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Dolichol, an end product of the mevalonate pathway, has been proposed as a biomarker of aging, but its biological role, not to mention its catabolism, has not been fully understood. UV-B radiation was used to induce oxidative stress in isolated rat hepatocytes by the collagenase method. Effects on dolichol, phospholipid-bound polyunsaturated fatty acids (PL-PUFA) and known lipid soluble antioxidants [coenzyme Q (CoQ) and ?-tocopherol] were studied. The increase in oxidative stress was detected by a probe sensitive to reactive oxygen species (ROS). Peroxidation of lipids was assessed by measuring the release of thiobarbituric acid reactive substances (TBARS). Dolichol, CoQ, and ?-tocopherol were assessed by high-pressure liquid chromatography (HPLC), PL-PUFA by gas-liquid chromatography (GC). UV-B radiation caused an immediate increase in ROS as well as lipid peroxidation and a simultaneous decrease in the levels of dolichol and lipid soluble antioxidants. Decrease in dolichol paralleled changes in CoQ levels and was smaller to that in ?-tocopherol. The addition of mevinolin, a competitive inhibitor of the enzyme 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoAR), magnified the loss of dolichol and was associated with an increase in TBARS production. Changes in PL-PUFA were minor. These findings highlight that oxidative stress has very early and similar effects on dolichol and lipid soluble antioxidants. Lower levels of dolichol are associated with enhanced peroxidation of lipids, which suggest that dolichol may have a protective role in the antioxidant machinery of cell membranes and perhaps be a key to understanding some adverse effects of statin therapy.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the deposition of extracellular amyloid-beta peptide (Aβ) and intracellular neurofibrillar tangles, associated with loss of neurons in the brain and consequent learning and memory deficits. Aβ is the major component of the senile plaques and is believed to play a central role in the development and progress of AD both in oligomer and fibril forms. Inhibition of the formation of Aβ fibrils as well as the destabilization of preformed Aβ in the Central Nervous System (CNS) would be an attractive therapeutic target for the treatment of AD. Moreover, a large number of studies indicate that oxidative stress and mitochondrial dysfunction may play an important role in AD and their suppression or reduction via antioxidant use could be a promising preventive or therapeutic intervention for AD patients. Many antioxidant compounds have been demonstrated to protect the brain from Aβ neurotoxicity. Ferulic acid (FA) is an antioxidant naturally present in plant cell walls with anti-inflammatory activities and it is able to act as a free radical scavenger. Here we present the role of FA as inhibitor or disaggregating agent of amyloid structures as well as its effects on biological models.

International audience; Curcumin is a natural polyphenol able to bind the amyloid beta peptide, which is related to Alzheimer's disease, and modify its self-assembly pathway. This paper focuses on a multi-disciplinary study that starts from the design of curcumin-like compounds with the key chemical features required for inhibiting amyloid beta aggregation, and reports the effects of these compounds on the in vitro aggregation of amyloid beta peptides. Chemoinformatic screening was performed through the calculation of molecular descriptors that were able to highlight the drug-like profile, followed by docking studies with an amyloid beta peptide fibril. The computational design underlined two different scaffolds that were easily synthesized in good yields. In vitro experiments, ranging from fluorescence spectroscopy and confocal microscopy up to small angle X-ray scattering, provided evidence that the synthesized compounds are able to modify the aggregation pattern of amyloid beta peptides both in the secondary structures, and in terms of the overall structure dimensions. The cytotoxic potential of the synthesized compounds was finally tested in vitro with a model neuronal cell line (LAN5). The overall view of this study suggests new concepts and potential difficulties in the design of novel drugs against diverse amyloidoses, including Alzheimer's disease

Conventional antimicrobial strategies have become increasingly ineffective due to the rapid emergence of antibiotic resistance among pathogenic bacteria. In order to overcome this problem, antimicrobial PhotoDynamic Therapy (PDT) is considered a promising alternative therapy. PDT has a broad spectrum of action and low mutagenic potential. It is particularly effective when microorganisms present endogenous photosensitizing pigments. Helicobacter pylori (Hp), a pathogen notoriously responsible of severe gastric infections (chronic gastritis, peptic ulcer, MALT lymphoma and gastric adenocarcinoma), produces and accumulates the photosensitizers protoporphyrin IX and coproporphyrin, thus it might be a suitable target of antimicrobial PDT. With the aim to design and develop an ingestible LED-based robotic pill for intragastric phototherapy, so that irradiation can be performed in situ without the use of invasive endoscopic light, photophysical studies on the Hp endogenous photosensitizers were carried out. These studies represent an important prerequisite in order to select the most effective irradiation conditions for Hp eradication. The photophysical characterization of Hp porphyrins, including their spectroscopic features in terms of absorption, steady-state and time-resolved fluorescence, was performed on bacterial extracts as well as within planktonic and biofilm growing Hp cells.

