Experimental Evidence Supports New Perspectives in Homeopathy.

The contentious debate between homeopathy and orthodox medicine has been due to the fact that homeopathy is founded on a heuristic philosophy that is not justified by contemporary scientific evidence. In this context, however, two pillars of the method, that is, serial dilution and succussion, are poorly understood in orthodox pharmacology. The experimental data collected in the last 10 years, by means of electronic microscopy, electron diffraction and DNA arrays investigations, are consistent with the presence of nanoparticles (nanoassociates) in homeopathic medicines and seem to provide a coherent view of the essence of the homeopathy discipline, superseding all previous speculative interpretations. An acceptance of this new evidence is here suggested to remove, in principle, the barrier that separates the conventional and homeopathic therapeutic methods, and to offer new and important perspectives on future health care.

Effects of Extreme Dilutions of Apis mellifica Preparations on Gene Expression Profiles of Human Cells.

Gene expression analysis has been employed in the past to test the effects of high dilutions on cell systems. However, most of the previous studies were restricted to the investigation of few dilutions, making it difficult to explore underlying mechanisms of action. Using whole-genome transcriptomic analysis, we investigated the effects of a wide range of Apis mellifica dilutions on gene expression profiles of human cells. RWPE-1 cells, a nonneoplastic adult human epithelial prostate cell line, were exposed to Apis mellifica preparations (3C, 5C, 7C, 9C, 12C, 15C, and 30C) or to the reference solvent solutions for 24 hours; nonexposed cells were also checked for gene expression variations. Our results showed that even the most diluted solutions retained the ability to trigger significant variations in gene expression. Gene pathway analysis revealed consistent variations in gene expression induced by Apis mellifica when compared to nonexposed reference cells but not to reference solvent solutions. Since the effects of Apis Mellifica at extreme dilutions did not show dose-effect relationships, the biological or functional interpretation of these results remains uncertain.

Hormetic effects of extremely diluted solutions on gene expression.

This paper summarizes the results of investigations showing how molecular biological tools, such as DNA-microarrays, can provide useful suggestions about the behaviour of human organisms treated with microamounts of drugs or homeopathic medicines. The results reviewed here suggest firstly that the action of drugs is not quenched by ultra-high dilution and proceeds through modulation of gene expressions. The efficacy of drug solutions seems to be maintained in ultra-highly diluted preparations, a fact which constitutes a challenge to the dogma of quantization of matter. The second and more important result is that the different gene expression profiles of cell systems treated with the same drugs at different dilutions suggest the existence of hormetic mechanisms. The gene expression profiles of cells treated with copper(II) sulfate, Gelsemium sempervirens and Apis mellifica, are characterized by the same common denominator of the concentration-dependent inversion of gene expression, which can justify at a molecular level the concept of simile adopted in homeopathy. The main conclusion we draw from these results is that these procedures provide new kinds of information and a tool for disclosing the mechanisms involved in hormetic effects. The application of these effects to modern medicine may allow researchers to conceive unprecedented therapeutic applications or to optimize the currently used ones in the framework of a low-dose pharmacology based on a reliable experimental platform.

Thermal and optical control of electronic states in a single layer of switchable paramagnetic molecules.

The control of the charge distribution in a monolayer of switchable cobalt-dioxolene complexes undergoing Valence Tautomerism (VT) has been achieved by means of thermal and optical stimuli. Thiol-derivatised VT molecules have been grafted on polycrystalline gold surface as monolayers from solution. X-ray photoelectron spectroscopy and time-of-flight secondary ions mass spectrometry evidenced the formation of a covalent bond between intact VT molecules and the surface and excluded the presence of physisorbed molecules. X-ray absorption spectroscopy revealed that the temperature- and light-induced conversion profiles of the monolayer closely reproduce the ones found for the crystalline phase. This study demonstrates that a wet chemistry based approach allows to transfer switchable paramagnetic molecules at the nanoscale, widening the playground to develop new hybrid molecular based architectures for novel technologies.

