P2X7 receptor as a novel drug delivery system to increase the entrance of hydrophilic drugs into cells during photodynamic therapy.

The second-generation photosensitizer methylene blue (MB) exhibits photochemical and photophysical properties suitable for photodynamic therapy (PDT)-based cancer treatment. However, the clinical application of MB is limited because of its high hydrophilicity, which hinders its penetration into tumor tissues. Therefore, new methods to improve the entry of MB into the cytoplasm of target cells are necessary. Because MB has a mass of 319 Da, transient pores on the plasma membrane, such as the pore induced by the P2X7 receptor (P2X7R) that allows the passage of molecules up to 900 Da, could be used. Using MTT viability assays, flow cytometry experiments, and fluorescence microscopy, we evaluated the toxicity and phototoxicity of MB and potentiation effects of ATP and MB on cell death processes in the J774 cell line (via a P2X7-associated pore). We observed that treatment with 5 µM MB for 15 min promoted the rate of entry of MB into the cytoplasm to 4.7 %. However, treatment with 5 µM MB and 1 mM ATP for the same amount of time increased this rate to 90.2 %. However, this effect was inhibited by pretreatment with a P2X7 antagonist. We used peritoneal macrophages and a cell line that does not express P2X7R as controls. These cells were more resistant to PDT with MB under the same experimental conditions. Taken together, these results suggest the use of the pore associated with P2X7R as a drug delivery system to increase the passage of hydrophilic drugs into cells that express this receptor, thus facilitating PDT.

The P2X7 receptor: shifting from a low- to a high-conductance channel - an enigmatic phenomenon?

The general structure of the P2X7 receptor (P2X7R) is similar to the structure of other P2X receptor family members, with the exception of its C terminus, which is the longest of this family. The P2X7R activates several intracellular signaling cascades, such as the calmodulin, mitogen-activated protein kinase and phospholipase D pathways. At low concentrations of ATP (micromolar range), P2X7R activation opens a cationic channel, similarly to other P2X receptors. However, in the presence of high concentrations of ATP (millimolar range), it opens a pathway that allows the passage of larger organic cations and anions. Here, we discuss both the structural characteristics of P2X7R related to its remarkable functions and the proposed mechanisms, including the dilation of the endogenous pore and the integration of another channel. In addition, we highlight the importance of P2X7R as a therapeutic target.

Structural and molecular modeling features of P2X receptors.

Currently, adenosine 5'-triphosphate (ATP) is recognized as the extracellular messenger that acts through P2 receptors. P2 receptors are divided into two subtypes: P2Y metabotropic receptors and P2X ionotropic receptors, both of which are found in virtually all mammalian cell types studied. Due to the difficulty in studying membrane protein structures by X-ray crystallography or NMR techniques, there is little information about these structures available in the literature. Two structures of the P2X4 receptor in truncated form have been solved by crystallography. Molecular modeling has proven to be an excellent tool for studying ionotropic receptors. Recently, modeling studies carried out on P2X receptors have advanced our knowledge of the P2X receptor structure-function relationships. This review presents a brief history of ion channel structural studies and shows how modeling approaches can be used to address relevant questions about P2X receptors.

A consensus segment in the M2 domain of the hP2X(7) receptor shows ion channel activity in planar lipid bilayers and in biological membranes.

The P2X(7) receptor (P2X(7)R) is an ATP-gated, cation-selective channel permeable to Na(+), K(+) and Ca(2+). This channel has also been associated with the opening of a non-selective pore that allows the flow of large organic ions. However, the biophysical properties of the P2X(7)R have yet to be characterized unequivocally. We investigated a region named ADSEG, which is conserved among all subtypes of P2X receptors (P2XRs). It is located in the M2 domain of hP2X(7)R, which aligns with the H5 signature sequence of potassium channels. We investigated the channel forming ability of ADSEG in artificial planar lipid bilayers and in biological membranes using the cell-attached patch-clamp techniques. ADSEG forms channels, which exhibit a preference for cations. They are voltage independent and show long-term stability in planar lipid bilayers as well as under patch-clamping conditions. The open probability of the ADSEG was similar to that of native P2X(7)R. The conserved part of the M2 domain of P2X(7)R forms ionic channels in planar lipid bilayers and in biological membranes. Its electrophysiological characteristics are similar to those of the whole receptor. Conserved and hydrophobic part of the M2 domain forms ion channels.

Predictions suggesting a participation of beta-sheet configuration in the M2 domain of the P2X(7) receptor: a novel conformation?