Antimicrobial PhotoDynamic Therapy (aPDT) is an emerging strategy aimed at the eradication of bacterial infections, with a special focus on antibiotic-resistant bacteria. This method is easy to apply, not expensive and particularly interesting in case of bacteria that spontaneously produce the required photosensitizers. In the framework of a project aimed at the development of an ingestible pill for the application of aPDT to gastric infections by Helicobacter pylori, a LED-based illuminating prototype (LED-BIP) was purposely designed in order to evaluate the photodamage induced by light of different wavelengths on porphyrin-producing bacteria. This short paper reports about temperature tests performed to assess the maximum exposure time and light dose that can be administered to bacterial cultures inside LED-BIP without reaching temperatures exceeding the physiological range.

We have performed an in vitro study to investigate the molecular basis of the aggregation kinetic of 1-40 β-amyloid peptides (Aβ and the possibility of affecting this aggregation process using an exogenous natural polycyclic pigment, hypericin (Hyp). The effect of Hyp on the self-assembly process at different times of the aggregation kinetic has been investigated utilizing a chaperon-like molecule, α-crystallin. Circular dichroism and fluorescence results suggest that Hyp can associate to precursors of the mature fibrils and perturb the aggregation process through intermolecular interactions with the Aβ peptides.Structured summaryMINT-6742984: 1-40 beta-amyloid peptide (uniprotkb:P05067) and 1-40 beta-amyloid peptide (uniprotkb:P05067) bind (MI:0407) by circular dichroism (MI:0016)MINT-6743002: 1-40 beta-amyloid peptide (uniprotkb:P05067) and 1-40 beta-amyloid peptide (uniprotkb:P05067) bind (MI:0407) by light scattering (MI:0067)MINT-6743012: 1-40 beta-amyloid peptide (uniprotkb:P05067) and 1-40 beta-amyloid peptide (uniprotkb:P05067) bind (MI:0407) by classical fluorescence spectroscopy (MI:0017)

Curcumin is a natural polyphenol able to bind the amyloid beta peptide, which is related to Alzheimer's disease, and modify its self-assembly pathway. This paper focuses on a multi-disciplinary study that starts from the design of curcumin-like compounds with the key chemical features required for inhibiting amyloid beta aggregation, and reports the effects of these compounds on the in vitro aggregation of amyloid beta peptides. Chemoinformatic screening was performed through the calculation of molecular descriptors that were able to highlight the drug-like profile, followed by docking studies with an amyloid beta peptide fibril. The computational design underlined two different scaffolds that were easily synthesized in good yields. In vitro experiments, ranging from fluorescence spectroscopy and confocal microscopy up to small angle X-ray scattering, provided evidence that the synthesized compounds are able to modify the aggregation pattern of amyloid beta peptides both in the secondary structures, and in terms of the overall structure dimensions. The cytotoxic potential of the synthesized compounds was finally tested in vitro with a model neuronal cell line (LAN5). The overall view of this study suggests new concepts and potential difficulties in the design of novel drugs against diverse amyloidoses, including Alzheimer's disease.

