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Single-molecule localization microscopy (SMLM) has allowed the observation of various molecular structures in cells beyond the diffraction limit using organic dyes. In principle, the SMLM resolution depends on the precision of photoswitching fluorophore localization, which is inversely correlated with the square root of the number of photons released from the individual fluorophores. Thus, increasing the photon number by using highly bright fluorophores, such as quantum dots (QDs), can theoretically fundamentally overcome the current resolution limit of SMLM. However, the use of QDs in SMLM has been challenging because QDs have no photoswitching property, which is essential for SMLM, and they exhibit nonspecificity and multivalency, which complicate their use in fluorescence imaging. Here, we present a method to utilize QDs in SMLM to surpass the resolution limit of the current SMLM utilizing organic dyes. We confer monovalency, specificity, and photoswitchability on QDs by steric exclusion via passivation and ligand exchange with ptDNA, PEG, and casein as well as by DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) via automatic thermally driven hybridization between target-bound docking and dye-bound complementary imager strands. QDs are made monovalent and photoswitchable to enable SMLM and show substantially better photophysical properties than Cy3, with higher fluorescence intensity and an improved resolution factor. QD-PAINT displays improved spatial resolution with a narrower full width at half maximum (FWHM) than DNA-PAINT with Cy3. In summary, QD-PAINT shows great promise as a next-generation SMLM method for overcoming the limited resolution of the current SMLM., Microscopy: Seeing individual molecules inside cells with improved resolution A new microscopy method called QD-PAINT will allow researchers to see inside cells with nanoscale precision. The method combines two existing technologies, DNA-PAINT and steric exclusion, to show a proof-of-concept of a proper utilization of quantum dots (QDs) in single-molecule localization microscopy (SMLM) with improved resolution. In the last decade, SMLM using organic dyes allowed observation of previously invisible biological phenomena, such as individual proteins, but resolution was limited to around 20 nm. QDs emit much brighter fluorescence than organic dyes and could theoretically improve the resolution of SMLM. Sung Ho Ryu and Do-Hyeon Kim at Pohang University of Science and Technology in South Korea adapted QDs for SMLM, conferring them with photoswitchability and specificity, and improved SMLM resolution to about 8 nm. The researchers suggest the method could be fine-tuned for even higher resolution.

Multicolor fluorescence imaging is a powerful tool visualizing the spatiotemporal relationship among biomolecules. Here, we report that commonly employed organic dyes exhibit a blue-conversion phenomenon, which can produce severe multicolor image artifacts leading to false-positive colocalization by invading predefined spectral windows, as demonstrated in the case study using EGFR and Tensin2. These multicolor image artifacts become much critical in localization-based superresolution microscopy as the blue-converted dyes are photoactivatable. We provide a practical guideline for the use of organic dyes for multicolor imaging to prevent artifacts derived by blue-conversion., Blue-conversion, a photooxidative conversion leading to the hypsochromic shift of absorption and emission spectra, occurs in popular organic dyes under conventional laser illumination and produces severe artifacts in multicolor fluorescence imaging.

SummaryInsulin is a key regulator of energy metabolism in peripheral tissues but also functions as a growth factor. Insulin binding to the insulin receptor (IR) leads to autophosphorylation of intracellular tyrosine residues, which simultaneously initiates a multitude of signals and functions. In contrast, some artificial (non-insulin) ligands for the IR result in biased agonism, selectively activating the PI3K/AKT pathway and metabolic effects without activating mitogen-activated protein kinase (MAPK) pathway and mitogenic effects. However, the precise mechanism of biased agonism at the receptor level remains unclear. The biased agonist IR-A48 aptamer selectively induces mono-phosphorylation of Tyr1150 residue (m-pY1150) in the kinase domain of IR. Hence, we hypothesized that IR autophosphorylation is a stepwise process in which formation of m-pY1150 represents an intermediate step. To explore this idea, we used hybrid receptors in which insulin can bind only one of the two binding sites of the dimeric receptor. Asymmetric insulin binding selectively induced symmetric m-pY1150 in both kinase domains. Moreover, the juxtamembrane domain, which interacts with the kinase domain, restricted the full activation of IR, and symmetric m-pY1150 played a crucial role in the rearrangement of intracellular domains to release this restriction. Our findings demonstrate that the symmetry of insulin binding to a dimeric receptor determines the stepwise autophosphorylation of IR. Furthermore, considering that the degree of ligand symmetry on a receptor depends mainly on ligand concentration, our results suggest that IR may play metabolic-biased roles in peripheral tissues dependent on local insulin concentrations.