Exploring the effects of homeopathic Apis mellifica preparations on human gene expression profiles.

BACKGROUND: Diluted preparations obtained from Apis mellifica are reported in the homeopathic literature to have anti-inflammatory activity. The present study was designed to explore the effects on global gene expression profiles of human cells by means of microarrays, using Apis mellifica mother tincture (TM) and its 3C, 5C, 7C dynamized dilutions; the technique employed allowed us to study the changes in gene expression at concentrations much lower than those associated with pharmacological responses. METHODS: An RWPE-1 cell line (human immortalized prostate epithelial cells) was used to study the effects on global gene expression by transcriptomic analysis. RESULTS: Apis mellifica TM and its 3C, 5C, 7C dynamized dilutions modulated hundreds of genes; using cluster analysis we observed groups of genes up- or down-regulated with similar expression profiles among treatments; other genes showed opposite regulation profiles at low and high dilutions of Apis mellifica, suggesting a hormetic response. In particular, genes involved in cytokine expression, inflammatory processes, anti-oxidative responses and proteasome degradation were differentially, and sometimes divergently expressed by the TM or by Apis mellifica 3C, 5C and 7C dilutions. We confirmed these data by RT-PCR analyses on 5 selected candidate genes (IL1ß, CD46, ATF1, UBE2Q2 and MT1X). CONCLUSIONS: Apis mellifica TM modifies gene expression in human cells and has inhibitory effects on regulatory processes of inflammation; in addition, extremely diluted dynamized dilutions (3C, 5C and 7C) still exert significant effects on genes involved in inflammation and oxidative stress.

Magnetic and spectroscopic investigation of thermally and optically driven valence tautomerism in thioether-bridged dinuclear cobalt-dioxolene complexes.

A series of dinuclear cobalt complexes of general formula [Co(Mentpa)(diox-S-diox)Co(Mentpa)](PF6)2·MeOH (n = 0, 2, 3) was prepared through the synthesis of the bis-bidentate ligand 6,6'-((1,4-phenylenebis(methylene))bis(sulfanediyl))bis(3,5-di-tert-butyl-benzene-1,2-diol) (diox-S-diox). The ancillary ligands Mentpa are obtained by the tripodal tris(2-pyridylmethyl)amine (tpa) ligand through successive introduction of methyl groups into the 6 position of the pyridine moieties. As expected, the steric hindrance induced by this substitution modulates the redox properties of the metal acceptor, determining the charge distribution of the metal-dioxolene adduct at room temperature. Magnetic measurements and X-ray photoelectron and X-ray absorption spectroscopies indicate that the charge distributions low-spin-Co(III)-catecholate and high-spin-Co(II)-semiquinonate characterize the complexes formed by the tpa and Me3tpa tetradentate ligands, respectively. The complex formed by the Me2tpa ligand undergoes a thermal- and light-induced interconversion of the two states, in agreement with the existence of a valence tautomeric equilibrium. All complexes were stable and behaved reproducibly under X-ray irradiation. This work points out a fast and simple chemical approach to structurally and electronically modify the catechol ring while leaving its coordination capabilities unaffected. These findings afford a robust chemical method to prepare sulfur-functionalized dioxolene ligands as new molecular bricks for chemical functionalization of noble metal surfaces with this class of molecular switches.

Valence tautomerism in co-dioxolene complexes: static and time-resolved infrared spectroscopy study.

