Sensitivity of Xylella fastidiosa subsp. pauca Salento-1 to light at 410 nm.

All over the world, from America to the Mediterranean Sea, the plant pathogen Xylella fastidiosa represents one of the most difficult challenges with many implications at ecological, agricultural, and economic levels. X. fastidiosa is a rod-shaped Gram-negative bacterium belonging to the family of Xanthomonadaceae. It grows at very low rates and infects a wide range of plants thanks to different vectors. Insects, through their stylets, suck a sap rich in nutrients and inject bacteria into xylem vessels. Since, until now, no antimicrobial treatment has been successfully applied to kill X. fastidiosa and/or prevent its diffusion, in this study, antimicrobial blue light (aBL) was explored as a potential anti-Xylella tool. Xylella fastidiosa subsp. pauca Salento-1, chosen as a model strain, showed a certain degree of sensitivity to light at 410 nm. The killing effect was light dose dependent and bacterial concentration dependent. These preliminary results support the potential of blue light in decontamination of agricultural equipment and/or plant surface; however, further investigations are needed for in vivo applications.

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

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

Polydopamine-Coated Liposomes for Methylene Blue Delivery in Anticancer Photodynamic Therapy: Effects in 2D and 3D Cellular Models.

Photodynamic therapy (PDT) is a therapeutic option for cancer, in which photosensitizer (PS) drugs, light, and molecular oxygen generate reactive oxygen species (ROS) and induce cell death. First- and second-generation PSs presented with problems that hindered their efficacy, including low solubility. Thus, second-generation PSs loaded into nanocarriers were produced to enhance their cellular uptake and therapeutic efficacy. Among other compounds investigated, the dye methylene blue (MB) showed potential as a PS, and its photodynamic activity in tumor cells was reported even in its nanocarrier-delivered form, including liposomes. Here, we prepared polydopamine (PDA)-coated liposomes and efficiently adsorbed MB onto their surface. lipoPDA@MB vesicles were first physico-chemically characterized and studies on their light stability and on the in vitro release of MB were performed. Photodynamic effects were then assessed on a panel of 2D- and 3D-cultured cancer cell lines, comparing the results with those obtained using free MB. lipoPDA@MB uptake, type of cell death induced, and ability to generate ROS were also investigated. Our results show that lipoPDA@MB possesses higher photodynamic potency compared to MB in both 2D and 3D cell models, probably thanks to its higher uptake, ROS production, and apoptotic cell death induction. Therefore, lipoPDA@MB appears as an efficient drug delivery system for MB-based PDT.

Supramolecular Biohybrid Construct for Photoconversion Based on a Bacterial Reaction Center Covalently Bound to Cytochrome c by an Organic Light Harvesting Bridge.

A supramolecular construct for solar energy conversion is developed by covalently bridging the reaction center (RC) from the photosynthetic bacterium Rhodobacter sphaeroides and cytochrome c (Cyt c) proteins with a tailored organic light harvesting antenna (hCy2). The RC-hCy2-Cyt c biohybrid mimics the working mechanism of biological assemblies located in the bacterial cell membrane to convert sunlight into metabolic energy. hCy2 collects visible light and transfers energy to the RC, increasing the rate of photocycle between a RC and Cyt c that are linked in such a way that enhances proximity without preventing protein mobility. The biohybrid obtained with average 1 RC/10 hCy2/1.5 Cyt c molar ratio features an almost doubled photoactivity versus the pristine RC upon illumination at 660 nm, and ∼10 times higher photocurrent versus an equimolar mixture of the unbound proteins. Our results represent an interesting insight into photoenzyme chemical manipulation, opening the way to new eco-sustainable systems for biophotovoltaics.

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

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

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

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

Encapsulation of Curcumin-Loaded Liposomes for Colonic Drug Delivery in a pH-Responsive Polymer Cluster Using a pH-Driven and Organic Solvent-Free Process.

The present study aimed to develop and optimize liposome formulation for the colonic delivery of biologically active compounds. A strategy to facilitate such targeting is to formulate liposomes with a polymer coating sensitive to the pH shifts in the gastrointestinal tract. To this end, liposomes encapsulating curcumin-chosen as the biologically active compound model-and coated with the pH-responsive polymer Eudragit S100 were prepared and characterized. Curcumin was encapsulated into small unilamellar vesicles (SUVs) by the micelle-to-vesicle transition method (MVT) in a simple and organic solvent-free way. Curcumin-loaded liposomes were coated with Eudragit S100 by a fast and easily scalable pH-driven method. The prepared liposomes were evaluated for size, surface morphology, entrapment efficiency, stability, in vitro drug release, and curcumin antioxidant activity. In particular, curcumin-loaded liposomes displayed size lower than 100 nm, encapsulation efficiency of 98%, high stability at both 4 °C and 25 °C, high in vitro antioxidant activity, and a cumulative release that was completed within 200 min. A good Eudragit S100 coating which did not alter the properties of the curcumin-loaded liposomes was obtained. The present work therefore provides a fast and solvent-free method to prepare pH-responsive polymer-coated liposomes for the colonic delivery of biologically active compounds.