Background Current research has indicated that small natural compounds could interfere with β-amyloid fibril growth and have the ability to disassemble preformed folded structures. Ferulic acid (FA), which possesses both hydrophilic and hydrophobic moieties and binds to peptides/proteins, is a potential candidate against amyloidogenesis. The molecular mechanisms connected to this action have not been elucidated in detail yet. Methods Here the effects of FA on preformed fibrils are investigated by means of a concerted experimental–computational approach. Spectroscopic techniques, such as FTIR, fluorescence, size exclusion chromatography and confocal microscopy in combination with molecular dynamics simulations are used to identify those features which play a key role in the destabilization of the aggregates. Results Experimental findings highlight that FA has disruptive effects on the fibrils. The computational analysis suggests that dissociation of peptides from the amyloid superstructures could take place along the fibril axis and be primarily determined by the cooperative rupture of the backbone hydrogen bonds and of the Asp-Lys salt bridges. Conclusion FA clusters could induce a sort of stabilization and tightening of the fibril structure in the short term and its disruption in the long term, inhibiting further fibril re-assembly through FA screening effects. General significance The combination of experimental and computational techniques could be successfully used to identify the disrupting action of FA on preformed Aβ fibrils in water solution.

Dolichol, the polyisoprenoid lipid found in all eukaryotic cells and suggested to represent a biomarker of aging, is inserted into cell membranes, also in tissues exposed to light such as the skin. A general question about its physiological role is whether dolichol may play the role of a natural barrier for the noxious components of solar radiation. In order to clarify this point, we established that dolichol is a component of human sebum and we performed an " in vitro " study of the effects of UV radiation on the spectral properties of dolichol in isopropanol. Our data clearly show that, following UV irradiation, the optical absorption spectrum of dolichol undergoes remarkable modifications below 400 nm: a significant, strongly dose-dependent, increase of the optical density around 320 nm and a minor, very slightly dose-dependent, raise of the absorbance at 250 nm. On the contrary, UV irradiation causes only minor changes in HPLC profiles and the formation of photooxidative products can be considered negligible in our experimental conditions. These results suggest that dolichol can be considered an innate, unusually efficient and promising UV screen for skin protection.

Many freely motile microorganisms can perceive and transduce external photic stimuli to the motor apparatus, eventually moving, by means of various behavioural strategies, into environments in which the illumination conditions are the most favourable for their life. In different microorganisms, a wide range of chromophores operate as light detectors, each of them set in a special molecular pocket that, in its turn, can be linked to another component of the transduction chain. The diverse photosensors are organized in special (and in many cases dedicated) photoreceptor units or subcellular organelles. The main molecular mechanisms connecting the early event of photon absorption to the formation of the signalling state down to the dark steps of the transduction chain are discussed in a selected number of case examples. The possible importance of an intensive multidisciplinary approach to these problems in an evolutionary perspective is finally briefly outlined.

— The ciliate Blepharisma japonicum was exposed to artificial polychromatic and monochromatic UV radiation to evaluate the relative roles of UVB (280–315 nm UV radiation) and UVA (315–400 nm UV radiation) in altering its motility and photobehavior and to determine absolute weighting coefficients for these effects in the UVB range. Under polychromatic UV irradiation B. japonicum cells showed a severe reduction of cell speed and of the capability to respond to light stimuli. At low doses, however, UV caused a significant increase in the average velocity of a cell population. The UVB exclusion experiments indicated that UVA does not significantly alter motility and photoresponsiveness. The increase and the subsequent decrease in cell velocity was observed also under monochromatic irradiation at 281, 290 and 300 nm, whereas at 310 nm cells swim faster up to the highest photon flux density used. The cell capability of reacting to photic stimuli, conversely, steadily declined with increasing photon flux density at all the tested UVB wavelengths. The action spectra for the alteration of cell velocity and the impairment of photoresponsiveness show that the lower the irradiation wavelength, the more remarkable are the UVB effects and suggest different targets for the increase and the decrease in cell velocity.

A key question to answer studying the biological effects of ultraviolet radiation on planktonic micro-organisms is whether they can perceive UV-B radiation as a sensory signal, likewise they do with visible light. We have faced this problem performing an individual-cell analysis of Blepharisma japonicum photomotile responses to UV-B stimuli. Our results on spectral responsiveness and on the effects of a photoresponse inhibitor indicate that B. japonicum is capable to perceive and transduce UV-B radiation as an environmental sensory stimulus, which it escapes from gathering in shadowed areas. Similar UV-B avoidance motile reactions could serve as a behavioural defence mechanism contributing to avoid harmful overexposure to UV-B.