In this work, we studied the valence tautomerism process on two different Co-dioxolene complexes by means of transient infrared spectroscopy (TRIR). The molecules investigated are ls-Co(III)(Cat-N-BQ)(Cat-N-SQ) (DQ2) and [ls-Co(III)(tpy)(Cat-N-SQ)]PF6 (tpy), where Cat-NBQ = 2-(2-hydroxy-3,5-ditert-butylphenyl-imino)-4,6-ditert-butylcyclohexa-3,5-dienone, Cat-N-SQ is the dianionic radical analogue, and tpy = 2,2'-6-2″-terpyridine. DFT calculations of the harmonic frequencies for the two complexes allow us to pinpoint the normal modes to be used as markers of the semiquinonate and benzoquinonate isomers. The photoinduced one-electron charge transfer process from the radical semiquinonate ligand to the metal center leads to a ls-Co(II)(x)(Cat-N-BQ) electronic state (where x is the other ligand). Following this first step, an ultrafast ISC process (τ < 200 fs) takes places, yielding the benzoquinonate isomer (hs-Co(II)(x)(Cat-N-BQ)). In the experiments, we employed different excitation wavelengths on resonance with different absorption bands of the two samples. Excitation in the ligand-to-metal charge transfer (LMCT) band at ∼520 nm and in the semiquinonate band at ∼1000 nm induces the valence tautomerism (VT) in both samples. From the time evolution of the TRIR spectra, we determine the time constants of the vibrational cooling in the tautomeric state (7-14 ps) and the ground state recovery times (∼350 ps for tpy and ∼450 ps for DQ2). In contrast, when the pump frequency is set at 712 nm, on resonance with the benzoquinonate absorption band of the second active ligand of the DQ2, no electron transfer takes place: the TRIR spectra basically show only ground state bleaching bands and no marker band of the tautomeric conversion shows up.

Transient infrared spectroscopy: a new approach to investigate valence tautomerism.

In this work we present, to our knowledge for the first time, the results of a transient infrared spectroscopic study of the photoinduced valence tautomerism process in cobalt-dioxolene complexes with sub-picosecond time resolution. The molecular systems investigated were [Co(tpa)(diox)]PF(6) (1) and [Co(Me(3)tpa)(diox)]PF(6) (2), where diox = 3,5-di-tert-butyl-1,2-dioxolene; tpa = tris(2-pyridylmethyl)amine and Me(3)tpa its 6-methylated analogue. Complex (1) is present in solution as ls-Co(III)(catecholate) (1-CAT), while (2) as hs-Co(II)(semiquinonate) (2-SQ). DFT calculation of the harmonic frequencies for (1) and (2) allowed us to identify the vibrational markers of catecholate and semiquinonate redox isomers. Irradiation with 405 and 810 nm pulses (~35 fs) of (1-CAT) induces the formation of an intermediate excited species from which the ground state population is recovered with a time constant of 1.5 ± 0.3 ns. Comparing the 1 ns transient infrared spectrum with the experimental difference spectrum FTIR(2-SQ)-FTIR(1-CAT) and with the calculated difference spectrum IR(c)(1-SQ)-IR(c)(1-CAT) we are able to unequivocally identify the long lived species as the semiquinonate redox isomer of (1). On the other hand, no evidence of photoconversion is observed upon irradiation of (2) with 405 nm. Temporal evolution of transient spectra was analyzed with the combined approach consisting of singular values decomposition and global fitting (global analysis). After 405 and 810 nm excitation of (1-CAT), the semiquinonate excited species is formed on an ultrafast time scale (<200 fs) and cools down within the first 50 ps. Excitation of (2-SQ) with 405 nm wavelength produces a short lived excited state in which the semiquinonate nature of dioxolene is preserved and the ground state recovery is completed within 30 ps.

Extremely low copper concentrations affect gene expression profiles of human prostate epithelial cell lines.