Synthetic Antenna Functioning As Light Harvester in the Whole Visible Region for Enhanced Hybrid Photosynthetic Reaction Centers.

The photosynthetic reaction center (RC) from the Rhodobacter sphaeroides bacterium has been covalently bioconjugated with a NIR-emitting fluorophore (AE800) whose synthesis was specifically tailored to act as artificial antenna harvesting light in the entire visible region. AE800 has a broad absorption spectrum with peaks centered in the absorption gaps of the RC and its emission overlaps the most intense RC absorption bands, ensuring a consistent increase of the protein optical cross section. The covalent hybrid AE800-RC is stable and fully functional. The energy collected by the artificial antenna is transferred to the protein via FRET mechanism, and the hybrid system outperforms by a noteworthy 30% the overall photochemical activity of the native protein under the entire range of visible light. This improvement in the optical characteristic of the photoenzyme demonstrates the effectiveness of the bioconjugation approach as a suitable route to new biohybrid materials for energy conversion, photocatalysis, and biosensing.

Semiquinone oscillations as a tool for investigating the ubiquinone binding to photosynthetic reaction centers.

Semiquinone oscillations induced by light pulses in the presence of exogenous electron donors are a valuable source of information on the kinetics and thermodynamics of ubiquinone chemistry relevant to the QB site of the photosynthetic reaction center (RC). In previous attempts to achieve the quantitative interpretation of data, the ubiquinone concentration was considered constant during the experiment since it was much bigger than that of RC. In this work, we extended existing models to low ubiquinone concentrations revealing several hidden processes taking place during the ubiquinone photoreduction and enabling the evaluation of the ubiquinone binding constant K Q at the QB site. The proposed approach provides for the first time the evaluation of K Q without any preliminary treatment of ubiquinone extraction from the binding site, thereby better preserving its native structure.

Assessment of antioxidant and antiproliferative properties of spinach plants grown under low oxygen availability.

BACKGROUND: In the human diet, the consumption of fresh fruits and vegetables is important in maintaining good health and in preventing chronic diseases. It is known that plant-derived food is a powerful source of chemopreventive molecules, i.e. antioxidants, and spinach (Spinacia oleracea L., Chenopodiaceae) possesses a wide range of metabolites with such biological activity. Plant stress response could lead to the production of metabolites with high value for human health and this could be a tool to enhance the production of molecules with antioxidant activity in plants. RESULTS: Data reported in this paper confirm the antioxidant properties of spinach plants, and show a strong antiproliferative activity of leaf extract on HT-29 human cell line. Besides, the hypoxic stress seems to affect the pool of antioxidant molecules present in spinach leaves, as verified by means of HPLC-MS/MS analysis and the aluminium chloride and ABTS assays. CONCLUSION: Our findings represent a basis for improving the biological and pharmacological properties of spinach plants, including the use of different growth conditions to modulate the phytocomplex profile of spinach.

Hybrid assemblies of fluorescent nanocrystals and membrane proteins in liposomes.

Because of the growing potential of nanoparticles in biological and medical applications, tuning and directing their properties toward a high compatibility with the aqueous biological milieu is of remarkable relevance. Moreover, the capability to combine nanocrystals (NCs) with biomolecules, such as proteins, offers great opportunities to design hybrid systems for both nanobiotechnology and biomedical technology. Here we report on the application of the micelle-to-vesicle transition (MVT) method for incorporation of hydrophobic, red-emitting CdSe@ZnS NCs into the bilayer of liposomes. This method enabled the construction of a novel hybrid proteo-NC-liposome containing, as model membrane protein, the photosynthetic reaction center (RC) of Rhodobacter sphaeroides. Electron microscopy confirmed the insertion of NCs within the lipid bilayer without significantly altering the structure of the unilamellar vesicles. The resulting aqueous NC-liposome suspensions showed low turbidity and kept unaltered the wavelengths of absorbance and emission peaks of the native NCs. A relative NC fluorescence quantum yield up to 8% was preserved after their incorporation in liposomes. Interestingly, in proteo-NC-liposomes, RC is not denatured by Cd-based NCs, retaining its structural and functional integrity as shown by absorption spectra and flash-induced charge recombination kinetics. The outlined strategy can be extended in principle to any suitably sized hydrophobic NC with similar surface chemistry and to any integral protein complex. Furthermore, the proposed approach could be used in nanomedicine for the realization of theranostic systems and provides new, interesting perspectives for understanding the interactions between integral membrane proteins and nanoparticles, i.e., in nanotoxicology studies.