Interactions between membrane proteins are poorly understood despite their importance in cell signaling and drug development. Here, we present a co-immunoimmobilization assay (Co-II) enabling the direct observation of membrane protein interactions in single living cells that overcomes the limitations of currently prevalent proximity-based indirect methods. Using Co-II, we investigated the transient homodimerizations of epidermal growth factor receptor (EGFR) and beta-2 adrenergic receptor (β2-AR) in living cells, revealing the differential regulation of these receptors’ dimerizations by molecular conformations and microenvironment in a plasma membrane. Co-II should provide a simple, rapid, and robust platform for visualizing both weak and strong protein interactions in the plasma membrane of living cells., Author summary Protein–protein interactions govern cellular processes. The majority of these physical interactions previously identified are strong/permanent interactions, which typically remain unbroken even after purification. The weak/transient interactions between proteins have been implicated in the control of dynamic cellular process that maintain cellular homeostasis and trigger signaling cascades upon environmental changes. However, these interactions are poorly investigated, mainly due to the methodological limitations. Here, we have developed a co-immunoimmobilization assay called Co-II that enables the direct visualization of protein–protein interactions in the membrane of living cells at the single-molecule level. Co-II is based on the intuitive concept that if the protein of interest is immobilized, the interacting protein must be co-immobilized. The use of intrinsic protein diffusivity fundamentally overcomes the limitations of proximity-based methods. Using Co-II, we study the transient homodimerizations of EGFR and β2-AR in living cells, which have been implicated in several types of cancers and heart diseases. We show that the dimerization of these receptors is differently regulated by molecular conformations and the microenvironment in the plasma membrane.

We present a single-molecule diffusional-mobility-shift assay (smDIMSA) for analyzing the interactions between membrane and water-soluble proteins in the crowded membrane of living cells. We found that ligand-receptor interactions decreased the diffusional mobility of ErbB receptors and β-adrenergic receptors, as determined by single-particle tracking with super-resolution microscopy. The shift in diffusional mobility was sensitive to the size of the water-soluble binders that ranged from a few tens of kilodaltons to several hundred kilodaltons. This technique was used to quantitatively analyze the dissociation constant and the cooperativity of antibody interactions with the epidermal growth factor receptor and its mutants. smDIMSA enables the quantitative investigation of previously undetected ligand-receptor interactions in the intact membrane of living cells on the basis of the diffusivity of single-molecule membrane proteins without ligand labeling.

The purpose of this research is to study the implementation of instructional practices and the needs for improving study habits of underachievers, as a preliminary research to develop improving program for underachievers’ study habits. Firstly, past researches on the subject were analyzed to find out the concept and scope of study habits, and factors related to academic achievement. After arranging and integrating these factors, we have extracted the constructs and factors of improvement program for students' study habits. Secondly, the instructional practices in the field and needs for improvement program for students' study habits were analyzed through a survey. The online survey composed of structured questionnaire was conducted among teachers consisting of regular classroom teachers, special class teachers, school counselors, etc. The results were reflected in the contents and configuration of the program. The contents of survey was the degree of practice and needs of improvement program for students' study habits in schools, and the degree of practice and needs of standardized tests. According to the survey results, the efforts for improving the underachieved students' study habits were low level. This study focused on study habits showing high demands in the development of program and manual.Key words: Study Habit, Feasibilities, Desirabilities, The Underachieved StudentsCorresponding author : 02-3704-3527, syhahm@kice.re.kr

Resveratrol (RSV) is a natural polyphenol that has a beneficial effect on health, and resveratrol-induced autophagy has been suggested to be a key process in mediating many beneficial effects of resveratrol, such as reduction of inflammation and induction of cancer cell death. Although various resveratrol targets have been suggested, the molecule that mediates resveratrol-induced autophagy remains unknown. Here, we demonstrate that resveratrol induces autophagy by directly inhibiting the mTOR-ULK1 pathway. We found that inhibition of mTOR activity and presence of ULK1 are required for autophagy induction by resveratrol. In line with this mTOR dependency, we found that resveratrol suppresses the viability of MCF7 cells but not of SW620 cells, which are mTOR inhibitor sensitive and insensitive cancer cells, respectively. We also found that resveratrol-induced cancer cell suppression occurred ULK1 dependently. For the mechanism of action of resveratrol on mTOR inhibition, we demonstrate that resveratrol directly inhibits mTOR. We found that resveratrol inhibits mTOR by docking onto the ATP-binding pocket of mTOR (i.e., it competes with ATP). We propose mTOR as a novel direct target of resveratrol, and inhibition of mTOR is necessary for autophagy induction.