There is an emerging interest in small natural molecules for their potential therapeutic use in neurodegenerative disorders like Alzheimer's disease (AD). Ferulic acid (FA), an antioxidant phenolic compound present in fruit and vegetables, has been proposed as an inhibitor of beta amyloid (Aβ) pathological aggregation. Using fluorescence and Fourier transform infrared spectroscopy, electrophoresis techniques, chromatographic analysis, and confocal microscopy, we investigated the effects of FA in the early stages of Aβ fibrillogenesis in vitro. Our results show that FA interacts promptly with Aβ monomers/oligomers, interfering since the beginning with its self-assembly and finally forming amorphous aggregates more prone to destabilization. These findings highlight the molecular basis underlying FA antiamyloidogenic activity in AD.

Laser-driven electron accelerators based on the Laser Wakefield Acceleration process has entered a mature phase to be considered as alternative devices to conventional radiofrequency linear accelerators used in medical applications. Before entering the medical practice, however, deep studies of the radiobiological effects of such short bunches as the ones produced by laser-driven accelerators have to be performed. Here we report on the setup, characterization and first test of a small-scale laser accelerator for radiobiology experiments. A brief description of the experimental setup will be given at first, followed by an overview of the electron bunch characterization, in particular in terms of dose delivered to the samples. Finally, the first results from the irradiation of biological samples will be briefly discussed.

We present a laser-driven source of electron bunches with average energy 260 keV and picosecond duration, which has been setup for radiobiological tests covering the previously untested sub-MeV energy range. Each bunch combines high charge with short duration and sub-millimeter range into a record instantaneous dose rate, as high as 10(9) Gy s(-1). The source can be operated at 10 Hz and its average dose rate is 35 mGy s(-1). Both the high instantaneous dose rate and high level of relative biological effectiveness, attached to sub-MeV electrons, make this source very attractive for studies of ultrafast radiobiology on thin cell samples. The source reliability, in terms of shot-to-shot stability of features such as mean energy, bunch charge and transverse beam profile, is discussed, along with a dosimetric characterization. Finally, a few preliminary biological tests performed with this source are presented. .

the effects of artificial UV-B irradiation on motility, viability, photomotile responses and photoreceptor spectroscopic properties of the red ciliate Blepharisma japonicum were investigated. Both the mean velocity of the cells and the percentage of motile cells exhibiting step-up photophobic responses significantly decreased, increasing UV-B irradiation time. No marked difference with dark-kept control samples was observed in cells irradiated with visible light. Absorption and fluorescence measurements on cell suspensions indicated that no UV-B induced bleaching or spectroscopically detectable damage of the photoreceptor chromophore took place. The experimental results suggest that the UV-B target is a component of the photosensory transduction chain different from the photopigment itself and that cell vitality is damaged by a direct effect of UV-B, without any significant contribution from a pigment-sensitized photodynamic reaction.

Intrinsic fluorescence of peptides and proteins is extensively used to monitor their specific interactions with several natural and synthetic molecules known to have wide-ranging beneficial or detrimental effects on health. A consequence of these interactions would be a significant decrease of the fluorescence emission intensity of Tyrosine (Tyr) and/or Tryptophan (Trp) residues in the protein due to structural rearrangements of proteic microenvironment. However fluorescence quenching can be also caused by "trivial" artefacts. In this study we examined the effect of Ferulic acid (FA) on Tyr fluorescence. FA is a natural anti-oxidant suggested to bind to and to modify the structural properties of several proteins thus altering their biological activities. Fluorescence spectroscopy experiments on Tyr and on proteins containing Tyr and no Trp like beta amyloid peptides and Insulin were performed. Our results suggest that Tyr fluorescence loss can mainly result from an inner filter effect rather than from specific interactions with FA.

Biomolecular self-assembly is a fundamental process in all organisms. As primary components of the life molecular machinery, proteins have a vast array of resources available to them for self-assembly in a functional structure. Protein self-assembly, however, can also occur in an aberrant way, giving rise to non-native aggregated structures responsible for severe, progressive human diseases that have a serious social impact. Different neurodegenerative disorders, like Huntington’s, Alzheimer’s, and spongiform encephalopathy diseases, have in common the presence of insoluble protein aggregates, generally termed “amyloid,” that share several physicochemical features: a fibrillar morphology, a predominantly beta-sheet secondary structure, birefringence upon staining with the dye Congo red, insolubility in common solvents and detergents, and protease resistance. Conformational constrains, hydrophobic and stacking interactions can play a key role in the fibrillogenesis process and protein–protein and peptide–peptide interactions—resulting in self-assembly phenomena of peptides yielding fibrils—that can be modulated and influenced by natural biomolecules. Small organic molecules, which possess both hydrophilic and hydrophobic moieties able to bind to peptide/protein molecules through hydrogen bonds and hydrophobic and aromatic interactions, are potential candidates against amyloidogenesis. In this review some significant case examples will be critically discussed.

