Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Structural and functional features of asthma participants with fixed airway obstruction using CT imaging and 1D computational fluid dynamics: A feasibility study

Abstract Asthma with fixed airway obstruction (FAO) is associated with significant morbidity and rapid decline in lung function, making its treatment challenging. Quantitative computed tomography (QCT) along with data postprocessing is a useful tool to obtain detailed information on airway structure, parenchymal function, and computational flow features. In this study, we aim to identify the structural and functional differences between asthma with and without FAO. The FAO group was defined by a ratio of forced expiratory volume in 1 s (FEV1) to forced vital capacity (FVC), FEV1/FVC <0.7. Accordingly, we obtained two sets of QCT images at inspiration and expiration of asthma subjects without (N = 24) and with FAO (N = 12). Structural and functional QCT‐derived airway variables were extracted, including normalized hydraulic diameter, normalized airway wall thickness, functional small airway disease, and emphysema percentage. A one‐dimensional (1D) computational fluid dynamics (CFD) model considering airway deformation was used to compare the pressure distribution between the two groups. The computational pressures showed strong correlations with the pulmonary function test (PFT)‐based metrics. In conclusion, asthma participants with FAO had worse lung functions and higher‐pressure drops than those without FAO

Enhancement of in vivo targeting properties of ErbB2 aptamer by chemical modification.

Aptamers have great potential for diagnostics and therapeutics due to high specificity to target molecules. However, studies have shown that aptamers are rapidly distributed and excreted from blood circulation due to nuclease degradation. To overcome this issue and to improve in vivo pharmacokinetic properties, inverted deoxythymidine (idT) incorporation at the end of aptamer has been developed. The goal of this study was to evaluate the biological characterization of 3'-idT modified ErbB2 aptamer and compare with that of unmodified aptamer via nuclear imaging. ErbB2-idT aptamer was labeled with radioisotope F-18 by base-pair hybridization using complementary oligonucleotide platform. The hyErbB2-idT aptamer demonstrated specific binding to targets in a ErbB2 expressing SK-BR-3 and KPL4 cells in vitro. Ex vivo biodistribution and in vivo imaging was studied in KPL4 xenograft bearing Balb/c nu/nu mice. 18F-hyErbB2-idT aptamer had significantly higher retention in the tumor (1.36 ± 0.17%ID/g) than unmodified 18F-hyErbB2 (0.98 ± 0.19%ID/g) or scrambled aptamer (0.79 ± 0.26% ID/g) at 1 h post-injection. 18F-hyErbB2-idT aptamer exhibited relatively slow blood clearance and delayed excretion by the renal and hepatobiliary system than 18F-hyErbB2 aptamer. In vivo PET imaging study showed that 18F-hyErbB2-idT aptamer had more stronger PET signals on KPL4 tumor than 18F-hyErbB2 aptamer. The results of this study demonstrate that attachment of idT at 3'-end of aptamer have a substantial influence on biological stability and extended blood circulation led to enhanced tumor uptake of aptamer

Study on ductile mode machining of single-crystal silicon by mechanical machining

Nano patterns on single-crystal silicon are generally manufactured by photolithography, which can form limited cross-sectional shapes such as U-shapes or rectangular channels. Though V-shaped patterns are widely used in the optical industries because they concentrate light, they are challenging to manufacture by conventional photolithography. Mechanical machining is useful in manufacturing various kinds of cross-sectional shapes including V-shapes with various apex angles, but is hard to apply to single-crystal silicon due to its brittle fracture. Here we suggest a novel way of mechanical machining of single-crystal silicon that suppresses brittle fracture below the critical point (the ductile-brittle transition point) as determined by nano-scratch testing. We find that the first drop point of the cutting force corresponds to a critical point and define the critical forces as the thrust force and the cutting force at the critical point. The critical forces are varied by the applied force per unit length, which is the possibility that the cutting tool interacts with mechanically weak atomic bonds. When the applied force per unit length is zero (a general condition of mechanical machining), the cutting speed does not affect the variation of the critical forces or the quality of the machined pattern. Based on analysis of the experimental results, we suggest that the single-crystal silicon can be mechanically machined without brittle fracture at high cutting speed if the thrust force is smaller than the critical force of zero applied force per unit length.clos

