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 title compound, C6H6N2O3·H2O, crystallizes with two formula units in the asymmetric unit. The mol­ecules are essentially planar with the nitro groups twisted slightly out of the ring planes [maximum deviations from the ring plane of 0.13 (2) and 0.22 (2) A in the two mol­ecules]. The respective O—N—C—C torsion angles are 6.0 (4) and 12.5 (4)°. In the crystal structure, mol­ecules are linked by inter­molecular N—H⋯O, C—H⋯O, O—H⋯O and O—H⋯N inter­actions into a three-dimensional network.

https://journals.iucr.org/e/issues/2010/10/00/bt5342/bt5342.pdf

2-Amino-4-nitro-phenol monohydrate.

A novel Schiff base compound was synthesized and characterized by IR spectroscopy and X-ray diffraction method. The compound crystallizes in monoclinic space group P21/c with Z = 4 in the unit cell. The ring systems are perfectly planar but the whole molecule is not planar. The dihedral angle between the thiophene and phenyl rings is 38.7(3)°. In the crystal, intermolecular C–H⋅⋅⋅O interactions link the molecules into a C(7) chain along the [20
$$\bar {1}$$

] direction. The crystal packing is also stabilized by N–O⋅⋅⋅π interaction. The molecular structure and vibrational frequencies of the title compound were calculated using the DFT/B3LYP method with the 6-311++G(d,p) basis set. The calculated results show that the optimized geometry can well reproduce the crystal structure, and the theoretical vibrational frequencies show good agreement with experimental results.

Crystal Structure, Spectroscopic and DFT Computational Studies of N-(4-Fluorophenyl)-1-(5-Nitrothiophen-2-yl)methanimine

In the title compound, C20H16N4O2S2, one of the thio­phene rings is disordered [occupancy ratio 0.710 (4):0.290 (4)] and the disorder is of the flip type. An intra­molecular C—H⋯O hydrogen bond generates a six-membered ring with an S(6) motif.

https://journals.iucr.org/e/issues/2010/02/00/dn2520/dn2520.pdf

1-Benzoyl-methyl-3-(2-thienylmeth-yl)-4-(2-thienylmethyl-eneamino)-1H-1,2,4-triazol-5(4H)-one.

The dihedral angle between the two rings in the title compound, C12H15N3O2, is 4903 (1)° The crystal structure is stabilized by inter­molecular N—H⋯O and C—H⋯O hydrogen bonds and π–π stacking inter­actions between the triazole rings with a centroid–centroid distance of 3394 A

https://journals.iucr.org/e/issues/2010/08/00/bt5301/bt5301.pdf

4-(4-Meth­oxy­pheneth­yl)-3-methyl-1H-1,2,4-triazol-5(4H)-one

In the crystal structure of the title compound, C20H20OP+·Cl−, the cations and anions are linked by inter­molecular C—H⋯Cl and O—H⋯Cl hydrogen bonds into chains running parallel to the b axis. In the cation, the hy­droxy­ethyl group is disordered over two orientations with site-occupancy factors of 0.554 (4) and 0.446 (4).

https://journals.iucr.org/e/issues/2011/03/00/rz2551/rz2551.pdf

Hasan Tanak

Papers

2-Amino-4-nitro-phenol monohydrate.

Crystal Structure, Spectroscopic and DFT Computational Studies of N-(4-Fluorophenyl)-1-(5-Nitrothiophen-2-yl)methanimine

1-Benzoyl-methyl-3-(2-thienylmeth-yl)-4-(2-thienylmethyl-eneamino)-1H-1,2,4-triazol-5(4H)-one.

4-(4-Methoxyphenethyl)-3-methyl-1H-1,2,4-triazol-5(4H)-one

(2-Hydroxyethyl)triphenylphosphonium chloride

Hasan Tanak

Papers

2-Amino-4-nitro-phenol monohydrate.

Crystal Structure, Spectroscopic and DFT Computational Studies of N-(4-Fluorophenyl)-1-(5-Nitrothiophen-2-yl)methanimine

1-Benzoyl-methyl-3-(2-thienylmeth-yl)-4-(2-thienylmethyl-eneamino)-1H-1,2,4-triazol-5(4H)-one.

4-(4-Meth­oxy­pheneth­yl)-3-methyl-1H-1,2,4-triazol-5(4H)-one

(2-Hy­droxy­eth­yl)triphenyl­phospho­nium chloride

4-(4-Methoxyphenethyl)-3-methyl-1H-1,2,4-triazol-5(4H)-one

(2-Hydroxyethyl)triphenylphosphonium chloride