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

Adult non–insulin requiring diabetes includes latent autoimmune diabetes of adults (LADA), distinguished from type 2 diabetes by the presence of islet autoantibodies. LADA China determined the characteristics of Chinese LADA. This nationwide, multicenter, clinic-based cross-sectional study was conducted in 46 university-affiliated hospitals in 25 Chinese cities. All 4,880 ketosis-free diabetic patients (<1 year postdiagnosis, without insulin therapy for >6 months, aged ≥30 years) had GAD antibody (GADA) and HLA-DQ genotype measured centrally with clinical data collected locally. GADA-positive subjects were classified as LADA. Of the patients, 5.9% were GADA positive with LADA. LADA showed a north-south gradient. Compared with GADA-negative type 2 diabetes, LADA patients were leaner, with lower fasting C-peptide and less metabolic syndrome. Patients with high GADA titers are phenotypically different from those with low GADA titers, while only a higher HDL distinguished the latter from those with type 2 diabetes. HLA diabetes–susceptible haplotypes were more frequent in LADA, even in those with low-titer GADA. HLA diabetes-protective haplotypes were less frequent in LADA. Our study implicates universal immunogenetic effects, with some ethnic differences, in adult-onset autoimmune diabetes. Autoantibody positivity and titer could be important for LADA risk stratification and accurate therapeutic choice in clinical practice.

Monodispersed surfactant-free MoS2 nanoparticles with sizes of less than 2 nm were prepared from bulk MoS2 by simple ultrasonication and gradient centrifugation. The ultrasmall MoS2 nanoparticles expose a large fraction of edge sites, along with their high surface area, which lead to attractive electrocatalytic activity for reduction of H2O2. An extremely sensitive H2O2 biosensor based on MoS2 nanoparticles with a real determination limit as low as 2.5 nM and wide linear range of 5 orders of magnitude was constructed. On the basis of this biosensor, the trace amount of H2O2 released from Raw 264.7 cells was successfully recorded, and an efficient glucose biosensor was also fabricated. Since H2O2 is a byproduct of many oxidative biological reactions, this work serves as a pathway for the application of MoS2 in the fields of electrochemical sensing and bioanalysis.

Monitoring autophagic flux is important for the analysis of autophagy. Tandem fluorescent-tagged LC3 (mRFP-EGFP-LC3) is a convenient assay for monitoring autophagic flux based on different pH stability of EGFP and mRFP fluorescent proteins. However, it has been reported that there is still weak fluorescence of EGFP in acidic environments (pH between 4 and 5) or acidic lysosomes. So it is possible that autolysosomes are labeled with yellow signals (GFP(+)RFP(+) puncta), which results in misinterpreting autophagic flux results. Therefore, it is desirable to choose a monomeric green fluorescent protein that is more acid sensitive than EGFP in the assay of autophagic flux. Here, we report on an mTagRFP-mWasabi-LC3 reporter, in which mWasabi is more acid sensitive than EGFP and has no fluorescence in acidic lysosomes. Meanwhile, mTagRFP-mWasabi-LC3ΔG was constructed as the negative control for this assay. Compared with mRFP-EGFP-LC3, our results showed that this reporter is more sensitive and accurate in detecting the accumulation of autophagosomes and autolysosomes. Using this reporter, we find that high-dose rapamycin (30 μM) will impair autophagic flux, inducing many more autophagosomes than autolysosomes in HeLa cells, while low-dose rapamycin (500 nM) has an opposite effect. In addition, other chemical autophagy inducers (cisplatin, staurosporine and Z18) also elicit much more autophagosomes at high doses than those at low doses. Our results suggest that the dosage of chemical autophagy inducers would obviously influence autophagic flux in cells.

The MutT enzyme (129 residues) catalyzes the hydrolysis of nucleoside triphosphates (NTP) by substitution at the rarely attacked beta-P, to yield NMP and pyrophosphate. It requires two divalent cations, forming an active E-M2+-NTP-M2+ complex. The solution structure of the free enzyme consists of a five-stranded mixed beta-sheet connected by loop I-alpha-helix I-loop II, by two tight turns, and by loop III and terminated by loop IV-alpha-helix II [Abeygunawardana, C., et al. (1995) Biochemistry 34, 14997-15005]. Assignments of backbone 15N and NH resonances and side chain 15N and NH2 resonances of the quaternary complex were made by 1H-15N HSQC titrations of the free enzyme with MgCl2 followed by equimolar AMPCPP/MgCl2. H(alpha) assignments were made by 1H-15N 3D TOCSY HSQC, and 1H-13C CT-HSQC spectra and backbone and side chain 1H and 13C assignments were made by 3D HCCH TOCSY experiments. Ligands donated by the protein to the enzyme-bound divalent cation, identified by paramagnetic effects of Co2+ and Mn2+ on CO(C)H spectra, are the carboxylate groups of Glu-56, -57, and -98 and the amide carbonyl of Gly-38. The solution structure of the complex was computed with XPLOR using a total of 2168 NOE and 83 phi restraints for the protein, 11 intramolecular NOEs for bound Mg2+ AMPCPP, 22 intermolecular NOEs between MutT and AMPCPP, and distances from the enzyme-bound Co2+ to the three phosphorus atoms of Co3+(NH3)4AMPCPP from paramagnetic effects of Co2+ on their T1 values. The fold of the MutT enzyme in the complex is very similar to that of the free enzyme, with minor changes in the metal and substrate binding sites. The adenine ring binds in a hydrophobic cleft, interacting with Leu-4 and Ile-6 on beta-strand A and with Ile-80 on beta-strand D. The 6-NH2 group of adenine approaches the side chain NH2 of Asn-119. This unfavorable interaction is consistent with the stronger binding by MutT of guanine nucleotides, which have a 6-keto group. The ribose binds with its hydroxyl groups oriented toward the solvent and its hydrophobic face interacting with Leu-4, Ile-6, and the gamma-CH2 of Lys-39 of loop I. The metal-triphosphate moiety appears to bind in the second coordination sphere of the enzyme-bound divalent cation. One of two intervening water ligands is well positioned to attack P(beta) with inversion and to donate a hydrogen bond to the conserved residue, Glu-53, which may deprotonate or orient the attacking water ligand. Lys-39 which is positioned to interact electrostatically with the alpha-phosphoryl group may facilitate the departure of the leaving NMP. On the basis of the structure of the quaternary complex, a mechanism of the MutT reaction is proposed which is qualitatively and quantitatively consistent with kinetic and mutagenesis studies. It is suggested that similar mechanisms may be operative for other enzymes that catalyze substitution at P(beta) of NTP substrates.

