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 insulin receptor substrate proteins IRS1 and IRS2 are key targets of the insulin receptor tyrosine kinase and are required for hormonal control of metabolism. Tissues from insulin-resistant and diabetic humans exhibit defects in IRS-dependent signalling, implicating their dysregulation in the initiation and progression of metabolic disease. However, IRS1 and IRS2 are regulated through a complex mechanism involving phosphorylation of >50 serine/threonine residues (S/T) within their long, unstructured tail regions. In cultured cells, insulin-stimulated kinases (including atypical PKC, AKT, SIK2, mTOR, S6K1, ERK1/2 and ROCK1) mediate feedback (autologous) S/T phosphorylation of IRS, with both positive and negative effects on insulin sensitivity. Additionally, insulin-independent (heterologous) kinases can phosphorylate IRS1/2 under basal conditions (AMPK, GSK3) or in response to sympathetic activation and lipid/inflammatory mediators, which are present at elevated levels in metabolic disease (GRK2, novel and conventional PKCs, JNK, IKKβ, mPLK). An emerging view is that the positive/negative regulation of IRS by autologous pathways is subverted/co-opted in disease by increased basal and other temporally inappropriate S/T phosphorylation. Compensatory hyperinsulinaemia may contribute strongly to this dysregulation. Here, we examine the links between altered patterns of IRS S/T phosphorylation and the emergence of insulin resistance and diabetes.

/pdf/regulation-of-insulin-sensitivity-by-serine-threonine-6zpwn70287.pdf

Regulation of insulin sensitivity by serine/threonine phosphorylation of insulin receptor substrate proteins IRS1 and IRS2

Spectroscopic investigations and quantum chemical computational study of (E)-4-methoxy-2-[(p-tolylimino)methyl]phenol.

The Schiff base compound, 2-[(4-Fluorophenylimino)methyl]-3,5-dimethoxyphenol, has been synthesized and characterized by IR, electronic spectroscopy, and X-ray single-crystal determination. Molecular geometry from X-ray experiment of the title compound in the ground state have been compared using the Hartree-Fock (HF) and density functional method (B3LYP) with 6–31G(d) basis set. Calculated results show that density functional theory (DFT) and HF can well reproduce the structure of the title compound. The energetic behavior of the title compound in solvent media has been examined using B3LYP method with the 6–31G(d) basis set by applying the polarizable continuum model (PCM). The total energy of the title compound decrease with the increasing polarity of the solvent. By using TD-DFT and TD-HF methods, electronic absorption spectra of the title compound have been predicted and a good agreement with the TD-DFT method and the experimental ones is determined. In addition, DFT calculations of the title compound, molecular electrostatic potential (MEP), natural bond orbital (NBO), and thermodynamic properties were performed at B3LYP/6–31G(d) level of theory.

Experimental and quantum chemical calculational studies on 2-[(4-Fluorophenylimino)methyl]-3,5-dimethoxyphenol.

Density functional calculations of the structure, atomic charges, molecular electrostatic potential and thermodynamic functions have been performed at B3LYP/6-31G(d,p) level of theory for the title compound (E)-2-[(2-hydroxy-5-nitrophenyl)-iminiomethyl]-4-nitrophenolate. The results show that the phenolate oxygen atom and all of the nitro group oxygen atoms have bigger negative charges, and the coordination ability of these atoms differs in different solvents. The energetic behavior of the title compound in solvent media has been examined using B3LYP method with the 6-31G(d,p) basis set by applying the Onsager method and the isodensity polarized continuum model (IPCM). The results obtained with these methods reveal that the IPCM method yielded a more stable structure than Onsager’s method. In addition, natural bond orbital and frontier molecular orbital analysis of the title compound were performed using the B3LYP/6-31G(d,p) method.

Density functional computational studies on (E)-2-[(2-Hydroxy-5-nitrophenyl)-iminiomethyl]-4-nitrophenolate.

