Dimerization of ß2-adrenergic receptor is responsible for the constitutive activity subjected to inverse agonism.

Dimerization of beta 2-adrenergic receptor (ß2-AR) has been observed across various physiologies. However, the function of dimeric ß2-AR is still elusive. Here, we revealed that dimerization of ß2-AR is responsible for the constitutive activity of ß2-AR generating inverse agonism. Using a co-immunoimmobilization assay, we found that transient ß2-AR dimers exist in a resting state, and the dimer was disrupted by the inverse agonists. A Gαs preferentially interacts with dimeric ß2-AR, but not monomeric ß2-AR, in a resting state, resulting in the production of a resting cAMP level. The formation of ß2-AR dimers requires cholesterol on the plasma membrane. The cholesterol did not interfere with the agonist-induced activation of monomeric ß2-AR, unlike the inverse agonists, implying that the cholesterol is a specific factor regulating the dimerization of ß2-AR. Our model not only shows the function of dimeric ß2-AR but also provides a molecular insight into the mechanism of the inverse agonism of ß2-AR.

Blue-conversion of organic dyes produces artifacts in multicolor fluorescence imaging.

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.

Improved resolution in single-molecule localization microscopy using QD-PAINT.

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.

Analysis of transient membrane protein interactions by single-molecule diffusional mobility shift assay.

Various repertoires of membrane protein interactions determine cellular responses to diverse environments around cells dynamically in space and time. Current assays, however, have limitations in unraveling these interactions in the physiological states in a living cell due to the lack of capability to probe the transient nature of these interactions on the crowded membrane. Here, we present a simple and robust assay that enables the investigation of transient protein interactions in living cells by using the single-molecule diffusional mobility shift assay (smDIMSA). Utilizing smDIMSA, we uncovered the interaction profile of EGFR with various membrane proteins and demonstrated the promiscuity of these interactions depending on the cancer cell line. The transient interaction profile obtained by smDIMSA will provide critical information to comprehend the crosstalk among various receptors on the plasma membrane.

The Prognosis of Glyphosate herbicide intoxicated patients according to their salt types

Purpose@#Glyphosate herbicide (GH) is a widely used herbicide and has been associated with significant mortality as poisoned cases increases. One of the reasons for high toxicity is thought to be toxic effect of its ingredient with glyphosate. This study was designed to determine differences in the clinical course with the salt-type contained in GH. @*Methods@#This was a retrospective study conducted at a single hospital between January 2013 and December 2017. We enrolled GH-poisoned patients visited the emergency department. According to salt-type, patients were divided into 4 groups: isopropylamine (IPA), ammonium (Am), potassium (Po), and mixed salts (Mi) groups. The demographics, laboratory variables, complications, and their mortality were analyzed to determine clinical differences associated with each salt-type. Addtionally, we subdivided patients into survivor and non-survivor groups for investigating predictive factors for the mortality. @*Results@#Total of 348 GH-poisoned patients were divided as follows: IPA 248, Am 41, Po 10, and Mi 49 patients. There was no difference in demographic or underlying disease history, but systolic blood pressure (SBP) was low in Po group. The ratio of intentional ingestion was higher in Po and Mi groups. Metabolic acidosis and relatively high lactate level were presented in Po group.As the primary outcome, the mortality rates were as follows: IPA, 26 (10.5%); Am, 2 (4.9%); Po, 1 (10%); and Mi, 1 (2%). There was no statistically significant difference in the mortality and the incidence of complications. Additionally, age, low SBP, low pH, corrected QT (QTc) prolongation, and respiratory failure requiring mechanical ventilation were analyzed as independent predictors for mortality in a regression analysis. @*Conclusion@#There was no statistical difference in their complications and the mortality across the GH-salt groups in this study.

Direct visualization of single-molecule membrane protein interactions in living cells.

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 (ß2-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.

Single particle tracking-based reaction progress kinetic analysis reveals a series of molecular mechanisms of cetuximab-induced EGFR processes in a single living cell.