We have determined the secondary structure of 1-40 beta-amyloid peptides by Fourier-transform infrared spectroscopy (FTIR) and characterized the peptide photophysical properties before and after self-assembly by using intrinsic tyrosine steady-state and time-resolved fluorescence. All measurements were performed in the presence and absence of hypericin (Hyp), an exogenous natural polycyclic pigment that has been shown to inhibit fibril formation and has also been used as a fluorescent probe. We monitored the time course of the aggregation process measuring 405 nm light diffusion at 90° and used thioflavin T to reveal the presence of fibrils. FTIR quantitative analysis evidenced a prevalent random conformation at t = 0 with and without Hyp. Fibrils showed a predominant parallel beta-sheet structure and a small percentage of alpha-helix. The results of fluorescence measurements showed that Hyp does significantly interact with peptides in beta-sheet conformation. In conclusion, hypericin does hinder the formation of fibrils, but the percentages of parallel beta-sheets were not significantly different from those found in samples not treated with Hyp.

The interaction of blepharismin (BP) and oxyblepharismin (OxyBP) with bovine alpha-crystallin (BAC) has been studied both by steady-state and femtosecond spectroscopy, with the aim of assessing the possible phototoxicity of these compounds toward the eye tissues. We showed that these pigments form with BAC potentially harmful ground-state complexes, the dissociation constants of which have been estimated to be 6 +/- 2 micromol L(-1) for OxyBP and 9 +/- 4 micromol L(-1) for BP. Irradiation with steady-state visible light of solutions of blepharismins in the presence of BAC proved to induce a quenching of both the pigment and the intrinsic protein fluorescences. These effects were tentatively rationalized in terms of structural changes of alpha-crystallin. On the other hand, femtosecond transient absorption spectroscopy was used to check the occurrence of any type I photoactivity of oxyblepharismin bound to alpha-crystallin. The existence of a particular type of fast photoinduced reaction, not observed in former studies with human serum albumin but present in the natural oxyblepharismin-binding protein, could here be evidenced but no specific reaction was observed during the first few nanoseconds after excitation. Partial denaturation of alpha-crystallin was however found to alter the excited-state behaviour of its complex with oxyblepharismin, making it partly resemble that of free oxyblepharismin in solution.

Different neurodegenerative disorders, like Huntington's, Alzheimer's, and spongiform encephalopathy diseases, have in common the presence of insoluble protein aggregates, generally termed "amyloid", that share several physicochemical features: a fibrillar morphology, a predominantly beta-sheet secondary structure, birefringence upon staining with the dye Congo red, insolubility in common solvents and detergents, and proteaseresistance. In the case of Alzheimer's disease (AD), in particular, senile extracellular deposits of beta-amyloid peptide (Abeta) fibrils (plaques) are observed. The conventional view has been that it is the amyloid aggregate that is pathological, but more recently an alternative view is emerging: the neurotoxic species would not be the insoluble aggregate itself, but rather soluble oligomeric species, including small globular structures, 2.7 to 6.0 nm in diameter, and curvilinear structures called "protofibrils". Conformational constrains and stacking interactions can play a key role in the fibrillogenesis process and protein-protein and peptide-peptide interactions -resulting in self-assembly phenomena of Abeta peptides yielding fibrils - can be modulated and influenced by small organic molecules. Polycyclic aromatic molecules are of special interest as they can disrupt the molecular architectures precursors of fibrils by means of aromatic interactions, like stacking interactions with tyrosine and phenylalanine residues of the beta-amyloid peptides.

