In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

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DNA intercalators represent an important class of compounds with a high potential as DNA-targeting drugs. In this review it is demonstrated that annelated quinolizinium derivatives such as coralyne and derivatives thereof intercalate into DNA and that this structural motif allows several variations of the substitution pattern without loss of intercalating properties. The commonly applied methods for the evaluation of the DNA association, mainly spectroscopic studies, are pointed out. In addition, studies on the biological activities of annelated quinolizinium derivatives, such as topoisomerase poisoning or cell toxicity, are highlighted.

A series of 1-aryl-5-(3′,4′,5′-trimethoxyphenyl) derivatives and their related 1-(3′,4′,5′-trimethoxyphenyl)-5-aryl-1,2,4-triazoles, designed as cis-restricted combretastatin analogues, were synthesized and evaluated for antiproliferative activity, inhibitory effects on tubulin polymerization, cell cycle effects, and apoptosis induction. Their activity was greater than, or comparable with, that of the reference compound CA-4. Flow cytometry studies showed that HeLa and Jurkat cells treated with the most active compounds 4l and 4o were arrested in the G2/M phase of the cell cycle in a concentration dependent manner. This effect was accompanied by apoptosis of the cells, mitochondrial depolarization, generation of reactive oxygen species, activation of caspase-3, and PARP cleavage. Compound 4l was also shown to have potential antivascular activity, since it induced endothelial cell shape change in vitro and disrupted the sprouting of endothelial cells in the chick aortic ring assay.

The absorption and steady-state emission properties of a series of N-alkyl- and N-aryl-9-aminoacridizinium derivatives and two 9-sulfanyl-substituted acridizinium derivatives were investigated. The N-alkyl derivatives and the 9-methylsulfanylacridizinium have an intense intrinsic fluorescence (phi(f) = 0.2-0.6), whereas the N-aryl-substituted compounds are virtually nonfluorescent in liquid solutions (phi(f) < or = 0.01). The emission intensity of the latter compounds significantly increases with increasing viscosity of the medium. It is demonstrated that the excited-state deactivation of the N-aryl-9-aminoacridizinium derivatives is due to two nonradiative processes: (i) torsional relaxation by rotation about the N-aryl bond and (ii) an electron-transfer process from an electron-donor substituted phenyl ring to the photoexcited acridizinium chromophore. The binding of several representative acridizinium derivatives to double-stranded DNA was studied by the spectrophotometric titrations and linear dichroism spectroscopy. The results give evidence that the prevailing binding mode is intercalation with binding constants in the range (0.5-5.0) x 10(5) M(-1) (in base pairs). Notably, the binding of most of the N-aryl-9-aminoacridizinium derivatives leads to a fluorescence enhancement by a factor of up to 50 upon binding to the biomacromolecules. Moreover, the addition of selected proteins, namely albumins, to N-(halogenophenyl)-9-aminoacridizinium ions in the presence of an anionic surfactant (sodium dodecyl sulfate) results in a 20-fold fluorescence enhancement. In each case, the emission enhancement is supposed to result from the hindrance of the torsional relaxation in the corresponding binding site of the biomacromolecule, which in turn suppresses the excited-state deactivation pathway.

This work concerns a combined photophysical, photochemical and photobiological study of three drugs (psychotherapeutic agents) of the phenothiazine series: perphenazine, fluphenazine hydrochloride and thioridazine hydrochloride. The excited-state properties were first investigated by stationary and time-resolved fluorimetry and by laser flash photolysis. The spectral description was assisted by quantum-mechanical calculations with the INDO/1-CI method. In organic media the lowest excited singlet state was found to decay by fluorescence (small quantum yield) and mainly by intersystem crossing to the lowest triplet state, which is responsible for oxygen photosensitization (high yields of singlet oxygen production) and photodegradation. A further decay pathway in aqueous solutions was the photoionization process, which led to the formation of the phenothiazine radical cations and the solvated electron. After the preliminary study of the photobehavior in organic solvents and in water, the phototoxicity of the three drugs was investigated on various biological substrates through a series of in vitro assays under UVA irradiation. Photohemolysis of mouse erythrocytes and phototoxicity on cultured murine fibroblasts were observed for all three compounds. Lipid photoperoxidation was then investigated using linoleic acid as the unsaturated lipid model and isolated red blood cell membranes. The drug-induced photodamage was also evaluated on proteins by measuring the photosensitizing cross-linking in erythrocyte ghosts. The combined approach proved to be useful in understanding the mechanism by which these phenothiazine derivatives induce skin photosensitization. In particular, the photophysical properties of the compounds under investigation and the results of the study on their phototoxicity are in agreement with a mechanism that involves the radical cation of the drugs as a main intermediate.

Receptor-specific nuclear targeting requires trifunctional metal complexes. We have synthesized [M(L(2)-pept)(L(1)-acr)(CO)(3)] (pept=peptide; acr=acridine-based agent) in which the fac-[M(CO)(3)](+) moiety (1st function, M=(99m)Tc, Re) couples an acridine-based nuclear-targeting agent (2nd function, L(1)-acr) and the specific cell-receptor-binding peptide bombesin (3rd function, L(2)-pept). The metal-mediated coupling is based on the mixed ligand [2+1] principle. The nuclear targeting agents have been derivatised with an isocyanide group for monodentate (L(1)) and bombesin (BBN) with a bidentate ligand (L(2)) for complexation to fac-[M(CO)(3)](+). For nuclear uptake studies, the model complexes [Re(L(2))(L(1)-acr)(CO)(3)] (L(2)=pyridine-2-carboxylic acid and pyridine-2,4-dicarboxylic acid) were synthesized and structurally characterized. We selected acridine derivatives as nuclear-targeting agents, because they are very good nucleus-staining agents and exhibit strong fluorescence. Despite the bulky metal complexes attached to acridine, all [Re(L(2))(L(1)-acr)(CO)(3)] showed high accumulation in the nuclei of PC3 and B16F1 cells, as evidenced by fluorescence microscopy. For radiopharmaceutical purposes, the (99m)Tc analogues have been prepared and radioactivity distribution confirmed the fluorescence results. Coupling of BBN to L(2) gave the receptor-selective complexes [M(L(2)-BBN)(L(1)-acr)(CO)(3)]. Whereas no internalization was found with B16F1 cells, fluorescence microscopy on PC3 cells bearing the BBN receptor showed high and rapid uptake by receptor-mediated endocytosis into the cytoplasm, but not into the nucleus.