After one decade of systematic fundamental research on graphene, it is generally agreed that many of the graphene superior physical properties are currently well understood. A major experimental challenge is now related to the high-quality graphene and graphene-based structures, which is a prerequisite to transform laboratory graphene science into viable technological applications. The full potential of laser-assisted methods in graphene productions has not yet been unlocked, despite that these methods offer a number of advantages. In their vast majority, studies of laser-assisted methods result in graphene of dubious quality. We present recent advances in our laboratory concerning the laser-assisted growth of graphene. In particular, we will present activities related to (i) the growth of epitaxial graphene on SiC(0001) using a continuous wave infrared CO2 laser (10.6 μm); (ii) production 3D porous graphene-like scaffolds through the decomposition of SiC micron-sized particles; and (iii) the growth of graphene films by decomposing various organic substances using pulsed lasers.

The low-cost supply of electricity using renewable energy sources, particularly solar, is today one of the pervasive challenges worldwide aimed at alleviating the quick depletion of natural resources and mitigating carbon dioxide emissions. Among the devices used for the exploitation of solar energy, photoelectrochemical (PEC) devices offers a promising way both for energy production and storage.

Metal oxide semiconductors such as TiO2 and ZnO are the best candidates to be used as anodes for PEC cells in view of their stability, for hydrogen production by water splitting or as electro-catalysts for the reduction of triiodide ions in dye sensitized solar cells (DSSCs). However, the wide bandgaps of these materials limit their light absorption only to the UV part of the solar spectrum, which strictly prevents the efficient exploitation of the solar energy in the visible region. Efforts are now directed to the growth of hybrid core/sheath nanostructure consisting of a metal oxide semiconductor core (ZnO) covered by transition metal di-chalcogenides layers (MX2, M: Mo, W, etc., and X: Se, S2, Te) and other chalcogenides such as ZnSe.

In this work, we explore the role of various chalcogenide-based thin films, of various thicknesses, fabricated as core/sheath structures, for increasing the solar-light-mediated PEC efficiency of the devices. Moreover, we examine the catalytic properties towards the I-/I3- redox couple, as a non-platinum counter electrode in a DSSC.

Two dimensional dichalcogenides has been intensively studied over the past few years due to its excellent mechanical, electrical and optical properties. Reliable and efficient wafer-scale CVD production methods are prerequisite for the fabrication of 2D-XS2 (X = Mo or W) based devices. A variety of scalable CVD routes to fabricate 2D-XS2 have been proposed in the past with the most common involving the sulfurization of XO3. Taking place in atmospheric pressure and requiring only XO3 and elemental sulfur as precursors, it is by far one of the simplest methods used to fabricate 2D-XS2. Yet, one of its main disadvantages is that the uniformity and nucleation density of the produced crystals is difficult to control. Although other synthetic routes that offer a greater degree of control exist, they tend to be more complex requiring low pressure systems and potentially more expensive or even toxic precursors.

In this work we show that it is feasible to fabricate single- and few-layered XS2 crystals in a simple, one-step atmospheric pressure CVD synthesis using Na2XO4 and elemental sulfur as precursors. Na2MoO4 in particular is widely available, water soluble and relatively safe, commonly used as a fertilizer. Na2WO4 is important as a catalyst in green oxidations. The thickness and size of the crystals can be controlled by the deposited amount of Na2XO4 precursor. The fabricated crystals are studied by means of X-ray photoelectron spectroscopy, atomic force microscopy, micro-Raman and micro-photoluminescence spectroscopies, and are compared with single-layer 2D-XS2 crystals that were exfoliated or transferred to other substrates. Through this comparison various spectral differences are found to arise from residual mechanical strain and/or doping levels induced by the interaction with the supporting substrate as well as the fabrication procedure. These strain and doping levels are quantified using an optical analysis published recently.

Nowadays, there is a frantic activity worldwide to exploit the unique optical properties of graphene and other 2D crystal materials in photonic applications ranging from solar cells and light-emitting devices to touch screens, photodetectors and ultrafast lasers. In this line, it has been recently shown experimentally that two-photon absorption in graphene is an extremely intense phenomenon. Moreover, graphene is an ideal memory material because of its transparency, conduction properties and solution processability. According to this, a novel technique for 3D optical data storage is proposed. Storage capacity as high as 10 TB in a 120-mm-diameter optical disk, namely three orders of magnitude greater than capacities achieved in conventional DVD disks, can be obtained.