Among chaperone-like functioning proteins, the lens alpha-crystallins are of particular interest because they are not renewed, and even minor alterations can hurt their function of maintaining the proper refractive index and avoiding cataract formation in the lens. Several reports have suggested the occurrence of remarkable structural modifications in lens proteins in the presence of endogenous and exogenous sensitizers upon exposure to light. In particular, it has been shown in vitro that hypericin, the active ingredient of Hypericum, can bind to and, in the presence of light, cause the photopolymerization of alpha-crystallin. On the basis of these results it has also been suggested that a subsequent significant impairment of the protein function can occur. Using absorption and emission spectroscopic techniques, as well as circular dichroism, we have studied the structural modifications of alpha-crystallin resulting from its interaction with hypericin after irradiation with visible light. To investigate the chaperone-like function of alpha-crystallin, the heat-induced aggregation kinetics of another lens protein, betaLow-crystallin, was monitored by measuring the apparent absorption due to scattering at 360 nm as a function of time, and no apparent damage to its functional role was observed. Spectroscopic results, on the contrary, show a prominent reduction in both tryptophan and hypericin fluorescence emission intensity after light irradiation, suggesting an alteration in the tryptophan microenvironment and a high degree of packing of the chromophore due to photoinduced modification of the molecular framework. Control experiments on alpha-crystallin structurally modified by light in the presence of hypericin indicated that the protein still retains its ability to chaperone both lens crystallins and insulin.

Among chaperone-like functioning proteins, the lens alpha-crystallins are of particular interest because they are not renewed, and even minor alterations can hurt their function of maintaining the proper refractive index and avoiding cataract formation in the lens. Several reports have suggested the occurrence of remarkable structural modifications in lens proteins in the presence of endogenous and exogenous sensitizers upon exposure to light. In particular, it has been shown in vitro that hypericin, the active ingredient of Hypericum, can bind to and, in the presence of light, cause the photopolymerization of alpha-crystallin. On the basis of these results it has also been suggested that a subsequent significant impairment of the protein function can occur. Using absorption and emission spectroscopic techniques, as well as circular dichroism, we have studied the structural modifications of alpha-crystallin resulting from its interaction with hypericin after irradiation with visible light. To investigate the chaperone-like function of alpha-crystallin, the heat-induced aggregation kinetics of another lens protein, betaLow-crystallin, was monitored by measuring the apparent absorption due to scattering at 360 nm as a function of time, and no apparent damage to its functional role was observed. Spectroscopic results, on the contrary, show a prominent reduction in both tryptophan and hypericin fluorescence emission intensity after light irradiation, suggesting an alteration in the tryptophan microenvironment and a high degree of packing of the chromophore due to photoinduced modification of the molecular framework. Control experiments on alpha-crystallin structurally modified by light in the presence of hypericin indicated that the protein still retains its ability to chaperone both lens crystallins and insulin.

Liposomes loaded with hypericin at different local concentrations were used as artificial models of biological light sensors. The steady state and time-resolved fluorescence of the chromophore were investigated in the presence of fluorescence quenchers which penetrate differently into and diffuse differently through lipid bilayers: iodide (I−), 9,10-anthraquinone-2,6-disulphonate (AQDS2−) and 9,10-anthraquinone-2-sulphonate (ACMS−). The results of the experiments with I− indicate that two of the three fluorescent species detected in our system, the long-lived and intermediate-lived species, are relatively close to the lipid-water interface. Anthraquinone compounds, which partially penetrate into the liposome, not only significantly quench the chromophore fluorescence emission, but also, apparently, affect the distribution of hypericin molecules in the vesicle, shifting the long-lived fluorescent molecules towards chemical-physical configurations characterized by shorter fluorescence lifetimes.

Any physiological phenomenon which is driven, triggered or modulated by sunlight in living organisms (from vision to photosynthesis, from photoreactivation to vitamin D biosynthesis, from phototropism to seed germination) is the final result of a series of physical, chemical, biophysical and biochemical processes which originate from the same “elementary” event: the absorption of a photon by the photoreceptor molecule.

