Chemical communication between synthetic and natural cells: a possible experimental design

The bottom-up construction of synthetic cells is one of the most intriguing and interesting research arenas in synthetic biology. Synthetic cells are built by encapsulating biomolecules inside lipid vesicles (liposomes), allowing the synthesis of one or more functional proteins. Thanks to the in situ synthesized proteins, synthetic cells become able to perform several biomolecular functions, which can be exploited for a large variety of applications. This paves the way to several advanced uses of synthetic cells in basic science and biotechnology, thanks to their versatility, modularity, biocompatibility, and programmability. In the previous WIVACE (2012) we presented the state-of-the-art of semi-synthetic minimal cell (SSMC) technology and introduced, for the first time, the idea of chemical communication between synthetic cells and natural cells. The development of a proper synthetic communication protocol should be seen as a tool for the nascent field of bio/chemical-based Information and Communication Technologies (bio-chem-ICTs) and ultimately aimed at building soft-wet-micro-robots. In this contribution (WIVACE, 2013) we present a blueprint for realizing this project, and show some preliminary experimental results. We firstly discuss how our research goal (based on the natural capabilities of biological systems to manipulate chemical signals) finds a proper place in the current scientific and technological contexts. Then, we shortly comment on the experimental approaches from the viewpoints of (i) synthetic cell construction, and (ii) bioengineering of microorganisms, providing up-to-date results from our laboratory. Finally, we shortly discuss how autopoiesis can be used as a theoretical framework for defining synthetic minimal life, minimal cognition, and as bridge between synthetic biology and artificial intelligence.Comment: In Proceedings Wivace 2013, arXiv:1309.712

The true story of Yeti, the "abominable" heterochromatic gene of drosophila melanogaster

The Drosophila Yeti gene (CG40218) was originally identified by recessive lethal mutation and subsequently mapped to the deep pericentromeric heterochromatin of chromosome 2. Functional studies have shown that Yeti encodes a 241 amino acid protein called YETI belonging to the evolutionarily conserved family of Bucentaur (BCNT) proteins and exhibiting a widespread distribution in animals and plants. Later studies have demonstrated that YETI protein: (i) is able to bind both subunits of the microtubule-based motor kinesin-I; (ii) is required for proper chromosome organization in both mitosis and meiosis divisions; and more recently (iii) is a new subunit of dTip60 chromatin remodeling complex. To date, other functions of YETI counterparts in chicken (CENtromere Protein 29, CENP-29), mouse (Cranio Protein 27, CP27), zebrafish and human (CranioFacial Development Protein 1, CFDP1) have been reported in literature, but the fully understanding of the multifaceted molecular function of this protein family remains still unclear. In this review we comprehensively highlight recent work and provide a more extensive hypothesis suggesting a broader range of YETI protein functions in different cellular processes

Pore-scale simulation of micro and nanoparticle transport in porous media

The transport and deposition of colloidal particles in saturated porous media are processes of considerable importance in many fields of science and engineering, including the propagation of contaminants and of microorganisms in aquifer systems and the use of micro- and nano-particles as reagents for groundwater remediation interventions. Colloid transport is a peculiar multi-scale problem: pore-scale phenomena and inter granular dynamics have an important impact on the larger-scale transport. In this thesis a microscale approach was used to gain a better understanding of the mechanisms underlying colloidal processes, such as deposition and aggregation. The research activity was carried out by performing numerical simulations through the FEM software, COMSOL Multiphysics®. The first part of the study focuses on the development of a new correlation equation to predict single collector efficiency, a key concept in filtration theory, which allows predicting particle deposition on a single spherical collector. By performing Eulerian and Lagrangian simulations in a simple geometry and by using an innovative approach to interpret the results, a new correlation equation to predict single collector efficiency has been formulated. A hierarchical approach to interpret the results was exploited. The proposed correlation equation presents innovative features, such as the validity for a wide range of parameters (also at very small Peclet numbers), the prediction of efficiency values always lower than unity, the total flux normalization and the analysis of the mutual interactions between the main transport mechanisms (advection, gravity and diffusion) and the steric effect. The final formula was also extended to include porosity and a reduced model was proposed. The second part of the study focuses on more realistic systems, characterized by a column of spherical collectors in series. The numerical simulations performed show the limits of the existing models to interpret the experimental data. Therefore, a more rigorous procedure to evaluate the filtration processes in presence of a series of collectors was developed

EMT/MET at the crossroad of stemness, regeneration and oncogenesis. The Ying-Yang equilibrium recapitulated in cell spheroids