Τα κελιά καυσίμου πολυμερικού ηλεκτρολύτη (Proton Exchange Membrane Fuel Cells, PEMFC) αποτελούν μια καθαρή πηγή ενέργειας και την ελκυστικότερη επιλογή για πολλές εφαρμογές, δεδομένου ότι χαρακτηρίζονται από απλότητα στην κατασκευή, υψηλή απόδοση και ευελιξία. Τα PEMFC υψηλής θερμοκρασίας, έχουν ορισμένα πλεονεκτήματα σε σχέση με τα state-of-the-art κελιά χαμηλών θερμοκρασιών, όπως απλουστεύσεις στο σύστημα, παραγωγή χρήσιμης θερμότητας (υψηλή συνολική απόδοση) και γρήγορη προσαρμογή σε δυναμικές μεταβολές κατανάλωσης ισχύος. Πολύ σημαντικά ζητήματα για τη καθιέρωση των κελιών καυσίμου στην αγορά είναι το κόστος, η αξιοπιστία τους και η μακροπρόθεσμα σταθερή λειτουργία τους. Σε αυτά τα συστήματα, οι ηλεκτροκαταλύτες βασισμένοι σε πλατίνα (Pt) έχουν αποδειχτεί ως οι πιο ενεργοί και ανθεκτικοί σε συνθήκες λειτουργίας τόσο της ανόδου όσο και της καθόδου ενός στοιχείου καυσίμου. Στόχο αποτελεί ο σχεδιασμός και η ανάπτυξη καταλυτικών στρωμάτων με δομές και αρχιτεκτονικές που να οδηγούν σε πιο ενεργές και σταθερές ηλεκτροχημικές διεπιφάνειες, με υψηλή εκμεταλλευσιμότητα και μειωμένα φορτία του ακριβού καταλύτη Pt.

Με βάση τα παραπάνω, θα παρουσιαστεί μια νέα προσέγγιση ανάπτυξης προηγμένων ηλεκτροκαταλυτών που ανοίγει το δρόμο για μία οικονομική και αποδοτική διάταξη παραγωγής ενέργειας. Η στρατηγική που έχει διαμορφωθεί είναι η εναπόθεση της Pt σε χημικά τροποποιημένους νανοσωλήνες άνθρακα. Η χημική τροποποίηση των υποστρωμάτων στοχεύει τόσο στη βελτίωση της διασποράς του καταλύτη πάνω σε αυτά, όσο και στην ενίσχυση της ηλεκτροχημικής διεπιφάνειας της αντίδρασης. Η απόδοση και η σταθερότητά των υλικών εξαρτάται σημαντικά από τα φυσικοχημικά χαρακτηριστικά τους, όπως η ειδική τους επιφάνεια, η κρυσταλλική τους δομή, το μέγεθος και το σχήμα των σωματιδίων και οι αλληλεπιδράσεις με το υπόστρωμα. Μειώνοντας το μέγεθος του μετάλλου σε συστάδες ή ακόμη και σε άτομα, μπορεί να αυξήσει σημαντικά τόσο την ενεργή επιφάνεια, όσο και τη δραστικότητα του καταλύτη λόγω της διαφοροποίησης ή ενίσχυσης των αλληλεπιδράσεων με το υπόστρωμα. Προς αυτήν την κατεύθυνση, έχουν αναπτυχθεί υλικά με ιδιαίτερα χαρακτηριστικά και αξιοσημείωτη απόδοση και σταθερότητα κάτω από απαιτητικές συνθήκες λειτουργίας ή τροφοδοσίας του κελιού, παρέχοντας τη δυνατότητα ελάττωσης της φόρτισης των ηλεκτροδίων σε ευγενές μέταλλο και διεύρυνσης των εφαρμογών των υλικών. Η πρόκληση έγκειται στην επίτευξη βέλτιστης διασποράς και επιφανειακής χημείας για την ταυτόχρονη προώθηση της καταλυτικής ενεργότητας και την αύξηση της εκμεταλλευσιμότητας του καταλύτη, καθώς και την διαμόρφωση μίας μορφολογίας σταθερής υπό τις συνθήκες λειτουργίας της εφαρμογής.

The rapid development of nanoscience and nanotechnology has created a plethora of new opportunities for nonlinear optics. The growing number of nanomaterials shown to exhibit remarkable nonlinear optical properties has greatly enhanced the design and fabrication of new nano/nano-scale optoelectronic and photonic devices. Among these nanomaterials, the family of carbon allotropes includes maybe the most representative products of nanotechnology: from 0D fullerenes, to 1D carbon nanotubes (CNTs), to 3D carbon nanostructures (graphite, C-dots, nano-diamonds, etc.) and then to 2D graphenes and their functionalized derivatives discovered most recently.

