J. Appl. Cryst.の発刊に際して

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.

For example, 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 versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase 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. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

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. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. 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 assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

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.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-28d1pi1jvo.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy

: The unpolarized absorption and circular dichroism spectra of the fundamental vibrational transitions of the chiral molecule, 4-methyl-2-oxetanone, are calculated ab initio. Harmonic force fields are obtained using Density Functional Theory (DFT), MP2, and SCF methodologies and a 5S4P2D/3S2P (TZ2P) basis set. DFT calculations use the Local Spin Density Approximation (LSDA), BLYP, and Becke3LYP (B3LYP) density functionals. Mid-IR spectra predicted using LSDA, BLYP, and B3LYP force fields are of significantly different quality, the B3LYP force field yielding spectra in clearly superior, and overall excellent, agreement with experiment. The MP2 force field yields spectra in slightly worse agreement with experiment than the B3LYP force field. The SCF force field yields spectra in poor agreement with experiment.The basis set dependence of B3LYP force fields is also explored: the 6-31G* and TZ2P basis sets give very similar results while the 3-21G basis set yields spectra in substantially worse agreements with experiment. jg

Ab initio calculation of vibrational absorption and circular dichroism spectra using density functional force fields

Vibrational Energy Distribution Analysis (VEDA): Scopes and limitations

The molecular geometry and vibrational frequencies of (7-chloro-2-oxo-2H-chromen-4-yl)-methyl diethylcarbamodithioate in the ground state have been calculated by using the Hartree–Fock and density functional theory methods (B3LYP and B3PW91) with 6-311++G(d,p) basis set. The results of the optimized molecular structure are presented and compared with the experimental X-ray data. The calculated vibrational frequencies are used to determine the types of molecular motions associated with each of the experimental bands observed. To investigate the nonlinear optics properties of the title compound, the polarizability and the first hyperpolarizability are calculated using the density functional B3LYP method. The predicted nonlinear optical properties of the title compound are greater than ones of urea. To determine conformational flexibility, molecular energy profile of the title compound is obtained by B3LYP calculations with respect to selected degree of torsional freedom, which is varied from −180◦ to +180◦ in steps of 10◦. Besides, molecular electrostatic potential, natural bond orbitals, natural atomic charges, frontier molecular orbitals, and several thermodynamic properties are performed by the density functional theory methods.

/pdf/molecular-and-electronic-analysis-of-7-chloro-2-oxo-2h-37ufrhifmy.pdf

Molecular and Electronic Analysis of (7-Chloro-2-oxo-2H-chromen-4-yl)-methyl diethylcarbamodithioate by DFT and HF Calculations

The crystal structure of the title compound, C14H12N2O3, was recently determined as a mixture of its neutral (OH containing) and zwitterionic (NH containing) forms, in a 0.60 (4): 0.40 (4) ratio using the X-ray determination. In this study, the title compound has been characterized by FT-IR and X-ray diffraction. The redetermination showed that the title compound has only enol (OH) form because of lack of the NH stretching vibration in FT-IR spectrum. In addition, the molecular structure and tautomerism of the title compound have been discussed.

Molecular and crystal structure of 2-{(E)-[(4-Methylphenyl)imino]methyl}-4-nitrophenol: A redetermination

In this study, Congo red removal from wastewater by a filtration method was studied via membranes obtained from polyacrylonitrile-co-poly(2-ethylhexylacrylate) copolymers having various monomer rat...

Congo red filtration by polyacrylonitrile-based copolymer membranes

The molecular structure of the title compound C16H13N3O3 was characterized by single crystal X-ray diffraction method. The compound crystallizes in the triclinic space group \(P\bar 1\) with Z = 4 in the unit cell. In the asymmetric unit of the title compound, there are two crystallographically independent molecules, designated A and B. In the crystal structure, the phenyl and benzoic acid ring systems are bridged by 1,2,4-triazole ring for both independent molecules. The ring systems are perfectly planar for both molecules but the whole molecule is not planar. The crystal structure is stabilized by N-H⋯O and O-H⋯O type classical intermolecular hydrogen bonds. In the crystal, N-H⋯O and O-H⋯O hydrogen bonds link the molecules into one-dimensional infinite chains along \([41\bar 2]\) direction. The crystal packing is also stabilized by C-H⋯π interactions.

Molecular and crystal structure of 3-benzyl-4-(4-carboxyphenyl)-4,5-dihydro-1H-1,2,4-triazol-5-one

A new tetradentate ligand incorporating dioxime moities has been synthesized. Its copper(II), nickel(II), and cobalt(II) complexes have been prepared and characterized by spectral methods. Elemental analyses and spectroscopic data of the metal complexes are consistent with the formation of the mononuclear copper(II), nickel(II) and cobalt(II) complexes. The crystal structure of the (2E, 2′E)-butane-2,3-dione 2,2′-{O 2,O 2′-[1,2-phenylenebis(methylene)]oxime compound (3) was determined by single-crystal x-ray diffraction technique. The free ligand and copper(II), nickel(II), and cobalt(II) complexes (5–7) were tested against the morphology of series bacteria. The Cu(II), Ni(II), and Co(II) complexes exhibit higher activity than dioxime ligand under identical experimental conditions.

Hasan Tanak

Papers

Molecular and Electronic Analysis of (7-Chloro-2-oxo-2H-chromen-4-yl)-methyl diethylcarbamodithioate by DFT and HF Calculations

Molecular and crystal structure of 2-{(E)-[(4-Methylphenyl)imino]methyl}-4-nitrophenol: A redetermination

Congo red filtration by polyacrylonitrile-based copolymer membranes

Molecular and crystal structure of 3-benzyl-4-(4-carboxyphenyl)-4,5-dihydro-1H-1,2,4-triazol-5-one

Synthesis and Structural and Biochemical Activity Studies of Dioxime Ligand and its Mononuclear Cu(II), Ni(II), and Co(II) Complexes