Fe5 C2 NPs exhibit a high contrast in magnetic resonance imaging (MRI), superior photoacoustic tomography improvements, and efficient photothermal therapy (PTT) due to their unique core/shell structure, with a magnetic core and carbon shell. By conjugating a new class of affinity proteins (ZHER2:342), they can target to tumor cells with low cytotoxicity, and kill them through laser irritation. It is also possible to ablate tumors under guidance by MRI and PTT without noticeable side effects.

Imaging-guided photothermal therapy (PTT) by combination of imaging and PTT has been emerging as a promising therapeutic method for precision therapy. However, the development of multicomponent nanoplatforms with stable structures for both PTT and multiple-model imaging remains a great challenge. Herein, we synthesized monodisperse Au-FeC Janus nanoparticles (JNPs) of 12 nm, which are multifunctional entities for cancer theranostics. Due to the broad absorption in the near-infrared range, Au-FeC JNPs showed a significant photothermal effect with a 30.2% calculated photothermal transduction efficiency under 808 nm laser irradiation in vitro. Owing to their excellent optical and magnetic properties, Au-FeC JNPs were demonstrated to be advantageous agents for triple-modal magnetic resonance imaging (MRI)/multispectral photoacoustic tomography (MSOT)/computed tomography (CT) both in vitro and in vivo. We found that Au-FeC JNPs conjugated with the affibody (Au-FeC-Z) have more accumulation and deeper penetration in tumor sites than nontargeting JNPs (Au-FeC-PEG) in vivo. Meanwhile, our results verified that Au-FeC-Z JNPs can selectively target tumor cells with low cytotoxicity and ablate tumor tissues effectively in a mouse model. In summary, monodisperse Au-FeC JNPs, used as a multifunctional nanoplatform, allow the combination of multiple-model imaging techniques and high therapeutic efficacy and have great potential for precision theranostic nanomedicines.

The MutT enzyme (129 residues) catalyzes the hydrolysis of normal and mutagenic nucleoside triphosphates, such as 8-oxo-dGTP, by substitution at the rarely attacked P-P, to yield NMP and pyrophosphate. Previous heteronuclear NMR studies of MutT have shown the secondary structure to consist of a five-stranded mixed P-sheet connected by the loop I-a-helix I-loop I1 motif, by two tight tums, and by loop 111, and terminated by loop IV-a-helix I1 (4) restraints and 34 backbone hydrogen bond restraints were used to determine the tertiary structure ofMutT by distance geometry, simulated annealing, and energy minimization with the program X-PLOR. The structure is globular and compact with the parallel portion of the P-sheet sandwiched between the two a-helices, forming an a f , ! ? fold. The essential divalent cation has previously been shown to bind near residues Gly-37, Gly-38, Lys-39, and Glu-57, and nucleotides have been shown to bind near residues Leu-54 and Val-58 by NMR relaxation methods [Frick et al. (1995) Biochemistry 34, 5577-55861. The tertiary structure of MutT shows these residues to be near each other along the loop I-helix I region of the enzyme. A cluster of five glutamate residues (41,53, 56,57, and 98) form a patch of strongly negative electrostatic potential likely constituting the metal binding site. This site is contiguous with a deep cleft between P-strands A, C, and D and loop I which may contribute to the nucleotide binding site. This location of the active site is consistent with mutagenesis studies and with sequence homologies among MutT-like pyrophosphohydrolases.The MutT enzyme, a pyrophosphohydrolase of 129 residues, catalyzes the unusual hydrolysis of nucleoside and deoxynucleoside triphosphates (NTP) by nucleophilic substitution at the rarely attacked ,!?-phosphorus, yielding pyrophosphate and a nucleotide (NMP) as products (Bhatnagar et al., 1991;Weber et al., 1992). Like other enzymes which catalyze substitution at the electron-rich ,!?-phosphorus, this enzyme requires two divalent cations for activity (Frick et al., 1994). The biological role of this enzyme is to prevent ' This work was supported in part by National Institutes of Health Grants DK28616 (to A.S.M.), GM18649 (to M.J.B.), and GM36358 (which supports A.G.G.).A complete listing of the distance restraints derived from the NOE data has been deposited at the Brookhaven Protein Data Bank (Chemistry Department, Brookhaven National Laboratory, Upton, NJ) together with the atomic coordinates of the family of 15 acceptable structures (file name IMUT).