The binding of quinone to the photosynthetic reaction centers: kinetics and thermodynamics of reactions occurring at the QB-site in zwitterionic and anionic liposomes.

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 QB-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 QB-depleted RC in the presence of increasing amounts of ubiquinone-10 over the temperature interval 6-35 °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 QB-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 °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 QB-site.

Evaluation of adhesion properties and antibacterial activities of the infant gut commensal Bifidobacterium bifidum PRL2010.

Bifidobacteria are extensively exploited by the food industry as health-promoting microorganisms. However, very little is known about the molecular mechanisms responsible for these beneficial activities, or the molecular players that sustain their ability to colonize and persist within the human gut. Here, we have investigated the enteric adaptation features of the gut commensal Bifidobacterium bifidum PRL2010, originally isolated from infant feces. This strain was able to survive under gastrointestinal challenges, while it was shown to adhere to human epithelial intestinal cell monolayers (Caco 2 and HT-29), thereby inhibiting adhesion of pathogenic bacteria such as Escherichia coli and Cronobacter sakazakii.

Purification of Recombinant Human Amphiphysin 1 and its N-BAR Domain.

Bin/Amphiphysin/Rvs (BAR) proteins are known as classical membrane curvature generators during endocytosis. Amphiphysin, a member of the N-BAR sub-family of proteins that contain a characteristic amphipathic sequence at the N-terminus of the BAR domain, is involved in clathrin-mediated endocytosis. Full-length amphiphysin contains a ~ 400 amino acid long disordered linker connecting the N-BAR domain and a C-terminal Src homology 3 (SH3) domain. We express and purify recombinant amphiphysin and its N-BAR domain along with an N-terminal glutathione-S-transferase (GST) tag. The GST tag allows extraction of the protein of interest using affinity chromatography and is removed in the subsequent protease treatment and ion-exchange chromatography steps. In the case of the N-BAR domain, cleavage of the GST tag was found to cause precipitation. This issue can be minimized by adding glycerol to the protein purification buffers. In the final step, size exclusion chromatography removes any potential oligomeric species. This protocol has also been successfully used to purify other N-BAR proteins, such as endophilin, Bin1, and their corresponding BAR domains. Graphical overview.

A simple strategy based on ATR-FTIR difference spectroscopy to monitor substrate intake and metabolite release by growing bacteria.

Attenuated total reflectance Fourier transform infrared (ATR-FTIR) difference spectroscopy has been employed for a variety of applications spanning from reaction mechanisms analysis to interface phenomena assessment. This technique is based on the detection of spectral changes induced by the chemical modification of the original sample. In the present study, we highlight the potential of the ATR-FTIR difference approach in the field of microbial biochemistry and biotechnology, reporting on the identification of main soluble species consumed and released by growing bacteria during the biohydrogen production process. Specifically, the mid-infrared spectrum of a model culture broth, composed of glucose, malt extract and yeast extract, was used as background to acquire the FTIR difference spectrum of the same broth as modified by Enterobacter aerogenes metabolism. The analysis of difference signals revealed that only glucose is degraded during hydrogen evolution in anaerobic conditions, while ethanol and 2,3-butanediol are the main soluble metabolites released with H2. This fast and easy analytical approach can therefore represent a sustainable strategy to screen different bacterial strains and to select raw and waste materials to be employed in the field of biofuel production.

Oxidoreductase activity of chromatophores and purified cytochrome bc1 complex from Rhodobacter sphaeroides: a possible role of cardiolipin.

Osmotic shock was used as a tool to obtain cardiolipin (CL) enriched chromatophores of Rhodobacter sphaeroides. After incubation of cells in iso- and hyper-osmotic buffers both chromatophores with a physiological lipid profile (Control) and with an almost doubled amount of CL (CL enriched) were isolated. Spectroscopic properties, reaction centre (RC) and reducible cytochrome (cyt) contents in Control and CL enriched chromatophores were the same. The oxidoreductase activity was found higher for CL enriched than for Control chromatophores, raising from 60 ± 2 to 93 ± 3 mol cyt c s(-1) (mol total cyt c)(-1). Antymicin and myxothiazol were tested to prove that oxidoreductase activity thus measured was mainly attributable to the cyt bc ( 1 ) complex. The enzyme was then purified from BH6 strain yielding a partially delipidated and almost inactive cyt bc ( 1 ) complex, although the protein was found to maintain its structural integrity in terms of subunit composition. The ability of CL in restoring the activity of the partially delipidated cyt bc ( 1 ) complex was proved in micellar systems by addition of exogenous CL. Results here reported indicate that CL affects oxidoreductase activity in the bacterium Rhodobacter sphaeroides both in chromatophore and in purified cyt bc ( 1 ) complex.

