Innovative Phosphorene Nanoplatform for Light Antimicrobial Therapy.

Over the past few years, antibiotic resistance has reached global dimensions as a major threat to public health. Consequently, there is a pressing need to find effective alternative therapies and therapeutic agents to combat drug-resistant pathogens. Photodynamic therapy (PDT), largely employed as a clinical treatment for several malignant pathologies, has also gained importance as a promising antimicrobial approach. Antimicrobial PDT (aPDT) relies on the application of a photosensitizer able to produce singlet oxygen (1O2) or other cytotoxic reactive oxygen species (ROS) upon exposure to appropriate light, which leads to cell death after the induced photodamage. Among different types of 2D nanomaterials with antimicrobial properties, phosphorene, the exfoliated form of black phosphorus (bP), has the unique property intrinsic photoactivity exploitable for photothermal therapy (PTT) as well as for PDT against pathogenic bacteria.

Fluorescence Lifetime Imaging Microscopy of Porphyrins in Helicobacter pylori Biofilms.

Bacterial biofilm constitutes a strong barrier against the penetration of drugs and against the action of the host immune system causing persistent infections hardly treatable by antibiotic therapy. Helicobacter pylori (Hp), the main causative agent for gastritis, peptic ulcer and gastric adenocarcinoma, can form a biofilm composed by an exopolysaccharide matrix layer covering the gastric surface where the bacterial cells become resistant and tolerant to the commonly used antibiotics clarithromycin, amoxicillin and metronidazole. Antimicrobial PhotoDynamic Therapy (aPDT) was proposed as an alternative treatment strategy for eradicating bacterial infections, particularly effective for Hp since this microorganism produces and stores up photosensitizing porphyrins. The knowledge of the photophysical characteristics of Hp porphyrins in their physiological biofilm microenvironment is crucial to implement and optimize the photodynamic treatment. Fluorescence lifetime imaging microscopy (FLIM) of intrinsic bacterial porphyrins was performed and data were analyzed by the 'fit-free' phasor approach in order to map the distribution of the different fluorescent species within Hp biofilm. Porphyrins inside bacteria were easily distinguished from those dispersed in the matrix suggesting FLIM-phasor technique as a sensitive and rapid tool to monitor the photosensitizer distribution inside bacterial biofilms and to better orientate the phototherapeutic strategy.

The in vitro Photoinactivation of Helicobacter pylori by a Novel LED-Based Device.

The rise of antibiotic resistance is the main cause for the failure of conventional antibiotic therapy of Helicobacter pylori infection, which is often associated with severe gastric diseases, including gastric cancer. In the last years, alternative non-pharmacological approaches have been considered in the treatment of H. pylori infection. Among these, antimicrobial PhotoDynamic Therapy (aPDT), a light-based treatment able to photoinactivate a wide range of bacteria, viruses, fungal and protozoan parasites, could represent a promising therapeutic strategy. In the case of H. pylori, aPDT can exploit photoactive endogenous porphyrins, such as protoporphyrin IX and coproporphyrin I and III, to induce photokilling, without any other exogenous photosensitizers. With the aim of developing an ingestible LED-based robotic pill for minimally invasive intragastric treatment of H. pylori infection, it is crucial to determine the best illumination parameters to activate the endogenous photosensitizers. In this study the photokilling effect on H. pylori has been evaluated by using a novel LED-based device, designed for testing the appropriate LEDs for the pill and suitable to perform in vitro irradiation experiments. Exposure to visible light induced bacterial photokilling most effectively at 405 nm and 460 nm. Sub-lethal light dose at 405 nm caused morphological changes on bacterial surface indicating the cell wall as one of the main targets of photodamage. For the first time endogenous photosensitizing molecules other than porphyrins, such as flavins, have been suggested to be involved in the 460 nm H. pylori photoinactivation.

Spectroscopic characterization and fluorescence imaging of Helicobacter pylori endogenous porphyrins.

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.

Dolichol: A Component of the Cellular Antioxidant Machinery.

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 α-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 α-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 α-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.

Ferulic Acid: A Hope for Alzheimer's Disease Therapy from Plants.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the deposition of extracellular amyloid-beta peptide (Aß) and intracellular neurofibrillar tangles, associated with loss of neurons in the brain and consequent learning and memory deficits. Aß 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 Aß fibrils as well as the destabilization of preformed Aß 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 Aß 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.

ß-Amyloid amorphous aggregates induced by the small natural molecule ferulic acid.

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 (Aß) 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 Aß fibrillogenesis in vitro. Our results show that FA interacts promptly with Aß 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.

A study for the cause of ferulic acid-induced quenching of tyrosine fluorescence and whether it is a reliable marker of intermolecular interactions or not.

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.

The effects of ferulic acid on ß-amyloid fibrillar structures investigated through experimental and computational techniques.

BACKGROUND: Current research has indicated that small natural compounds could interfere with ß-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 Aß fibrils in water solution.

Natural biomolecules and protein aggregation: emerging strategies against amyloidogenesis.

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.

Effects of hypericin on the structure and aggregation properties of ß-amyloid peptides.

We have determined the secondary structure of 1-40 ß-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 ß-sheet structure and a small percentage of α-helix. The results of fluorescence measurements showed that Hyp does significantly interact with peptides in ß-sheet conformation. In conclusion, hypericin does hinder the formation of fibrils, but the percentages of parallel ß-sheets were not significantly different from those found in samples not treated with Hyp.

In vitro perturbation of aggregation processes in beta-amyloid peptides: a spectroscopic study.

We have performed an in vitro study to investigate the molecular basis of the aggregation kinetic of 1-40 beta-amyloid peptides (Abeta 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, alpha-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 Abeta peptides.

Photosensitized structural modifications of the lens protein alpha-crystallin: do all modifications impair chaperone-like activity?

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.

Dolichol: a solar filter with UV-absorbing properties which can be photoenhanced.

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.

Photoreception and photomovements of microorganisms.

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.