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. 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 fl ux 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, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost 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 fi eld 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 delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so 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 autophagy assays, we hope to encourage technical innovation in the field

Availability of Nanopore sequences in the genome taxonomy for Vibrionaceae systematics: Rumoiensis Glade species as a test case

Whole genome sequence comparisons have become essential for establishing a robust scheme in bacterial taxonomy. To generalize this genome-based taxonomy, fast, reliable, and cost-effective gnome sequencing methodologies are required. MinION, the palm -sized sequencer from Oxford Nanopore Technologies, enables rapid sequencing of bacterial genomes using minimal laboratory resources. Here we tested the ability of Nanopore sequences for the genome-based taxonomy of Vibrionaceae and compared Nanopore- only assemblies to complete gnomes of fiw Rumoiensis clade species: Vibrio aphrogenes, V. algivorus, V. casei, V. litoralis, and V. rumoiensis. Comparison of overall gnome relatedness indices (OGRI) and multilocus sequence analysis (MLSA) based on Nanopore-only assembly and Illumina or hybrid assemblies revealed that errors in Nanopore-only assembly do not Influence average nucleotide identity (ANI), in silico DNA -DNA hybridization (DDH), G+C content, or MLSA tree topology in Vibrionaceae. Our results show that the genome sequences frorn Nanopore-based approach can be used for rapid species identification based on the OGRI and MLSA

Treatment of a Patient with Class I Malocclusion and Severe Tooth Crowding Using Invisalign and Fixed Appliances

The Invisalign system is widely used to treat mild to moderate tooth crowding. Recently, Invisalign appliances have been used in orthodontic patients with increasingly complex malocclusions. When using an Invisalign appliance to correct severe tooth crowding, root positions must be carefully controlled during extraction space closure. We report our treatment of a 34-year-old man who presented with a Class I relationship, a midline deviation, severe overjet, and severe tooth crowding in the maxillary and mandibular anterior regions. The treatment plan involved extraction of the maxillary and mandibular first premolars on both sides. We treated this patient with severe anterior tooth crowding using an Invisalign appliance combined with a fixed appliance with power arms and elastics. Treatment by Invisalign as an application of computational calculated result, especially in extraction cases, doctors should have professional skill and experience as an orthodontist

Association of circulating exosomal miR-122 levels with BAT activity in healthy humans

Brown adipose tissue (BAT) plays an important role in body fat accumulation and the regulation of energy expenditure. Since the role of miRNAs in the pathogenesis of obesity and related metabolic diseases is contentious, we analyzed exosomal miRNAs in serum of healthy subjects with special references to BAT activity and body fat level. Forty male volunteers aged 20-30 years were recruited. Their BAT activity was assessed by fluorodeoxyglucose positron emission tomography and computed tomography after 2 h of cold exposure and expressed as a maximal standardized uptake value (SUVmax). Exosomal miRNA levels was analyzed using microarray and real-time PCR analyses. The miR-122-5p level in the high BAT activity group (SUV >= 3) was 53% lower than in the low BAT activity group (SUVmax <3). Pearson's correlation analysis revealed that the serum miR-122-5p level correlated negatively with BAT activity and the serum HDL-cholesterol, and it correlated positively with age, BMI, body fat mass, and total cholesterol and trig lyceride serum levels. Multivariate regression analysis revealed that BAT activity was associated with the serum miR-122-5p level independently of the other parameters. These results reveal the serum exosomal miR-122-5p level is negatively associated with BAT activity independently of obesity

Group A streptococcus endocarditis in children: 2 cases and a review of the literature

Abstract Background Infective endocarditis (IE) is defined as endocarditis caused by microorganisms (bacteria or fungi) involving either the heart or great vessels. The clinical course of IE can be complicated by cardiac dysfunction and bacterial embolization to virtually any organ. Staphylococcus aureus and viridans group streptococci are the most common causative organisms, whereas group A Streptococcus (GAS) is less common. Although some GAS serotypes have been associated with severe disease, there are few reports of IE associated with GAS serotypes. Here, we report two cases of GAS endocarditis and review the associated literature. Case presentations Patient 1 was a previously healthy 14-year-old girl who developed bacteremia and disseminated intravascular coagulation secondary to left foot cellulitis. She was administered intravenous antibiotics. Two of three blood cultures grew Streptococcus pyogenes (T6 M6, emm6.104). Three days later, a new systolic ejection murmur was heard and echocardiography showed mitral regurgitation with mitral valve vegetation. Because of the resultant severity of the mitral regurgitation, she underwent mitral valve repair after 10 weeks of antibiotic treatment. Patient 2 was a 17-month old boy who presented with a fever. He had a history of spontaneous closure of a ventricular septal defect (VSD). He was started on intravenous antibiotics for possible bacteremia. Two consecutive blood cultures with an interval of more than 12 h grew S. pyogenes (T4 M4, emm4.0). Five days later, echocardiography showed vegetation on a membranous ventricular septal aneurysm. The patient responded well to antibiotics, and recovered fully with no complications. Conclusions Although both patients developed GAS endocarditis, patient 1 did not have any predisposing conditions for IE, and patient 2 had a only a low-risk predisposing condition, a VSD that had closed spontaneously at five months of age. We found twelve reports in the literature of GAS endocarditis with information on serotypes. All patients in these reports had GAS endocarditis caused by serotypes generally associated with milder infections, but no specific risk trends were identified. A greater accumulation of cases is necessary to more clearly elucidate the association between GAS IE and specific serotypes