The epithelial-to-mesenchymal transition (EMT) is an essential trans-differentiation process, which plays a critical role in embryonic development, wound healing, tissue regeneration, organ fibrosis, and cancer progression. It is the fundamental mechanism by which epithelial cells lose many of their characteristics while acquiring features typical of mesenchymal cells, such as migratory capacity and invasiveness. Depending on the contest, EMT is complemented and balanced by the reverse process, the mesenchymal-to-epithelial transition (MET). In the saving economy of the living organisms, the same (Ying-Yang) tool is integrated as a physiological strategy in embryonic development, as well as in the course of reparative or disease processes, prominently fibrosis, tumor invasion and metastasis. These mechanisms and their related signaling (e.g., TGF-β and BMPs) have been effectively studied in vitro by tissue-derived cell spheroids models. These three-dimensional (3D) cell culture systems, whose phenotype has been shown to be strongly dependent on TGF-β-regulated EMT/MET processes, present the advantage of recapitulating in vitro the hypoxic in vivo micro-environment of tissue stem cell niches and their formation. These spheroids, therefore, nicely reproduce the finely regulated Ying-Yang equilibrium, which, together with other mechanisms, can be determinant in cell fate decisions in many pathophysiological scenarios, such as differentiation, fibrosis, regeneration, and oncogenesis. In this review, current progress in the knowledge of signaling pathways affecting EMT/MET and stemness regulation will be outlined by comparing data obtained from cellular spheroids systems, as ex vivo niches of stem cells derived from normal and tumoral tissues. The mechanistic correspondence in vivo and the possible pharmacological perspective will be also explored, focusing especially on the TGF-β-related networks, as well as others, such as SNAI1, PTEN, and EGR1. This latter, in particular, for its ability to convey multiple types of stimuli into relevant changes of the cell transcriptional program, can be regarded as a heterogeneous "stress-sensor" for EMT-related inducers (growth factor, hypoxia, mechano-stress), and thus as a therapeutic target

Effects of feed restriction and re-feeding on body condition, plasma metabolites and intestinal brush border enzyme activity in rainbow trout Oncorhynchus mykiss

Recovery of body weight and condition as a consequence of liberal feeding after periods of fasting or feed shortage, is a well-known phenomenon in salmonid fish species. To what extent the adoption of culture protocols, alternating liberal feeding to severe feed restriction to exploit compensatory growth, could be a challenge in terms of metabolic adaptation and welfare, still remains questionable. In this context, the present study was aimed at evaluating some physiological responses of rainbow trout subjected to 3 weeks fasting or restricted feed ration and re-feeding over the following 2 weeks. Ninety-nine trout (body weight 109.1 \ub1 3.5 g) were randomly distributed among 3 tanks (0.5 m3) each supplied with 8 L min-1 of well water at a temperature of 12.7\ub10.1\ub0C. Fish groups were subjected to one of the following treatments: C, continuous feeding with a commercial trout diet at 1% body weight over 5 weeks; R, restricted ration (30% of C ration) over 3 weeks followed by 2 weeks feeding to visual satiety; F, fasting over 3 weeks followed by 2 weeks feeding to visual satiety. Three fish per group were euthanized at time 0 and after 1, 2, 4, 7, and 14 days during the re-feeding period and sampled for viscera, liver and mesenteric fat to calculate carcass yield, visceral organ or tissue weight and somatic indices. Blood and gut samples were also collected and analysed for plasma metabolites (glucose, lipid and protein levels) and the activity of intestinal brush border membrane (BBM) enzymes (disaccarases, alkaline phosphatase, \u3b3-glutamil transaminase) in different sections (pyloric caeca, foregut, hindgut). In comparison to the control group, i.e. continuously fed fish, a feed restriction or fasting over 3 weeks resulted in significantly reduced body and visceral to body weight ratio and in diminished activity of the intestinal BBM enzymes. Among plasma metabolites, only cholesterol was reduced. During the re-feeding period, liver and viscera weight increased more rapidly in groups R and F than in group C, resulting in significantly higher organ to body scores. The pattern of the activity of the intestinal BBM enzymes varied among the different tracts in a specific manner, but no differences were observed among treatments after 2 weeks of re-feeding. In conclusion, a period of 14 days of liberal feeding seems to be able to restore the metabolic status in rainbow trout previously subjected to fasting or restricted feed ration over 3 weeks

New architectural design of delivery room reduces morbidity in preterm neonates: a prospective cohort study