Remarkably, all these nano-carbons exhibit diverse and important nonlinear optical properties. For instance, carbon black suspensions show strong thermally-induced nonlinear scattering effect and hence optical limiting for intense ns laser pulses; fullerenes possess large third-order optical nonlinearities and reverse saturable absorption at certain wavelengths; CNTs show ultrafast 2nd- and 3rd-order nonlinearities and saturable absorption in the near infrared; and graphenes show ultrafast carrier relaxation time and ultra-broadband resonant NLO response.

However, the most important and fascinating advantage of these carbon nanomaterials is that they have tailorable chemical and electronic/optoelectronic properties by binding different functional materials, e.g., polymers, organic molecules, metallic nanoparticles, forming versatile materials for photonics. It is well establish today, that the bandgap of graphenes can be easily modulated following different strategies, providing photonic materials with custom tailored linear and nonlinear optical properties.

In this work, some examples from recent work performed in our group will be presented, concerning the nonlinear optical response of various carbon allotrope nanostructures under different excitation wavelengths ns, ps and fs laser radiations, as for instance different C-dots (doped and/or undoped), fullerene-porphyrin pseudorotaxanes, graphene oxides, fluorographenes, hydrogenated fluorographenes, and graphenes with a variety of chain- or aromatic ring- type organic molecules and/or decorated with metallic nanoparticles

Nowadays, food packaging materials exhibiting sensor-type properties play a key role in the quality of the food and the information/warning of the consumers; food packaging sector is mainly focused on new technologies for hybrid food packaging materials with “intelligent” characteristics (sensing ability, O2 scavenging, etc.). Numerous 3d coordination complexes exhibiting Spin Crossover Phenomenon (SCO) show temperature-dependent drastic colour changes accompanied by conversion of their spin states between low-spin (LS) and high-spin (HS).

The long term aim of our research is the synthesis and study of complexes, which could be potentially used as sensors mainly in food industry. More specifically, our research is mainly focused on the synthesis and characterization of low nuclearity Fe(II) coordination complexes exhibiting SCO phenomenon which could act as temperature sensors in refrigerated food packaging materials. Temperature dependent and low-frequency Raman spectroscopy play a significant role in our studies in order to meticulously study/examine the SCO phenomenon. The exploitation of SCO complexes for sensing applications in food packaging materials constitutes a challenging new goal.

Surface Enhanced Raman Scattering (SERS) is potentially a valuable technique for identification and quantification of molecular species when extremely low detection limits are required. The sensitivity of the method is manifested from the enhancement by many orders of magnitude of the intensity of Raman radiation by molecules in the immediate vicinity to nano-rough metal surfaces and nano-structured metal systems. The SERS enhancement factor depends strongly on the size and shape of the nanostructures that give rise to the effect. The enhancement of the scattered radiation becomes even stronger when resonance Raman phenomena are additionally involved. The design of novel SERS substrates that take into account both plasmonic and resonance Raman enhancements offer new possibilities for detection of specific molecular species at extremely low concentrations. The current work aims on maximizing the SE(R)RS -(R) standing for Resonance- signal of specific analytes, while sustaining the technique’s quantification capabilities. The latter is accomplished by optimizing the experimental parameters involved in the substrate preparation.

Photocatalytic wastewater purification and disinfection are, currently, important tasks in the field of environmental research depending, mainly, on the design and development on nanomaterials with high photocatalytic (PC) activity. Aside from the illustrious metal oxide TiO2, recently, ZnO emerges in many research studies as a very promising PC material for wastewater treatment, due to its low cost, high redox potential and favourable bandgap. In order to enhance the PC efficiency of the aforementioned semiconductors as well as to activate them to absorb more the visible part of the solar spectrum, it is challenging to understand the influence of material properties, such as defects, along with experimental conditions, such as pH of the solution on the material PC activity.

Regarding lattice defects, ball-milling of TiO2 and ZnO nanoparticles was employed in this study as it provides a means to introduce them in a controlled way. A number of techniques were employed to scrutinize the nature of defects (bulk or surface) and correlate their amount to the observed changes of the PC activity. Ball-milling appears to introduce mainly bulk defects on ZnO nanoparticles, whereas engenders phase-transformation effects on the crystal lattice (phase transition and partial amorphization) of TiO2 nanoparticles. The PC activity of both ZnO and TiO2 on the degradation of methylene blue (MB) in water found to decrease systematically with milling time. Furthermore, the effect of pH value of the MB solution on the PC activity of TiO2 and ZnO was investigated. Experiments indicated that the PC efficiency of TiO2 was higher at both alkaline and acidic conditions compared to neutral conditions, and the maximum photodegradation rate of MB was observed at alkaline conditions. Regarding ZnO, no respectable PC activity changes were observed between neutral and alkaline conditions of the MB solution.