A Rare Case of Familial Schwannomatosis Showing Intrafamilial Variability with Identification of a Shared Novel Germline SMARCB1 Mutation

Schwannomatosis is characterized by the presence of multiple schwannomas without landmarks of NF2. It is considered the rarest form of neurofibromatosis (NF). Here, we report the first case of familial schwannomatosis with regard to the segmental/generalized phenotype, in which the proband and the daughter present a distinct phenotype in this classification. The proband presents a generalized, painless, extradural type of schwannomatosis, while the daughter shows a segmental, painful, intradural type of schwannomatosis. Whole-exome sequencing of the affected individuals revealed a shared novel SMARCB1 gene mutation (c.92A &gt; G, p.Glu31Gly) despite the clinical variability. We thus suggest two points in the diagnosis of familial schwannomatosis: The identified novel germline SMARCB1 variant can be reflective of a phenotypical progression from a segmental to a generalized type of schwannomatosis, or an intrafamilial variability in inherited schwannomatosis, which was not reported in previous literature. The specific combination of somatic NF2 mutations may be a major factor in regulating the severity and scope of the resulting phenotype in schwannomatosis

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Aptamers have great potential for diagnostics and therapeutics due to high specificity to target molecules. However, studies have shown that aptamers are rapidly distributed and excreted from blood circulation due to nuclease degradation. To overcome this issue and to improve in vivo pharmacokinetic properties, inverted deoxythymidine (idT) incorporation at the end of aptamer has been developed. The goal of this study was to evaluate the biological characterization of 3’-idT modified ErbB2 aptamer and compare with that of unmodified aptamer via nuclear imaging. ErbB2-idT aptamer was labeled with radioisotope F-18 by base-pair hybridization using complementary oligonucleotide platform. The hyErbB2-idT aptamer demonstrated specific binding to targets in a ErbB2 expressing SK-BR-3 and KPL4 cells in vitro. Ex vivo biodistribution and in vivo imaging was studied in KPL4 xenograft bearing Balb/c nu/nu mice. 18F-hyErbB2-idT aptamer had significantly higher retention in the tumor (1.36 ± 0.17%ID/g) than unmodified 18F-hyErbB2 (0.98 ± 0.19%ID/g) or scrambled aptamer (0.79 ± 0.26% ID/g) at 1 h post-injection. 18F-hyErbB2-idT aptamer exhibited relatively slow blood clearance and delayed excretion by the renal and hepatobiliary system than 18F-hyErbB2 aptamer. In vivo PET imaging study showed that 18F-hyErbB2-idT aptamer had more stronger PET signals on KPL4 tumor than 18F-hyErbB2 aptamer. The results of this study demonstrate that attachment of idT at 3’-end of aptamer have a substantial influence on biological stability and extended blood circulation led to enhanced tumor uptake of aptamer.</div

Characterization of ErbB2 aptamer.

Secondary structure predictions of ErbB2, RC-ErbB2-idT and hyErbB2 aptamer. The equilibrium dissociation constant (Kd) values of ErbB2 aptamer to recombinant ErbB2 protein were determined by ELONA assay.</p

Fig 3 -

Cell binding assay of ErbB2 aptamers using flow cytometry and fluorescence spectroscopy (A) The specific binding capacity of ErbB2 aptamers to human breast cancer cells was investigated by flow cytometry. ErbB2-positive SK-BR-3 and KPL4, and ErbB2-negative MCF-7 cells were stained with Cy5-labeled ErbB2 aptamer, hyErbB2-idT aptamer, scrambled ErbB2 (ScrErbB2) aptamer or cODN. (B) Confocal microscopy analysis with Cy5-ErbB2 or Cy5-hyErbB2-idT on SK-BR-3, KPL4, and MCF-7 cells at 4°C. Aptamers are visualized in red. (C) The binding properties of Cy5-ScrErbB2 aptamer were also measured on KPL4 and MCF-7 cells for comparison. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue).</p

Representative <i>in vivo</i> PET images of ¹⁸F-hyErbB2, ¹⁸F-hyErbB2-idT and ¹⁸F-hyScrErbB2 aptamer in athymic nude mice bearing KPL4 tumors.

Coronal images were acquired at 1 h after injection. The white arrows indicate the location of the tumor. Uptake values are shown as mean %ID/g.</p