Scanning experiments have shown that the putative TM2 domain of the P2X(7) receptor (P2X(7)R) lines the ionic pore. However, none has identified an alpha-helix structure, the paradigmatic secondary structure of ion channels in mammalian cells. In addition, some researchers have suggested a beta-sheet conformation in the TM2 domain of P2X(2). These data led us to investigate a new architecture within the P2X receptor family. P2X(7)R is considered an intriguing receptor because its activation induces nonselective large pore formation, in contrast to the majority of other ionic channel proteins in mammals. This receptor has two states: a low-conductance channel (approximately 10 pS) and a large pore (> 400 pS). To our knowledge, one fundamental question remains unanswered: Are the P2X(7)R channel and the pore itself the same entity or are they different structures? There are no structural data to help solve this question. Thus, we investigated the hydrophobic M2 domain with the aim of predicting the fitted position and the secondary structure of the TM2 segment from human P2X(7)R (hP2X(7)R). We provide evidence for a beta-sheet conformation, using bioinformatics algorithms and molecular-dynamics simulation in conjunction with circular dichroism in different environments and Fourier transform infrared spectroscopy. In summary, our study suggests the possibility that a segment composed of residues from part of the M2 domain and part of the putative TM2 segment of P2X(7)R is partially folded in a beta-sheet conformation, and may play an important role in channel/pore formation associated with P2X(7)R activation. It is important to note that most nonselective large pores have a transmembrane beta-sheet conformation. Thus, this study may lead to a paradigmatic change in the P2X(7)R field and/or raise new questions about this issue.

Pore forming channels as a drug delivery system for photodynamic therapy in cancer associated with nanoscintillators.

According to the World Health Organization (WHO), cancer is one of main causes of death worldwide, with 8.2 million people dying from this disease in 2012. Because of this, new forms of treatments or improvement of current treatments are crucial. In this regard, Photodynamic therapy (PDT) has been used to successfully treat cancers that can be easily accessed externally or by fibre-optic endoscopes, such as skin, bladder and esophagus cancers. In addition, this therapy can used alongside radiotherapy and chemotherapy in order to kill cancer cells. The main problem in implementing PDT is penetration of visible light deeper than 10 mm in tissues, due to scattering and absorption by tissue chromophores. Unfortunately, this excludes several internal organs affected by cancer. Another issue in this regard is the use of a selective cancer cell-photosensitizing compound. Nevertheless, several groups have recently developed scintillation nanoparticles, which can be stimulated by X-rays, thereby making this a possible solution for light production in deeper tissues. Alternative approaches have also been developed, such as photosensitizer structure modifications and cell membrane permeabilizing agents. In this context, certain channels lead to transitory plasma membrane permeability changes, such as pannexin, connexin hemmichannels, TRPV1-4 and P2×7, which allow for the non-selective passage of molecules up to 1,000 Da. Herein, we discuss the particular case of the P2×7 receptor-associated pore as a drug delivery system for hydrophilic substances to be applied in PDT, which could also be carried out with other channels. Methylene blue (MB) is a low cost dye used as a prototype photosensitizer, approved for clinical use in several other clinical conditions, as well as photodynamic therapy for fungi infections.

Johrei Effects on Water: A Pilot Study by Counting Drops.

BACKGROUND: Water is a key ingredient in the creation and sustainment of life. Moreover, water may be a key vehicle in the processes of energy healing, such as in the preparation of homeopathic remedies and spiritual treatments. Given these properties, the purpose of this study was to investigate whether the application of Johrei to water could lead to significant changes in the hydrodynamic behaviour of the fluid. METHODS: Four regular Johrei practitioners (P1, P2, P3 and P4) were selected for this study. Dripping water produced at the tip of a capillary was used as the hydrodynamic behaviour model. This behaviour was modelled mathematically, and tuning parameters φ4 and τ were used to assess significant differences in the dripping water samples that were subjected to Johrei compared with the samples that were not so treated. The tuning parameters were obtained using the Levenberg-Marquardt fitting algorithm. The data sets for each Johrei practitioner and the control experiment were analysed using ANOVA and a paired t-test. RESULTS: The mathematical model exhibited an excellent fit to our data, generating correlation coefficients (r) greater than or equal to 0.999. Significant differences were observed in both τ (P1 and P2, P < 0.05 and P < 0.01, respectively) and φ4 (P2, P < 0.01). As expected, no significant difference for the control experiment (without Johrei) was observed. CONCLUSIONS: Our results indicated a statistically significant change in the hydrodynamic behaviour of water correlated with Johrei treatment for 50% of the participating Johrei practitioners.

Johrei, a Japanese healing technique, enhances the growth of sucrose crystals.

The effect of Johrei treatment on the crystallization of sucrose from supersaturated solutions was studied in comparison with the crystallization in untreated solutions. This work was performed assuming that Johrei enhances the natural mechanisms of equilibrium restoration in biological and nonbiological systems. The crystallization in Johrei-treated solutions as judged by statistical analysis was found to be faster than the crystallization in untreated solutions. A discussion is presented about the mechanisms possibly involved.