Cellular processes occur through the orchestration of multi-step molecular reactions. Reaction progress kinetic analysis (RPKA) can provide the mechanistic details to elucidate the multi-step molecular reactions. However, current tools have limited ability to simultaneously monitor dynamic variations in multiple complex states at the single molecule level to apply RPKA in living cells. In this research, a single particle tracking-based reaction progress kinetic analysis (sptRPKA) was developed to simultaneously determine the kinetics of multiple states of protein complexes in the membrane of a single living cell. The subpopulation ratios of different states were quantitatively (and statistically) reliably extracted from the diffusion coefficient distribution rapidly acquired by single particle tracking at constant and high density over a long period of time using super-resolution microscopy. Using sptRPKA, a series of molecular mechanisms of epidermal growth factor receptor (EGFR) cellular processing induced by cetuximab were investigated. By comprehensively measuring the rate constants and cooperativity of the molecular reactions involving four EGFR complex states, a previously unknown intermediate state was identified that represents the rate limiting step responsible for the selectivity of cetuximab-induced EGFR endocytosis to cancer cells.

[Natural Transition Of Endemicity Of Malayan Flariasis In Inland Korea: Pattern Of Change In Microfilaria Rate Among Inhabitants Of Yongpung (Former Yongju) Area During The Period Of The Last Seven Years]

With a purpose to find out natural transition of endemicity of Malayan filariasis in inland Korea, a survey was conducted in June 1980 in Isan-Myeon of Yongpung-Gun (former Yongju-Gun) where an epidemiological investigation had been carried out in 1973 without any control activities such as chemotherapy. Five sample villages were surveyed for microfilaremia by 20 microliter night blood examination among inhabitants and the results of the surveys conducted in 1973 and 1980 were compared to determine natural transition of the endemicity of malayan filariasis during the period of the last 7 years. 1. The current microfilaria rate among inhabitants in the 5 villages was 2.2 % on the average (male: 1.6 %, female: 2.8 %) from 370 persons examined. By village, the rates were 5.9 % (number of persons examined: 34) in Baranggol, 0 % (30) in Guitonggl, 4.2 % (72) in Alseonggol, 0 %(65) in Jangjagol and 1.8 % (169) in Saehae. 2. Extremely low microfilaria rate was noted in young age groups. By age group, no positive case was found in those age groups below 30~39 years except 10~14 age group in which 2 positives (4.4 %) were found. Two positives each were found in the respective age groups of 40~49(3.2 %), 50~59 (4.4 %) and over 60 (3.4 %). 3. In evaluation of the natural transition of the endemicity during the period of the last 7 years, the microfilaria rate turned out from 13.1 % in 1973 to 2.2 % in 1980. The difference in the microfilaria rate was 10.9 % and the natural reduction rate per year was 1.6 % on the average. 4. From the examination of 35 cases which had revealed microfilaremia 7 years ago, 85.7 %(30) of them were found to have converted to microfilaria negatives. On the other hand, from the 151 cases which had revealed no microfilaria in 1973, only 0.7 % (1) of them was found to have converted to microfilaria positive. 5. In the intensity of microfilaremia, the number of microfilaria/20 microliter blood per positive case was 11.0 in 1973 and 9.1 in 1980. The number of microfilaria/20 microliter per examinee was 1.4 in 1973 and 0.2 in 1980, thus reduced to 1/7 during the period of the 7 years. 6. The retarding endemicity of malayan filariasis in inland Korea was considered to be resulted in by the gradual increase of environmental factors in relation to ecology of vector mosquitoes, which adversely affect to the transmission of malayan filariasis. Followings are suggested to be the factors which control the transmission of the malayan filariasis in this area: 1) Inhabitants are the only natural final host of Brugia malayi infection in this area, 2) Gradual elevation of living standards of the inhabitants, 3) Gradual awakening of consciousness and behavior among inhabitants to protect themselves from mosquito biting using such as mosquito nets and insecticide sprays, 4) Preference of animal bait of vector mosquitoes, Anopheles sinensis, 5) Increase in number of domestic animals and fowls being raised in the village areas which play a major part of blood donors to vector mosquitoes, and 6) Relatively short (3~4 months) period of mosquito season in a year.