During the last years, laser induced plasmas have become a very popular approach for obtaining information about the elemental composition and its temporal and spatial variations of a large variety of samples. In this context, Laser Induced Breakdown Spectroscopy (LIBS) is today among the most popular techniques for elemental analysis because of its experimental simplicity and the unique advantages it exhibits. LIBS is probably the most rapid analytical technique for elemental analysis, capable of analyzing in real time all kinds of matter (i.e. solid, liquid, or gaseous; single-crystalline, or amorphous; conducting or dielectric), while it can operate in-situ and/or remotely, in laboratory and also in harsh and dangerous environments where human access/presence is prohibited, being capable to provide accurate, qualitative and quantitative information about the elemental content of a sample, with high spatial and temporal resolution, without requiring time consuming sample conditioning procedures, and needing only very small amounts of a sample.

In this work, some selected examples are presented from our recent work concerning the application of LIBS: i) in metallurgy (e.g. for the simultaneous on-line monitoring of different metals in molten, high temperature metallurgical samples for production control and quality assurance), ii) for the study of the spatiotemporal evolution and the diagnostics of combustion processes (e.g. the on-line and in-situ spatial and time-resolved mapping of the fuel in different flames) and iii) for the detection of metals and non-metallic species (e.g. S, P, C, etc.) in soils, plants, food, etc. in combination with VUV spectroscopy.

A transmission environmental scanning electron microscope with use of a scintillation gaseous detection device has been implemented. This corresponds to a transmission scanning electron microscope but with addition of a gaseous environment acting both as environmental and detection medium. A commercial type of low vacuum machine has been employed together with appropriate modifications to the detection configuration. This involves controlled screening of various emitted signals in conjunction with a scintillation gaseous detection device already provided with the machine for regular surface imaging. Dark field and bright field imaging has been obtained along with other detection conditions. With a progressive series of modifications and tests, the theory and practice of a novel type of microscopy is briefly shown now ushering further significant improvements and developments in electron microscopy as a whole.

Nitrate is a pollutant of major and rapidly increasing concern in drinking water worldwide. Long exposure to high levels of nitrates in drinking water has been shown to cause serious health problems in infants and the older population. Therefore, World Health Organization and the European Community have set a maximum contaminant level of 50 mg NO3- /L which is equal to 11.3 mg NO3-N /L (WHO, 2011). Various treatment technologies have been proposed (chemical, physicochemical, electrochemical and biological) with varying degrees of efficiency, cost and ease of operation. In this work both electrochemical and biological processes were examined for nitrate removal from drinking water using various operating conditions

Specifically, electrochemical removal of nitrates from water was studied using Al, Cu and stainless steel (SS) as cathodes and Ti/IrO2-Pt, Ti/Pt, Ti/Ru-Pt and Ti/IrO2 as anodes in an undivided cell. Experiments in laboratory-scale reactor (of volume 0.150 L) were carried out to treat synthetic nitrate solutions with initial nitrate nitrogen concentrations of 10-100 mg/L through batch mode without using additional pH buffers. Various operating variables such as applied current density (8.3 mA/cm2 to 83.3 mA/cm2), concentration of NaCl (0.1 to 1.0 g/L) and time were tested for their effects on nitrate removal. Results of the present study showed that the maximum nitrate removal of 0.974 gNO-3-N/Ld was obtained (without ammonia production) using Ti/IrO2-Pt and Al as anode and cathode arrangement for initial NO3-N concentration of 100 mg/L and 1.0 g/L NaCl. On the other hand, SS failed to achieve nitrate removal even for high NaCl concentration and current density.

Additionally, suspended growth bioreactors were used to investigate nitrate removal from contaminated drinking water through hydrogenotrophic denitrification with hydrogen produced from electrolysis of water and electric energy provided by a solar cell. A mixed culture originating from a wastewater treatment plant was used to inoculate three different reactors operating under batch (two of them) and continues mode. The first batch reactor (of volume 1 L) was operated with additional nutrients (KH2PO4, Na2PO4), while the second one (of same volume of 1L) without using any extra nutrients. For initial nitrate concertation of 50 mgNO-3-N/L the maximum denitrification rates achieved for the two batch reactors were 0.063 and 0.018 gNO-3-N/Ld, respectively. The reactor under continuous operation (of volume 1.165 L) was operated under two different organic loads (14.4 mg/Ld and 28.8 mg/Ld) without adding any extra nutrients. The maximum denitrification rates achieved under continues operation were 0.0123 and 0.0247 gNO-3-N/Ld for the loadings of 14.4 mg/Ld and 28.8 mg/Ld, respectively.