In photoresponsive ciliates, like Blepharisma japonicum and Stentor coeruleus, the photoreceptor pigments responsible for photomotile reactions are hypericin-type chromophores packed in highly osmiophilic subpellicular granules. Lipopsomes loaded with hypericin can constitute a simple model system, appropriate for understanding the primary light-induced molecular events triggering the sensory chain in these microorganisms. Optical absorption, steady-state and time-resolved fluorescence and pulsed photoacoustic calorimetry have been used to measure spectral distributions, fluorescence lifetimes, radiative and radiationless transition quantum yields of hypericin when assembled into egg L-alpha-phosphatidylcholine liposomes. With respect to hypericin ethanol solutions, both absorption and fluorescence maxima are 5 nm red shifted when the pigment is inserted into the lipidic microenvironment, regardless of the hypericin local concentration. Increasing by 100 times the hypericin local concentration decreases the relative fluorescence quantum yield by a factor of around 150 and the fraction of thermally released energy, conversely, increases from 0.6 to 0.9. From the analysis of fluorescence lifetimes and their relative amplitudes it appears that a subnanosecond living component is predominant at the highest hypericin local concentrations.

Conventional antimicrobial strategies have become increasingly ineffective due to the emergence of antibiotic resistance among pathogenic bacteria. In order to overcome this problem, antimicrobial PhotoDynamic Therapy (PDT) is considered an alternative promising therapy. PDT has a broad spectrum of action and low mutagenic potential and is particularly effective when microorganisms present endogenous photosensitizing pigments. Helicobacter pylori (Hp), a pathogen notoriously responsible for severe gastric infections (chronic gastritis, peptic ulcer, MALT lymphoma and gastric adenocarcinoma), might be a suitable target of antimicrobial PDT as produces and accumulates the photosensitizers protoporphyrin IX and coproporphyrins. With the aim to design and develop an ingestible LED-based robotic pill for intragastric phototerapy so that irradiation can be performed in situ without the use of invasive endoscopic light, the action spectra for Hp photokilling will be determined. The photophysical properties of the Hp porphyrins will be studied. Hp culture samples will be irradiated at various wavelengths and intensities to determine the most efficient light conditions. Framework project: "CapsuLight"(CUP B52I14005760002) financed by Regione Toscana, Bando FAS Salute 2014 (Italy).

Helicobacter pylori (Hp) is among the most common infective agents in humans and is responsible for several gastric infections; moreover, its antibiotic resistance is raising new challenges. Antimicrobial photodynamic therapy (PDT) is a promising technique based on the use of non-toxic photosensitizers that, under the effect of a selected visible light, can generate cytotoxic reactive oxygen species; PDT is particularly effective when the target microorganism presents endogenous photosensitizers. It has been shown that the photosensitizers protoporphyrin IX and coproporphyrin I are endogenously produced in Hp, making it a suitable target for PDT. Aiming at the production of an ingestible LED-based robotic pill for intragastric PDT able to perform in situ irradiation without the use of endoscopic devices, the effectiveness of Hp photokilling in models has to be determined. We studied the distribution of porphyrins within bacteria suspensions and biofilms and adapted a protocol for porphyrins extraction from bacteria to characterize the spectroscopic features of the extracts. Project: "CapsuLight - Design of an ingestible robotic pill based on LED sources for the treatment of gastrointestinal disorders" (CUP B52I14005760002) financed by Regione Toscana Bando FAS Salute 2014 (Italy). P-809 Biomechanical studies with ultrasound in cell biology V. Bentivegna1, F. Stewart1, S. Cochran2, I. N¨athke1 1Cell and Developmental Biology, School of Life Sciences, University of Dundee, Dundee, UK; 2School of Engineering, University of Glasgow, Glasgow, UK During tissue development and cancer progression, cells undergo mechanical changes and respond differently to physical cues from their environment. Usually measuring physical properties of biological samples relies on direct physical contact with the sample. We propose ultrasound as a versatile tool for imaging and also exerting forces on cells that does not require direct contact. Using a 40 MHz microultrasound transducer, we compared radiofrequency signals with optical images of 3D tissue structures. This revealed that the size of spherical structures correlates with the ultrasound RF signal. Ultrasound overestimates the size of hollow cysts by 33%, while no such offset was observed in solid spheroids. Early results also suggest that microultrasound can reveal mechanical properties. At lower frequencies (~ 4 MHz) and higher intensities, ultrasound can compress cell layers via acoustic radiation pressure and without the need to directly contact the sample. This allowed investigating how cells react to external mechanical forces. Developing this approach further will permit comparison of mechanical properties of tissue at different stages of cancer progression and increase our understanding of how mechanical forces direct