Although cellular copper metabolism is tightly regulated through a variety of copper transport proteins and chaperones, disturbances in copper homeostasis are involved in several pathological disorders. The aim of this study was to evaluate the effects of extremely low copper concentrations on gene expression profiles of a line of human prostate epithelial cells (RWPE) which grows in the absence of fetal calf serum, a source of variable and unpredictable copper. Cells were exposed to copper(II) sulfate for 24h at concentrations varying from 10(-6) to 10(-17)M and untreated reference cells were exposed to the same volume of copper-free water. Relative gene expression variations between copper-treated and control cells were studied with microarray technology using the Whole Human Genome Array from Agilent. Microarray data demonstrated that copper added to the medium varied gene expression at all concentrations tested. Many genes belonging to functional gene families were modulated by copper, some dose-dependently. Amongst these genes metallothioneins (MT1A and MT2A) were over-expressed at all copper concentrations, MT1M was up-regulated between 10(-6) and 10(-9)M, while MT1B, MT1E, MT1G and MT1H were up-regulated between 10(-6) and 10(-14)M. The heat shock protein (HSP) gene family showed similar behavior: some HSP genes were constantly up-regulated by copper (HSP90Ad, HSP90B1 and HSPD1) and others only at higher concentrations (HSP90AB1 and HSPA8). Reverse-transcription-PCR analysis, performed on four different genes on five biological replicates for selected genes, on each copper concentration tested, confirmed the trend observed in microarray results. In conclusion, we unexpectedly observed a modulation of gene expression even at extremely diluted copper concentrations, similar to that induced by toxic concentrations, possibly as a result of very tight control of free copper(II) levels inside the cells.

Coexistence of two thermally induced intramolecular electron transfer processes in a series of metal complexes [M(Cat-N-BQ)(Cat-N-SQ)]/[M(Cat-N-BQ)2] (M = Co, Fe, and Ni) bearing non-innocent catechol-based ligands: a combined experimental and theoretical study.

The different thermally induced intermolecular electron transfer (IET) processes that can take place in the series of complexes [M(Cat-N-BQ)(Cat-N-SQ)]/[M(Cat-N-BQ)(2)], for which M = Co (2), Fe (3) and Ni(4), and Cat-N-BQ and Cat-N-SQ denote the mononegative (Cat-N-BQ(-)) or dinegative (Cat-N-SQ(2-)) radical forms of the tridentate Schiff-base ligand 3,5-di-tert-butyl-1,2-quinone-1-(2-hydroxy-3,5-di-tert-butylphenyl)imine, have been studied by variable-temperature UV/Vis and NMR spectroscopies. Depending on the metal ion, rather different behaviors are observed. Complex 2 has been found to be one of the few examples so far reported to exhibit the coexistence of two thermally induced electron transfer processes, ligand-to-metal (IET(LM)) and ligand-to-ligand (IET(LL)). IET(LL) was only found to take place in complex 3, and no IET was observed for complex 4. Such experimental studies have been combined with ab initio wavefunction-based CASSCF/CASPT2 calculations. Such a strategy allows one to solicit selectively the speculated orbitals and to access the ground states and excited-spin states, as well as charge-transfer states giving additional information on the different IET processes.

X-ray absorption spectroscopy as a probe of photo- and thermally induced valence tautomeric transition in a 1:1 cobalt-dioxolene complex.

The role of the cobalt ion in the entropy- and optically-driven valence tautomeric (VT) interconversion exhibited by the [Co(Me(2)tpa)(DTBdiox)](PF(6))C(6)H(5)CH(3) complex (Me(2)tpa = bis (6-methyl-(2-pyridylmethyl))(2-pyridylmethyl)amine, DBdiox = 3,5-ditertbutyl-dioxolene) is established by means of X-ray absorption spectroscopy (XAS). Analysis of the pre-edge features at 6 and 300 K in the Co K-edge XAS spectra using a ligand field multiplet approach allows us to obtain detailed information on the electronic structures of the metal ion in the two redox isomers. The temperature dependence of the spectra confirms the occurrence of a thermally induced VT transition and suggests that nucleation and distortion of the phase boundaries take place during the process. Moreover, optically induced metastable state formation is monitored at low temperatures--with a high degree of reproducibility--without changing the position of measurement on the sample during the experiment. This result paves the way for the use of such a highly sensitive technique for the investigation of photoswitchable materials in non-crystalline and nanostructured environments.

Tuning the charge distribution and photoswitchable properties of cobalt-dioxolene complexes by using molecular techniques.