Each discussed treatment method was found to have its own advantages and limitations, in terms of implementation simplicity, efficiency and cost. In terms of removal efficiency, electrochemical denitrification was found to be very efficient. However, they are high in operating costs because much of electrochemical energy is consumed in undesirable side reactions. Also, nitrate-N removal through electrochemical process may be accompanied by toxic byproducts’ generation, such as ammonia and nitrite. Therefore, is very difficult to applied alone at field scale operations. Contrary, at hydrogenotrophic denitrification, H2 is an excellent autotrophic choice because of its clean nature and low biomass yield, as well as that it does not persist in the treated water and no further steps are required to remove either excess substrates or its derivatives. However, the selection of the most appropriate treatment option depends on various key factors specific to the needs and priorities of individual water systems.

People with chronic kidney failure require hemodialysis in order to survive, but this process only provides a limited removal of protein-bound uremic toxins (PBT) , which leads to considerable morbidity and mortality rates. One proposed solution already demonstrated by experiments is the application of a bioartificial kidney device. Its primary component is a hybrid membrane, a hollow fiber dialysis membrane where a monolayer of immortalized living proximal tubule epithelial kidney cells (ciPTEC) is attached, performing active removal of PBT’s using Organic Ion Transporters (OAT) attached on the surface of the cells. However, the mechanisms involved are not fully understood yet. A 3D computer model and a parametric study of a single hollow fiber with a ciPTEC monolayer attached was built to study the main transport and reaction mechanisms and the role of critical parameters.

In the last 8 years we have prepared and studied a number of targeted liposomes, as nanoparticle based drug delivery systems for brain-located pathologies, and especially for Alzheimers Disease (AD). Most studies were carried out with liposomes which were decorated with ligands, to target: (i) the blood-brain-barrier (BBB) (two ligands, an anti-transferrin monoclonal antibody and a peptide derivative of APOe3 [1], were used), and (ii) the amyloid deposits (Mab against Aβ peptides, and curcumin derivatives (such as TREG [2,3]). The former liposomes were prepared by optimized methodologies that ensured formation of stable liposomes with nanoscale dimensions (hydrodynamic diameter <200 nm), high yield of targeting ligand immobilization on their surface and high production yield. After additionally demonstrating that the optimal targeted liposomes had adequate integrity for in vitro and in vivo testing, a number of in vitro and in vivo studies were carried out to evaluate their BBB targeting potential as well as their affinity for Aβ peptides (by measuring their capability to inhibit amyloid peptide aggregation, and decrease the cytotoxicity of amyloid peptides towards neural cells). For evaluation of the liposome BBB targeting potential in vitro, the hCMEC/D3 cellular model of the BBB was used. In vivo studies were also carried out by live animal imaging after iv administration of DiR-labelled liposomes, in normal mice and APP/PS1 double transgenic mice and their wild-type (WT) littermates. Interesting conclusions have been drawn about the effect of the curcumin derivative addition on the BBB-targeted liposomes, in respect to their capability to target the brain [4, 5]. Recently, various types of cellular vesicles, were constructed using liposome-technology engineering methodologies, and were tested (in vitro) under identical conditions (with those used in the case of liposomes) on the hCMEC/D3 cellular BBB model. The results of these latter studies are very interesting [6], suggesting that exploitation of the targeting components of cellular (or perhaps exosomal) vesicles may potentially lead to construction of superior types of drug-carriers for theragnosis of brain localized diseases.

References

[1] E Markoutsa et al, Pharm Res 31:1275-1289 (2014)

[2] S Mourtas et al, Biomaterials, 32: 6, 1635-1645 (2011)

[3] M Taylor et al, Nanomedicine: NBM 7 (5), 541-550 (2011)

[4] K. Papadia et al, Eur. J. Phar. Sciences, 101, 140-148 (2017)

[5] K. Papadia et al, Eur. J. Phar. Sciences, 102, 180-187 (2017)

[6] Papadia K, Basta A, Antimisiaris SG. Liposome-like vesicles of cellular origin to target the brain: Potential inspiration for design of advanced ligand-targeted liposomes? Under publication.

We have developed a series of disposable, dipstick-type DNA biosensors that enable visual detection of DNA/RNA sequences either by naked eye or by using a smartphone. Their advantages are: simplicity, low cost, portability, no need for specialized equipment and elimination of multiple pipetting, incubation and washing steps. For genotyping, the interrogated sequence is subjected to amplification and allele-discrimination reactions. The product is applied to the sample area of the biosensor, which is then immersed in the appropriate buffer. The appearance of a characteristic colored line at the test zone indicates the presence of the ‘normal’ or ‘mutant’ allele in the sample. The biosensors were applied successfully to SNP genotyping, mutation detection as well as pathogen detection in clinical samples. Furthermore the biosensors were applied to the DNA-based verification of species/varietal origin of food and agricultural products.