Dried seasoned filefish (Thamnaconus modestus) was irradiated at 0–10 kGy and the identification of irradiation treatment was investigated by analyzing the characteristics of thermoluminescence (TL), hydrocarbon (HC), and 2-alkylacyclobutanone (2-ACB). The TL (TL1), glow curve of the irradiated samples peaked at approximately 150 °C with high intensity, but that of the non-irradiated samples peaked at about 300 °C with little intensity, thus making it possible to discriminate between irradiated and non-irradiated samples. Moreover, TL ratio (TL1/TL2), through normalization steps, enhanced the reliability of the TL1 results. Six kinds of HCs and three kinds of 2-ACBs quantitatively determined for the samples linearly increased in proportion to irradiation doses. In particular, two HCs like 1-hexadecane and 1,7-hexadecadine, and three ACBs, such as 2-dodecylcyclobutanone, 2-(5′-tetradeceyl)cyclobutanone, and 2-tetradecylcyclobutanone, were identified only in the irradiated samples as radiation-induced markers.

Commercial packages of sliced–dried Pollack (64 kg) were gamma-irradiated at 5 kGy and transported along with the non-irradiated control from Korea to India via both air and ocean cargos. The samples received at the Bhabha Atomic Research Centre were stored at ambient temperature up to 6 months after irradiation and periodically examined for their qualities. Sensory evaluation by 35 panel members from the Central Institute of Fisheries Education, Mumbai, India showed that 60% of the panelists preferred irradiated Pollack to the non-irradiated one. Irradiated samples showed an acceptable quality over 6 months at ambient temperature from the hygienic and organoleptic point of view. Irradiated samples could be identified from the non-irradiated ones by the analysis of thermoluminescence and hydrocarbons. This trial proved the feasibility of inter-country transportation of irradiated dried Pollack.

Hydrocarbons induced by gamma-irradiation of green, black, and oolong teas were analyzed to determine whether the hydrocarbons can be used as markers for detecting post-irradiation of these teas. The samples were irradiated at 0, 2.5, 5, 7.5, and 10 kGy. Detection was attempted by extracting fat from the teas, separation of hydrocarbons with florisil column chromatography, and identification of hydrocarbons by gas chromatography-mass spectroscopy (GC-MS). Concentration of hydrocarbons increased with the irradiation dose. The major hydrocarbons in irradiated green, black, and oolong teas were 1-tetradecence (14:1), pentadecane (15:0), 1,7-hexadecadiene (16:2), 1-hexadecene (16:1), 8-heptadecene (17:1), and heptadecane (17:0). Radiation-induced hydrocarbons in teas were 1,7- hexadecadiene and 8-heptadecene. These compounds were not detected in non-irradiated samples, so the hydrocarbons (16:2, 17:1) can be used as markers for detecting post-irradiation of the teas. Furthermore, detection of hydrocarbons after 12 months storage at room temperature remains a suitable method for identifying irradiated teas.

mTOR complex 1 (mTORC1) is a multiprotein complex that integrates diverse signals including growth factors, nutrients, and stress to control cell growth. Raptor is an essential component of mTORC1 that functions to recruit specific substrates. Recently, Raptor was suggested to be a key target of regulation of mTORC1. Here, we show that Raptor is phosphorylated by JNK upon osmotic stress. We identified that osmotic stress induces the phosphorylation of Raptor at Ser-696, Thr-706, and Ser-863 using liquid chromatography-tandem mass spectrometry. We found that JNK is responsible for the phosphorylation. The inhibition of JNK abolishes the phosphorylation of Raptor induced by osmotic stress in cells. Furthermore, JNK physically associates with Raptor and phosphorylates Raptor in vitro, implying that JNK is responsible for the phosphorylation of Raptor. Finally, we found that osmotic stress activates mTORC1 kinase activity in a JNK-dependent manner. Our findings suggest that the molecular link between JNK and Raptor is a potential mechanism by which stress regulates the mTORC1 signaling pathway.

Phospholipase D (PLD) is involved in diverse cellular processes including cell movement, adhesion, and vesicle trafficking through cytoskeletal rearrangements. However, the mechanism by which PLD induces cytoskeletal reorganization is still not fully understood. Here, we describe a new link to cytoskeletal changes that is mediated by PLD2 through direct nucleotide exchange on RhoA. We found that PLD2 induces RhoA activation independent of its lipase activity. PLD2 directly interacted with RhoA, and the PX domain of PLD2 specifically recognized nucleotide-free RhoA. Finally, we found that the PX domain of PLD2 has guanine nucleotide-exchange factor (GEF) activity for RhoA in vitro. In addition, we verified that overexpression of the PLD2-PX domain induces RhoA activation, thereby provoking stress fiber formation. Together, our findings suggest that PLD2 functions as an upstream regulator of RhoA, which enables us to understand how PLD2 regulates cytoskeletal reorganization in a lipase activity-independent manner.