The triazole compound 4-(3-(1H-imidazol-1-yl)propyl)-5-methyl-2H-1,2,4-triazol-3(4H)-one monohydrate has been synthesised and characterised by 1H-NMR, 13C-NMR, IR, and X-ray single-crystal determination. The compound crystallizes in the triclinic space group with a = 9.0366(7) A, b = 11.5690(8) A, c = 12.0571(9) A, α = 110.733(6)°, β = 94.172(6)°, γ = 98.085(6)° and Z = 4. In addition to the molecular geometry from X-ray determination, the molecular geometry, vibrational frequencies and gauge, including atomic orbital (GIAO) 1H- and 13C-NMR chemical shift values of the title compound in the ground state, were calculated using the density functional method (B3LYP) with the 6-31G(d) basis set. The calculated results show that the optimised geometries can well reproduce the crystal structure, and the theoretical vibrational frequencies and chemical shift values show good agreement with experimental values. The energetic behaviour of the title compound in solvent media was examined using the B3LYP method with...

An experimental and DFT computational study on 4-(3-(1H-imidazol-1-yl)propyl)-5-methyl-2H-1,2,4-triazol-3(4H)-one monohydrate

The Schiff base compound, N-n-Decyl-2-oxo-5-nitro-1-benzylidene-methylamine, has been -synthesized and characterized by IR, electronic spectroscopy, and X-ray single-crystal determination. Molecular geometry from X-ray experiment of the title compound in the ground state have been compared using the Hartree-Fock (HF) and density functional method (B3LYP) with 6-31G(d) basis set. Calculated results show that density functional theory (DFT) at B3LYP/6-31G(d) level can well reproduce the structure of the title compound. To investigate the solvent effect for the atomic charge distributions of the title compound, self-consistent reaction field theory with Onsager reaction field model was used. In addition, DFT calculations of the title compound, molecular electrostatic potential and thermodynamic properties were performed at B3LYP/6-31G(d) level of theory.

Theoretical modeling and experimental studies on N-n-decyl-2-oxo-5-nitro-1-benzylidene-methylamine.

Density functional calculations of the structure, molecular electrostatic potential and thermodynamic functions have been performed at B3LYP/6-31G(d) level of theory for the title compound of N -2-Methoxyphenyl-2-oxo-5-nitro-1-benzylidenemethylamine. To investigate the tautomeric stability, optimization calculations at B3LYP/6-31G(d) level were performed for the enol and keto forms of the title compound. Calculated results reveal that the enol form of the title compound is more stable than its keto form. The predicted non-linear optical properties of the title compound are much greater than ones of p-Nitroaniline. The changes of thermodynamic properties from the monomers to title compound with the temperature ranging from 200 K to 450 K have been obtained using the statistical thermodynamic method. At 298.15 K the change of Gibbs free energy for the formation reaction of the title compound is 30.654 kJ/mol. The title compound cannot be spontaneously produced from the isolated monomers at room temperature. The tautomeric equilibrium constant is computed as 0.0192 at 298.15 K for enol-imine ↔ keto-amine tautomerization of the title compound. In addition, natural bond orbital analysis of the title compound were performed using the B3LYP/6-31G(d) method.

Metin Yavuz

Papers

Experimental and quantum chemical calculational studies on 2-[(4-Fluorophenylimino)methyl]-3,5-dimethoxyphenol.

Density functional computational studies on (E)-2-[(2-Hydroxy-5-nitrophenyl)-iminiomethyl]-4-nitrophenolate.

An experimental and DFT computational study on 4-(3-(1H-imidazol-1-yl)propyl)-5-methyl-2H-1,2,4-triazol-3(4H)-one monohydrate

Theoretical modeling and experimental studies on N-n-decyl-2-oxo-5-nitro-1-benzylidene-methylamine.

Density functional modelling studies on N-2-Methoxyphenyl-2-oxo-5-nitro-1-benzylidenemethylamine