Antimicrobial and antifouling coatings have been developed using copolymers bearing covalently bound and electrostatically attached quaternized ammonium groups and their blends with different polymeric matrixes1,2. Additionally, reactive blending of copolymers with complementary functional groups was applied in order to develop coatings and control their antimicrobial activity and antifouling action in real conditions3. Different copolymers with poly(4-vinylbenzyl chloride-co-acrylic acid), P(VBC-co-AAx), and poly(sodium 4-styrenesulfonate-co-glycidyl methacrylate), P(SSNa-co-GMAx) units, were synthesized via free radical copolymerization and further modified by the incorporation of biocidal units either covalently (vinyl benzyl dimethylhexadecylammonium chloride, VBCHAM) or electrostatically bound (cetyltrimethylammonium 4-styrene sulfonate, SSAmC16). The crosslinking reaction of the carboxylic group of acrylic acid (AA) with the epoxide group of glycidyl methacrylate (GMA) of these reactive antimicrobial copolymers was explored after solution casting and curing at various temperatures. Antimicrobial tests revealed that the crosslinked membranes presented strong antimicrobial activity against S. aureus and P. aeruginosa. Finally, a specific coating combination was applied on aquaculture nets and examined after emerging in the sea water for 15 and 35 days, showing high antifouling action as compared to blank nets.

References:

1. E. Kougia, M. Tselepi, G. Vasilopoulos, G.Ch. Lainioti, N.D. Koromilas, D. Druvari, G. Bokias, A. Vantarakis, J.K. Kallitsis, Molecules 20, 21313–21327 (2015)

2. N. D. Koromilas G. Ch. Lainioti G. Vasilopoulos A. Vantarakis, J. K. Kallitsis, Polymer Chemistry 7, 3562-3575 (2016)

3. D. Druvari, N. Koromilas, G. Lainioti, G. Bokias, G. Vasilopoulos, A. Vantarakis, I. Baras, N. Dourala, J. Kallitsis, ACS Applied Materials & Interfaces, 8(51), 35593–35605 (2016)

Acknowledgements

This research has been co-financed by the European Union (European Social Fund – ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF 2007-2013)-Research Funding Program: THALES. Investing in knowledge society through the European Social Fund. Project title: “Development of Novel Functional Copolymers and Surfaces with Permanent and/or Controlled released biocidal species” (MIS: 379523).

This work focuses in the in silico reconstruction of the protein-protein interaction network of Streptomyces lividans based on available information from experimentally supported protein interactions in other bacteria and bioinformatics analysis. Streptomycetes exhibit unique metabolic diversity and enzymatic capabilities. They can secrete secondary metabolites that are valuable for industrial and pharmaceutical purposes making them a valuable resource for synthetic biology and metabolic engineering applications. The latter can lead to the production of minimal strains with increased cell stability in long-term fermentation and enhanced metabolic potential for the biosynthesis of heterologous molecules [1,2]. Still, the incorporation of information from omic analyses and the reconstruction of gene regulation, protein and metabolic networks in the context of systems biology approaches, are expected to enhance the knowledge about the S. lividans regulatory machinery, providing significant leads for directed metabolic engineering actions. This is one of the major objectives of the collaborative European FP7 project STREPSYNTH “Rewiring the Streptomyces cell factory for cost-effective production of biomolecules”, a task of which is the presented study.

Currently, there is not any protein-protein interaction (PPI) network reconstructed either experimentally or in silico for any Streptomyces. A limited number (25) of experimentally identified protein protein interactions (PPIs) are reported in publicly available databases (MPIDB, https://omictools.com/microbial-protein-interaction-database-2-tool; MINT, mint.bio.uniroma2.it/; and IntAct, http://www.ebi.ac.uk/intact/) for Steptomycetes in general, only one of which has been associated specifically with S. lividans. In the presented work, we reconstructed in silico the core S. lividans PPI network, starting from the recently published Escherichia coli (strain K12) direct PPI network [3]. After identifying the orthologue to S. lividans genes in the E. coli, we used the STRING PPI database (https://string-db.org/) to predict ~2200 PPIs among these proteins, based on available information from all bacteria stored in STRING. The reconstructed network was analyzed for its topology to determine its connectivity structure and the role of the various protein classes. In order to determine additional data to expand the reconstructed S. lividans PPI map, we identified in all available experimentally supported bacterial direct protein interactomes the PPIs of which both interactors have orthologues in E. coli. Specifically, we examined the interactomes of Campylobacter jejuni, Mycobacterium tuberculosis, Treponema pallidum, Synechocystis sp., Mesorhizobium loti, Helicobacter pylori. Interestingly, through these comparisons, we were able to in silico predict 675 additional PPIs and 162 additional proteins-nodes in the E. coli K12 PPI map, extending thus the available experimental information. We plan to further expand the predicted S. lividans PPI network through comparison with reported interactomes for bacteria closer to Streptomycetes in the evolution tree. The reconstructed S. lividans PPI network is to be integrated the metabolic network and omic data from various molecular levels of cellular function within the collaborative European FP7 project towards a better understanding of this bacterium physiology.

