discussed.

The construction of artificial cells based on the encapsulation of chemical reacting systems inside lipid vesicles is rapidly progressing in recent years. Several groups are currently interested in synthesizing such simple cell models for biotechnological purposes or for investigating origin of life scenarios. Within this context, the properties of lipid vesicles (e.g., their stability, permeability, growth dynamics, potential to host reactions or undergo division processes…) play a central role, in combination with the dynamics of the encapsulated chemical or biochemical networks. Thus, from a theoretical standpoint, it is very important to develop deterministic equations in order to explore first - and specify later - the conditions that allow the robust implementation of these complex chemically reacting systems, as well as their controlled reproduction. Due to their intrinsic compartmentalized nature, the population of reacting molecules can be very low in terms of number of molecules so that their behaviour can be highly affected by stochastic effects both in the time course of their reactions and in their occupancy distribution among the vesicle population. In this contribution we report our mathematical approaches to model artificial cell systems in this complex scenario, with emphasis on the issue of primitive cell (protocell) systems.

Macromolecular hybrid structures were prepared in which two types of enzymes, horseradish peroxidase (HRP) and bovine erythrocytes Cu,Zn-superoxide dismutase (SOD), were linked to a fluorescently labeled, polycationic, dendronized polymer (denpol). Two homologous denpols of first and second generation were used and compared, and the activities of HRP and SOD of the conjugates were measured in aqueous solution separately and in combination. In the latter case the efficiency of the two enzymes in catalyzing a two-step cascade reaction was evaluated. Both enzymes in the two types of conjugates were highly active and comparable to free enzymes, although the efficiency of the enzymes bound to the second-generation denpol was significantly lower (up to a factor of 2) than the efficiency of HRP and SOD linked to the first-generation denpol. Both conjugates were analyzed by atomic force microscopy (AFM), confirming the expected increase in object size compared to free denpols and demonstrating the presence of enzyme molecules localized along the denpol chains. Finally, giant phospholipid vesicles with diameters of up to about 20 μm containing in their aqueous interior pool a first-generation denpol−HRP conjugate were prepared. The HRP of the entrapped conjugate was shown to remain active toward externally added, membrane-permeable substrates, an important prerequisite for the development of vesicular multienzyme reaction systems.

Self-assembled monolayers (SAMs) derived of 4-methoxy-terphenyl-300,500-dimethanethiol (TPDMT) and 4-methoxyterphenyl- 400-methanethiol (TPMT) have been prepared by chemisorption from solution onto gold thin films and nanoparticles. The SAMs have been characterized by spectroscopic ellipsometry, Raman spectroscopy and atomic force microscopy to determine their optical properties, namely the refractive index and extinction coefficient, in an extended spectral range of 0.75-6.5 eV. From the analysis of the optical data, information on SAMs structural organization has been inferred. Comparison of SAMs generated from the above aromatic thiols to well-known SAMs generated from the alkanethiol dodecanethiol revealed that the former aromatic SAMs are densely packed and highly vertically oriented, with a slightly higher packing density and a absence of molecular inclination in TPMT/Au. The thermal behavior of SAMs has also been monitored using ellipsometry in the temperature range 25-500 C. Gold nanoparticles functionalized by the same aromatic thiols have also been discussed for surface enhanced Raman spectroscopy applications. This study represents a step forward tailoring the optical and thermal behavior of surfaces as well as nanoparticles.

Liposomes represent a versatile biomimetic environment for studying the interaction between integral membrane proteins and hydrophobic ligands. In this paper, the quinone binding to the Q(B)-site of the photosynthetic reaction centers (RC) from Rhodobacter sphaeroides has been investigated in liposomes prepared with either the zwitterionic phosphatidylcholine (PC) or the negatively charged phosphatidylglycerol (PG) to highlight the role of the different phospholipid polar heads. Quinone binding (K (Q)) and interquinone electron transfer (L (AB)) equilibrium constants in the two type of liposomes were obtained by charge recombination reaction of Q(B)-depleted RC in the presence of increasing amounts of ubiquinone-10 over the temperature interval 6-35 A degrees C. The kinetic of the charge recombination reactions has been fitted by numerically solving the ordinary differential equations set associated with a detailed kinetic scheme involving electron transfer reactions coupled with quinone release and uptake. The entire set of traces at each temperature was accurately fitted using the sole quinone release constants (both in a neutral and a charge separated state) as adjustable parameters. The temperature dependence of the quinone exchange rate at the Q(B)-site was, hence, obtained. It was found that the quinone exchange regime was always fast for PC while it switched from slow to fast in PG as the temperature rose above 20 A degrees C. A new method was introduced in this paper for the evaluation of constant K (Q) using the area underneath the charge recombination traces as the indicator of the amount of quinone bound to the Q(B)-site.

Here we investigate the structural evolution of TX100 micelles upon loading with several linear and cyclic alkanes by DLS, PGSE-NMR, 2D NOESY NMR, viscosity measurements, and molecular dynamic simulations. Our results confirm that TX100 alone forms spherical, onion-like micelles made of several partially interpenetrating surfactant layers where the polyethylene glycol chains are in contact with the tetramethyl-butyl-phenyl moieties. Loading with non-penetrating oils larger than decane induces a decrease in micellar size and hydration because the alkane molecules compete with both water and tetramethyl-butyl-phenyl groups for the polyethylene glycol chains. This results in the partial peeling of the “onion” and in the dehydration of polyethylene glycol chains so that the micelles increase in number and decrease in size upon alkane loading. In contrast, small and penetrable oils (mainly cyclo-alkanes) first swell the onion-like micelles (inducing an increase in size) and only above a critical oil/surfactant ratio does the oil induce the weakening of the multilayer structure and the dehydration of polyethylene glycol chains found in long linear alkanes.

The so-called Ribocell (ribozymes-based cell) is a theoretical cellular model that has been proposed some years ago as a possible minimal cell prototype. It consists in a self-replicating minimum RNA genome coupled with a self-reproducing lipid vesicle compartment. This model assumes the existence of two hypothetical ribozymes one able to catalyze the conversion of molecular precursors into lipids and the second one able to replicate RNA strands. Therefore, in an environment rich both of lipid precursors and activated nucleotides, the ribocell can self-reproduce if the genome self-replication and the compartment self-reproduction mechanisms are somehow synchronized. The aim of this contribution is to explore the feasibility of this model with in silico simulations using kinetic parameters available in literature.

In previous works we have explored the dynamics of chemically reacting proto-cellular systems, under different experimental conditions and kinetic parameters, by means of our stochastic simulation platform ‘ENVIRONMENT’. In this paper we, somehow, turn the question around: accepting some broad modeling assumptions, we investigate the conditions under which simple protocells will spontaneously settle into a stationary reproducing regime, characterized by a regular growth/division cycle and the maintenance of a certain standard size and chemical composition across generations. In the first part, starting from purely geometric considerations, the condition for stationary reproduction of a protocell will be expressed in terms of a growth control coefficient (). Then, an explicit relationship, the osmotic synchronization condition, will be analytically derived under a set of kinetic simplifications and taking into account the osmotic pressure balance operating across the protocell membrane. In the second part of the paper, this general condition that constrains different molecular/kinetic parameters and features of the system (reaction rates, permeability coefficients, metabolite concentrations, system volume) will be applied to different cases of self-producing vesicles, predicting the stationary protocell size or lifetime. Finally, in order to test the validity of our analytic results and predictions, the case study is contrasted with data obtained through both stochastic and deterministic computational algorithms.