Light induced transmembrane proton gradient in artificial lipid vesicles reconstituted with photosynthetic reaction centers.

Photosynthetic reaction center (RC) is the minimal nanoscopic photoconverter in the photosynthetic membrane that catalyzes the conversion of solar light to energy readily usable for the metabolism of the living organisms. After electronic excitation the energy of light is converted into chemical potential by the generation of a charge separated state accompanied by intraprotein and ultimately transmembrane proton movements. We designed a system which fulfills the minimum structural and functional requirements to investigate the physico/chemical conditions of the processes: RCs were reconstituted in closed lipid vesicles made of selected lipids entrapping a pH sensitive indicator, and electron donors (cytochrome c2 and K4[Fe(CN)6]) and acceptors (decylubiquinone) were added to sustain the photocycle. Thanks to the low proton permeability of our preparations, we could show the formation of a transmembrane proton gradient under illumination and low buffering conditions directly by measuring proton-related signals simultaneously inside and outside the vesicles. The effect of selected ionophores such as gramicidin, nigericin and valinomycin was used to gain more information on the transmembrane proton gradient driven by the RC photochemistry.

Isolation of Squarebop I bacteriorhodopsin from biomass of coastal salterns.

Squarebop I bacteriorhodopsin is a light-activated proton pump present in the membranes of the archeon Haloquadratum walsbyi, a square-shaped organism representing 50-60% of microbial population in the crystallizer ponds of the coastal salterns. Here we describe: (1) the operating mode of a bioreactor designed to concentrate the saltern biomass through a microfiltration process based on polyethersulfone hollow fibers; (2) the isolation of Squarebop I bacteriorhodopsin from solubilized biomass by means of a single chromatographic step; (3) tightly bound lipids to the isolated and purified protein as revealed by MALDI-TOF/MS analysis; (4) the photoactivity of Squarebop I bacteriorhodopsin isolated from environmental samples by flash spectroscopy. Yield of the isolation process is 150 µg of Squarebop I bacteriorhodopsin from 1l of 25-fold concentrated biomass. The possibility of using the concentrated biomass of salterns, as renewable resource for the isolation of functional bacteriorhodopsin and possibly other valuable bioproducts, is briefly discussed.

Nanosized optoelectronic devices based on photoactivated proteins.

Molecular nanoelectronics is attracting much attention, because of the possibility to add functionalities to silicon-based electronics by means of intrinsically nanoscale biological or organic materials. The contact point between active molecules and electrodes must present, besides nanoscale size, a very low resistance. To realize Metal-Molecule-Metal junctions it is, thus, mandatory to be able to control the formation of useful nanometric contacts. The distance between the electrodes has to be of the same size of the molecule being put in between. Nanogaps technology is a perfect fit to fulfill this requirement. In this work, nanogaps between gold electrodes have been used to develop optoelectronic devices based on photoactive proteins. Reaction Centers (RC) and Bacteriorhodopsin (BR) have been inserted in nanogaps by drop casting. Electrical characterizations of the obtained structures were performed. It has been demonstrated that these nanodevices working principle is based on charge separation and photovoltage response. The former is induced by the application of a proper voltage on the RC, while the latter comes from the activation of BR by light of appropriate wavelengths.

Anti-proliferative activity and chemoprotective effects towards DNA oxidative damage of fresh and cooked Brassicaceae.

Epidemiological evidence shows that regular consumption of Brassicaceae is associated with a reduced risk of cancer and heart disease. Cruciferous species are usually processed before eating and the real impact of cooking practices on their bioactive properties is not fully understood. We have evaluated the effect of common cooking practices (boiling, microwaving, and steaming) on the biological activities of broccoli, cauliflower and Brussels sprouts. Anti-proliferative and chemoprotective effects towards DNA oxidative damage of fresh and cooked vegetable extracts were evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and Comet assays on HT-29 human colon carcinoma cells. The fresh vegetable extracts showed the highest anti-proliferative and antioxidant activities on HT-29 cells (broccoli>cauliflower = Brussels sprouts). No genotoxic activity was detected in any of the samples tested. The cooking methods that were applied influenced the anti-proliferative activity of Brassica extracts but did not alter considerably the antioxidant activity presented by the raw vegetables. Raw, microwaved, boiled (except broccoli) and steamed vegetable extracts, at different concentrations, presented a protective antioxidative action comparable with vitamin C (1 mm). These data provide new insight into the influence of domestic treatment on the quality of food, which could support the recent epidemiological studies suggesting that consumption of cruciferous vegetables, mainly cooked, may be related to a reduced risk of developing cancer.