References

1. Chater KF, Biró S, Lee KJ, Palmer T, Schrempf H. (2010) The complex extracellular biology of Streptomyces. FEMS Microbiol Rev. 34(2):171-98.

2. Anné J, Maldonado B,Van Impe J,Van Mellaert L,Bernaerts K. Anné et al., Recombinant protein production and streptomycetes. J Biotechnol. 2012 158(4):159-672012

3. Rajagopala S.V., Sikorski P., Kumar A, et al. 2014. The binary protein-protein interaction landscape of Escherichia coli. Nat Biotechnol 3:285-90.

Acknowledgements

The research leading to these results has received funding from:

1) European Commission’s Seventh Framework Programme (FP7/2007-2013) under the grant agreement STREPSYNTH Rewiring the Streptomyces cell factory for cost-effective production of biomolecules (project n° 613877).

2) Stavros Niarchos Foundation Postdoctoral fellowship to Dr. Ch. Chasapis from FORTH/ICE-HT, titled “Structural Systems Biology for Metabolic Engineering Applications” in the context of the research project “ ARCHERS: Advancing Young Researchers’ Human Capital in Cutting Edge Technologies in the Preservation of Cultural Heritage and the Tackling of Societal Challenges”.

The FRA10A fragile site causative gene, FRA10AC1, encodes a well-conserved nuclear protein with yet, unknown function (Sarafidou et al., Genomics, 84:69, 2004). The FRA10AC1 position in the reconstructed human protein interactome (Klapa et al., 2013; www.pickle.gr) suggests its involvement in splicing and/or other related pre-mRNA processes (Galliopoulou et al, in prep.). To elucidate the biological role of FRA10AC1, we developed HeLa cells with impaired FRA10AC1 expression and performed integrated transcriptomic, proteomic and metabolomic analyses. The respective profiles were acquired using GeneChip® U133 Plus2.0 arrays, LC-MS with iTRAQ-labeling and GC-MS, capturing information for ~20400 transcripts, ~6400 proteins, and ~130 metabolite derivatives, respectively. After normalization and filtering, 5153 proteins and 69 metabolites were further considered. Multivariate statistical analysis indicated that the FRA10AC1 downregulation affected strongly the abundance of ~2000 transcripts with unique UniProt_IDs, 560 proteins and 32 metabolites. Its overexpression had a strong impact on ~1540 transcripts with unique UniProt_IDs, 215 proteins and 16 metabolites. Ontological analysis of the differentially expressed transcripts and proteins, indicated that most are implicated in mRNA processing and metabolism, alternative splicing, transcription and translation, strongly supporting a role for FRA10AC1 in pre-mRNA processing. The educated integration of all three omic profiles indicated that the aberrant FRA10AC1 expression affects the cytoskeleton and cell membrane organization, the endoplasmic reticulum and Golgi apparatus function, the cell signaling through protein phosphorylation and response to glucocorticoids, the glucose and cholesterol homeostasis, the oxidative phosphorylation and the anti-inflammatory response activity. Interestingly, the observed alterations can be linked with certain neurological disorders.

OBJECTIVE: The major objective of medical genetics concerns the elucidation of the genetic architecture of the diseases. In the era of systems and network medicine, this effort could be greatly assisted by the investigation of the known disease-gene associations through the protein-protein interaction network (protein interactome) of human. Protein interactions being fundamental for the regulation and catalysis of all major cellular processes, the interactome provides a representation of the intracellular molecular function interconnectivity. In this context, the specific aims of this study are: (a) the determination of a high-reliability gene-disease association set to be used as the basis of our investigation, (b) the projection of this dataset on the human protein interactome and (c) the analysis of the network to reveal biological associations and molecular mechanisms underlying the genetic architecture of the diseases. Integrating this information with available GWAS data for multifactorial diseases can significantly upgrade their information content towards the determination of elementary molecular mechanisms and potential genetic markers for early and accurate diagnosis. This approach will be demonstrated in the context of hypertension.