A series of cobalt complexes [Co(Me(n)tpa)(diox)]PF(6)sol (diox=3,5-di-tert-butyl-1,2-dioxolene; sol=ethanol, toluene; tpa=tris(2-pyridylmethyl)amine) were prepared by using tripod-like Me(n)tpa (n=0, 1, 2, 3), derived from tpa by successive introduction of methyl groups into the 6-position of the pyridine moieties, as an ancillary ligand. The steric hindrance induced by this substitution modulates the redox properties of the metal acceptor, thus determining the charge distribution of the metal-dioxolene moiety at room temperature. All of these complexes were characterised by using diffractometric studies, electronic spectroscopic analysis, and magnetic susceptibility measurements. In the solid state, the [Co(Me(n)tpa)(diox)](+) ions (n=0, 1) can be described as diamagnetic cobalt(III)-catecholato derivatives, whereas a cobalt(II)-semiquinonato description seems appropriate for the paramagnetic [Co(Me(3)tpa)(diox)](+) complex. The complex [Co(Me(2)tpa)(diox)]PF(6)C(2)H(5)OH undergoes entropy-driven valence tautomeric interconversion at room temperature. Optically induced valence tautomerism was observed by irradiation of [Co(Me(n)tpa)(diox)]PF(6) complexes (n=0, 1, 2) at cryogenic temperatures. The different relaxation kinetics of the photoinduced metastable phases are related to the respective free-energy changes of the interconversion, as estimated by cyclic voltammetric experiments at room temperature, and to the different lattice interactions, as supported by structural data. These results show the importance of molecular techniques for controlling the relaxation properties of photoinduced metastable species. At the same time, this behaviour strongly suggests that this paradigm exhibits intrinsic limits because of the less controllable factors that affect the process.

Unprecedented optically induced long-lived intramolecular electron transfer in cobalt-dioxolene complexes.

The observation of very long lifetimes of the metastable states of two cobalt-dioxolene complexes undergoing photoinduced and high T(c) thermally-induced valence tautomer interconversion opens new research perspectives.

Valence tautomerism interconversion triggers transition to stable charge distribution in solid polymeric cobalt-polyoxolene complexes.

The reaction of 3,3,3',3'-tetramethyl-5,5',6,6'-tetrahydroxy-1,1'spiro-bis(indane), L, with cobalt salts in the presence of diazine ligands affords polymeric derivatives of general formula [CoL(diazine)]infinity . nH2O, L being coordinated as a mixed-valence SQ-Cat species. The bipiridyl and o-phenantroline derivatives have been characterised by means of magnetic, EPR and calorimetric measurements. The characterisation showed that both systems are obtained in a metastable charge distribution, presumably trapped due to the class I character of the mixed valence form of the ligand. The occurrence of entropy-driven valence tautomeric interconversion induces the transition to the most stable charge distribution. The photomagnetic characterisation evidenced that valence tautomeric process can be photoinduced and that the lifetime of the photoinduced metastable state, obtained in low yield, is of ca. 2 x 10(5) s at 9 K.

The influence of ligand field effects on the magnetic exchange of high-spin Co(II)-semiquinonate complexes.

[Co(Me(4)cyclam)(tropolonate)](PF(6)) was synthesised and structurally characterised. Its electronic and W-band EPR spectra have been analysed by means of the angular overlap calculation of the Spin Hamiltonian parameters that provided also a satisfactory reproduction of the temperature dependence of the magnetic susceptibility. The present results can be interpreted assuming a pseudo-octahedral character for the Co(II) center. This prompted us to reconsider the model formerly used for the analysis of the magnetic coupling between hs-Co(II) and the paramagnetic o-semiquinonate ligand in the corresponding derivatives [Co(Me(4)cyclam)(PhenSQ)](PF(6)) and [Co(Me(4)cyclam)(DTBSQ)](PF(6)). These results indicate that the effect of the magnetic coupling is active only below 50 K and that a more refined model of exchange coupling between Co(II) and semiquinonato ligands is needed to quantitatively analyze the magnetic behaviour of this class of systems.