Background: A multidisciplinary committee composed of a panel of experts, including a member of the American Academy of Pediatrics and American Institute of Architects, has suggested that the delivery room (DR) and the neonatal intensive care units (NICU) room should be directly interconnected. We aimed to investigate the impact of the architectural design of the DR and the NICU on neonatal outcome. Methods: Two cohorts of preterm neonates born at < 32weeks of gestational age, consecutively observed during 2years, were compared prospectively before (Cohort 1: "conventional DR") and after architectural renovation of the DR realized in accordance with specific standards (Cohort 2: "new concept of DR"). In Cohort 1, neonates were initially cared for a conventional resuscitation area, situated in the DR, and then transferred to the NICU, located on a separate floor of the same hospital. In Cohort 2 neonates were assisted at birth directly in the NICU room, which was directly connected to the DR via a pass-through door. The primary outcome of the study was morbidity, defined by the proportion of neonates with at least one complication of prematurity (i.e., late-onset sepsis, patent ductus arteriosus, intraventricular hemorrhage, periventricular leukomalacia, bronchopulmonary dysplasia, retinopathy of prematurity and necrotizing enterocolitis). Secondary outcomes were mortality and duration of hospitalization. Statistical analysis was performed using standard methods by SPSS software. Results: We enrolled 106 neonates (56 in Cohort 1 and 50 in Cohort 2). The main clinical and demographic characteristics of the 2cohorts were similar. Moderate hypothermia (body temperature ≤ 35.9° C) was more frequent in Cohort 1 (57%) compared with Cohort 2 (24%, p = 0.001). Morbidity was increased in Cohort 1 (73%) compared with Cohort 2 (44%, p = 0.002). No statistically significant differences in mortality and median duration of hospitalization were observed between the 2 cohorts of the study. Conclusions: If realized according to the proposed architectural standards, renovation of DR and NICU may represent an opportunity to reduce morbidity in preterm neonates

Effect of efflux pump inhibition on Pseudomonas aeruginosa transcriptome and virulence

Efflux pumps of the resistance-nodulation-cell-division (RND) family increase antibiotic resistance in many bacterial pathogens, representing candidate targets for the development of antibiotic adjuvants. RND pumps have also been proposed to contribute to bacterial infection, implying that efflux pump inhibitors (EPIs) could also act as anti-virulence drugs. Nevertheless, EPIs are usually investigated only for their properties as antibiotic adjuvants, while their potential anti-virulence activity is seldom taken into account. In this study it is shown that RND efflux pumps contribute to Pseudomonas aeruginosa PAO1 pathogenicity in an insect model of infection, and that the well-characterized EPI Phe-Arg-β-naphthylamide (PAβN) is able to reduce in vivo virulence of the P. aeruginosa PAO1 laboratory strain, as well as of clinical isolates. The production of quorum sensing (QS) molecules and of QS-dependent virulence phenotypes is differentially affected by PAβN, depending on the strain. Transcriptomic and phenotypic analyses showed that the protection exerted by PAβN from P. aeruginosa PAO1 infection in vivo correlates with the down-regulation of key virulence genes (e.g. genes involved in iron and phosphate starvation). Since PAβN impacts P. aeruginosa virulence, anti-virulence properties of EPIs are worthy to be explored, taking into account possible strain-specificity of their activit

Southern Area of the Greek Agora in Kos. Archaeological park project.

The project, included in a degree thesis held at the Polytechnic of Bari, is a plan for an archaeological park set in the area of the Hellenistic agora of Kos (Greece). The proposal is based on a preliminary historical research, in these years carried out by prof. G. Rocco e M. Livadiotti, focused on the reconstruction of the ancient form of the large agora (IV-II cent. B.C.). The study analyses the remains of building structures and architectural fragments discovered in six different diggings. Our project goals are: 1) to allow visitors to understand the original plan of the city, although the deep contrast with the current urban tissue; 2) to redevelop the six archaeological sites analysed in order to make the remains of the ancient agora accessible to the public and, moreover, to allow to consider them as elements of a single monument. The planning is developed at urban scale and involves enlargement and redefinition of the excavated portions, creation of accesses and footbridges and eventually anastylosis in situ of some remains. Other relevant features of the project are: 1) the building of retaining walls made of concrete covered with a earth mixture which makes wall surface similar to the ground; 2) the insertion of 246-2 vegetation in some specific areas which, on one hand, functions as enclosure and visual barrier to modern city and, on the other, it works as instrument to highlight the most important streets of the ancient city. Since it’s not allowed to build in archaeological sites, it’s chosen to use green-system to configure three dimensional space. Therefore, control and knowledge of the specific form of woody species play an important role in the project

On the failure of upscaling the single-collector efficiency to the transport of colloids in an array of collectors

Defining the removal efficiency of a filter is a key aspect for colloid transport in porous media. Several efforts were devoted to derive accurate correlations for the single-collector removal efficiency, but its upscaling to the entire porous medium is still a challenging topic. A common approach involves the assumption of deposition being independent of the history of transport, that is, the collector efficiency is uniform along the porous medium. However, this approach was shown inadequate under unfavorable deposition conditions. In this work, the authors demonstrate that it is not adequate even in the simplest case of favorable deposition. Computational Fluid Dynamics (CFD) simulations were run in a vertical array of 50 identical spherical collectors. Particle transport was numerically solved by analyzing a broad range of parameters. The results evidenced that when particle deposition is not controlled by Brownian diffusion, nonexponential concentration profiles are retrieved, in contrast with the assumption of uniform efficiency. If sedimentation and interception dominate, the efficiency of the first sphere is significantly higher compared to the others, and then declines along the array down to an asymptotic value. Finally, a correlation for the upscaled removal efficiency of the entire array was derived