PRIMARY SOURCES AND METHODS: Gene-disease associations were mined from the Online Mendelian Inheritance in Man (OMIM) [1] and Universal Protein Resource (UniProt) [2] databases. The final dataset was determined from the intersection of the two primary sources after appropriate normalization. Finally, these associations were linked with the human protein interactome of the knowledge metadatabase PICKLE (www.pickle.gr), [3] & [4], developed by our group. Currently, PICKLE records ~121,000 interactions for ~14,200 proteins, i.e. ~70% of the human proteome. GWAS data for hypertension were collected from the GWAS-Catalog [5] and additional literature search.

RESULTS: The analysis of the collected gene-disease associations through the human protein interactome indicates that most of the disease proteins are peripheral nodes characterized by a small number of direct interactions. The proteins that are considered essential for life are mainly central nodes with a high number of interactions. Mutations in these proteins are rarely associated with mendelian diseases, but mostly with diseases of somatic cells, such as cancer. Proteins associated with the same disease or similar diseases are usually adjacent in the network. Finally, distinct diseases associated with overlapping molecular pathways share clinical features, and vice versa.

In the case of hypertension, the combined analysis of GWAS data and the human protein interactome identified two distinct relevant protein sub-networks; a central one, and another the members of which are directly associated with at least a member of the former. Interestingly, four major biological processes associated with hypertension are connected through this network topology.

CONCLUSIONS: The integrated analysis of a high-reliability gene-disease association set through the human protein interactome can further our understanding of the molecular pathophysiology of inherited diseases, and specific multifactorial diseases in particular, leading to the identification of novel biomarkers, and of potential targets for drug repurposing or novel drug designing.

References

[1] http://www.omim.org/;

[2] http://www.uniprot.org/

[3] Klapa et al.(2013). BMC_Systems Biol.:2;7:96.

[4] Gioutlakis, Klapa and Moschonas (2017) (under revision)

[5] https://www.ebi.ac.uk/gwas/

The new technologies for high-throughput biomolecular analysis (also known as “omics”) enabled the holistic study of biological systems by modeling the structure and regulation of biomolecular networks at various levels of cellular function. The integration of these analyses and methodologies of bioinformatics and systems biology with physiological studies of organs, animal models and human involving biochemical and behavioral analysis protocols, have led to the development of a new research field, that of Systems Physiology. In the case of animal models, in particular, one of the objectives and current challenges is the holistic analysis of the molecular physiology at the body level, through multi-tissue studies. The final goal is the reconstruction of a comprehensive model that can describe the interaction between tissues and with the blood circulation. These models could provide significant information about the physiology of the entire organism towards the identification of molecular mechanisms underlying complex pathophysiologies that affect various organs and/or are due to the combined deregulation of interacting pathways between tissues. There is currently a limited number of such studies at any molecular level of cellular function, as they are challenging both experimentally and computationally [1].

In this study, we present a sex-comparative integrated metabolomic analysis of the effect of adult-onset-hypothyroidism (AOH) on the metabolic physiology of five brain regions (i.e. cortex, cerebellum, midbrain, striatum and hippocampus) and one peripheral tissue (heart) in a chemical induction Balb-c/J mouse model [2]. The brain had for long been considered as metabolically nonresponsive to perturbations in the thyroid hormone (TH) levels and thus was not among the main tissues studies in the context of hypothyroidism. However, a large number of studies have provided evidence that the mammalian brain is a TH target tissue. Our group was the first to validate this fact through a Gas Chromatography-Mass Spectrometry (GC-MS) metabolomic study of the cerebellum tissue in the particular AOH mouse model [2]. Here, we expand this study to investigate collectively the brain regional variation of AOH effect in both sexes along with any potential differences in the response of the heart physiology to the particular dysfunction. We follow an optimized tissue perfusion, collection and quenching protocol [3] and the final metabolomic results are analyzed using the software platform for GC-MS metabolomic data deposition, normalization and unknown peak identification platform, M-IOLITE, that has been developed by our group (miolite2.iceht.forth.gr) [4].

References

1. Torell, F., Bennett, K., Cereghini, S., et. al. (2015). PlOS One, 10(6), e0129260.

2. Maga-Nteve C, Vasilopoulou CG, Constantinou C, Margarity M, Klapa MI. 2017. J Chromatogr B Analyt Technol Biomed Life Sci. 1041:158-166

3. Vasilopoulou CG, Margarity M and Klapa MI. 2016. Front Physiol. 7: 183.

4. Maga-Nteve C and Klapa MI. 2016. IFAC-PapersOnLine 49-26 (2016) 286–288.