Jungyun Park

Transient Receptor Potential Cation Channel V1 (TRPV1) Is Degraded by Starvation- and Glucocorticoid-Mediated Autophagy

A mammalian cell renovates itself by autophagy, a process through which cellular components are recycled to produce energy and maintain homeostasis. Recently, the abundance of gap junction proteins was shown to be regulated by autophagy during starvation conditions, suggesting that transmembrane proteins are also regulated by autophagy. Transient receptor potential vanilloid type 1 (TRPV1), an ion channel localized to the plasma membrane and endoplasmic reticulum (ER), is a sensory transducer that is activated by a wide variety of exogenous and endogenous physical and chemical stimuli. Intriguingly, the abundance of cellular TRPV1 can change dynamically under pathological conditions. However, the mechanisms by which the protein levels of TRPV1 are regulated have not yet been explored. Therefore, we investigated the mechanisms of TRPV1 recycling using HeLa cells constitutively expressing TRPV1. Endogenous TRPV1 was degraded in starvation conditions; this degradation was blocked by chloroquine (CLQ), 3MA, or downregulation of Atg7. Interestingly, a glucocorticoid (cortisol) was capable of inducing autophagy in HeLa cells. Cortisol increased cellular conversion of LC3-I to LC-3II, leading autophagy and resulting in TRPV1 degradation, which was similarly inhibited by treatment with CLQ, 3MA, or downregulation of Atg7. Furthermore, cortisol treatment induced the colocalization of GFP-LC3 with endogenous TRPV1. Cumulatively, these observations provide evidence that degradation of TRPV1 is mediated by autophagy, and that this pathway can be enhanced by cortisol.

Clearance of Damaged Mitochondria Through PINK1 Stabilization by JNK and ERK MAPK Signaling in Chlorpyrifos-Treated Neuroblastoma Cells

Mitochondrial quality control and clearance of damaged mitochondria through mitophagy are important cellular activities. Studies have shown that PTEN-induced putative protein kinase 1 (PINK1) and Parkin play central roles in triggering mitophagy; however, little is known regarding the mechanism by which PINK1 modulates mitophagy in response to reactive oxygen species (ROS)-induced stress. In this study, chlorpyrifos (CPF)-induced ROS caused mitochondrial damage and subsequent engulfing of mitochondria in double-membrane autophagic vesicles, indicating that clearance of damaged mitochondria is due to mitophagy. CPF treatment resulted in PINK1 stabilization on the outer mitochondrial membrane and subsequently increased Parkin recruitment from the cytosol to the abnormal mitochondria. We found that PINK1 physically interacts with Parkin in the mitochondria of CPF-treated cells. Furthermore, a knockdown of PINK1 strongly inhibited the LC3-II protein level by blocking Parkin recruitment. This indicates that CPF-induced mitophagy is due to PINK1 stabilization in mitochondria. We observed that PINK1 stabilization was selectively regulated by ROS-mediated c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling activation but not p38 signaling. In the mitochondria of CPF-exposed cells, pretreatment with specific inhibitors of JNK and ERK1/2 significantly decreased PINK1 stabilization and Parkin recruitment and blocked the LC3-II protein level. Specifically, JNK and ERK1/2 inhibition also dramatically blocked the interaction between PINK1 and Parkin. Our results demonstrated that PINK1 regulation plays a critical role in CPF-induced mitophagy. The simple interpretation of these results is that JNK and ERK1/2 signaling regulates PINK1/Parkin-dependent mitophagy in the mitochondria of CPF-treated cells. Overall, this study proposes a novel molecular regulatory mechanism of PINK1 stabilization under CPF exposure.

Si wafer surface cleaning using laser-induced shock wave: a new dry cleaning methodology

It was investigated that a new dry cleaning methodology named laser shock cleaning could be applied to remove the particles from the wafer surface. The γ-alumina particles of 0.05 μm in diameter were deposited uniformly on bare Si wafer surface by aerosol spray method. The number of particles on wafer surface was measured by surface scanner before and after cleaning. It was found that most of the particles on the wafer surface were removed after dry shock cleaning using Nd:YAG laser. The average removal efficiency of the particles was over 90%, and the larger particles are easier to remove from the surface due to their smaller adhesion force. The removal efficiency of particles increased slightly as the particle size was to be larger.

Staufen1-mediated mRNA decay induces Requiem mRNA decay through binding of Staufen1 to the Requiem 3'UTR.

Requiem (REQ/DPF2) was originally identified as an apoptosis-inducing protein in mouse myeloid cells and belongs to the novel Krüppel-type zinc finger d4-protein family of proteins, which includes neuro-d4 (DPF1) and cer-d4 (DPF3). Interestingly, when a portion of the REQ messenger ribonucleic acid (mRNA) 3′ untranslated region (3′UTR), referred to as G8, was overexpressed in K562 cells, β-globin expression was induced, suggesting that the 3′UTR of REQ mRNA plays a physiological role. Here, we present evidence that the REQ mRNA 3′UTR, along with its trans-acting factor, Staufen1 (STAU1), is able to reduce the level of REQ mRNA via STAU1-mediated mRNA decay (SMD). By screening a complementary deoxyribonucleic acid (cDNA) expression library with an RNA–ligand binding assay, we identified STAU1 as an interactor of the REQ mRNA 3′UTR. Specifically, we provide evidence that STAU1 binds to putative 30-nucleotide stem–loop-structured RNA sequences within the G8 region, which we term the protein binding site core; this binding triggers the degradation of REQ mRNA and thus regulates translation. Furthermore, we demonstrate that siRNA-mediated silencing of either STAU1 or UPF1 increases the abundance of cellular REQ mRNA and, consequently, the REQ protein, indicating that REQ mRNA is a target of SMD.

Optimization of detection geometry for industrial SPECT by Monte Carlo simulations

The Korea Atomic Energy Research Institute (KAERI) has developed an industrial SPECT to investigate the fluid flow and mixing patterns in columns. It has been found that the industrial SPECT is indeed a very powerful tool to study the hydrodynamics in multiphase reactors. One of the practical issues in the development of industrial SPECTs is to achieve a required imaging resolution of an industrial SPECT with a minimum number of component detectors, the number of which is frequently limited by both the size of the detectors and the total cost of the imaging system. In the present study, a set of different geometries of industrial SPECTs were evaluated by Monte Carlo simulation using MCNPX to determine the minimum number of detectors that will provide a spatial resolution that corresponds to 10% of the cylindrical column diameter. Our results show that 11 and 12 detectors will satisfy the 10% resolution requirement for the 40 cm and 60 cm diameter columns, respectively, for the industrial SPECT and radioisotopes considered in the present study. The conclusion of this result is valid only for the case considered in the present study, but we believe that the same procedure can be applied to other industrial SPECTs for this kind of optimization.

Laser-induced shock wave removal of chemical-mechanical polishing slurries from silicon wafers

A new dry cleaning methodology named laser-induced shock cleaning has been applied to remove the chemical-mechanical polishing (CMP) slurries from silicon wafer surfaces. After CMP process using the slurries, the slurry particles should be removed from the surface in order to avoid the circuit failure and enhance the yield. The well-distributed remaining silica particles were attempted to remove from the surface by using laser-induced plasma shock waves. In order to evaluate the cleaning performance quantitatively, the number of particles on the wafer surfaces were measured by surface scanner before and after cleaning. It was found that most of the silica particles on the wafer surface were removed after the treatment of laser-induced shock waves. The average removal efficiency of the particles was 99% over. It was found that cleaning performance is strongly dependent on a gap distance between laser focus and the surface and a suitable control of the gap is crucial for the successful removal of the particles.

Insulin Signaling Augments eIF4E-Dependent Nonsense-Mediated mRNA Decay in Mammalian Cells.

Nonsense-mediated mRNA decay (NMD) modulates the level of mRNA harboring a premature termination codon (PTC) in a translation-dependent manner. Inhibition of translation is known to impair NMD; however, few studies have investigated the correlation between enhanced translation and increased NMD. Here, we demonstrate that insulin signaling events increase translation, leading to an increase in NMD of eIF4E-bound transcripts. We provide evidence that (i) insulin-mediated enhancement of translation augments NMD and rapamycin abrogates this enhancement; (ii) an increase in AKT phosphorylation due to inhibition of PTEN facilitates NMD; (iii) insulin stimulation increases the binding of up-frameshift factor 1 (UPF1), most likely to eIF4E-bound PTC-containing transcripts; and (iv) insulin stimulation induces the colocalization of UPF1 and eIF4E in processing bodies. These results illustrate how extracellular signaling promotes the removal of eIF4E-bound NMD targets.

Influence of void on image quality of industrial SPECT

Industrial single photon emission computed tomography (SPECT) is a promising technique to determine the dynamic behavior of industrial process media and has been developed in the Korea Atomic Energy Research Institute. The present study evaluated the influence of a void, which is presence in multiphase reactors of industrial process, on the image quality of an industrial SPECT. The results are very encouraging; that is, the performance of the industrial SPECT system is little influenced by the presence of a void, which means that industrial SPECT is an appropriate tool to estimate the dynamic characteristics of the process media in a water-air phase bubble column with a static gas sparger.

Lin28B and miR-142-3p regulate neuronal differentiation by modulating Staufen1 expression

Staufen1 (STAU1) and Lin28B are RNA-binding proteins that are involved in neuronal differentiation as a function of post-transcriptional regulation. STAU1 triggers post-transcriptional regulation, including mRNA export, mRNA relocation, translation and mRNA decay. Lin28B also has multiple functions in miRNA biogenesis and the regulation of translation. Here, we examined the connection between STAU1 and Lin28B and found that Lin28B regulates the abundance of STAU1 mRNA via miRNA maturation. Decreases in the expression of both STAU1 and Lin28B were observed during neuronal differentiation. Depletion of STAU1 or Lin28B inhibited neuronal differentiation, and overexpression of STAU1 or Lin28B enhanced neuronal differentiation. Interestingly, the stability of STAU1 mRNA was modulated by miR-142-3p, whose maturation was regulated by Lin28B. Thus, miR-142-3p expression increased as Lin28B expression decreased during differentiation, leading to the reduction of STAU1 expression. The transcriptome from Staufen-mediated mRNA decay (SMD) targets during differentiation was analyzed, confirming that STAU1 was a key factor in neuronal differentiation. In support of this finding, regulation of STAU1 expression in mouse neural precursor cells had the same effects on neuronal differentiation as it did in human neuroblastoma cells. These results revealed the collaboration of two RNA-binding proteins, STAU1 and Lin28B, as a regulatory mechanism in neuronal differentiation.

Simultaneous Determination of Multiple microRNA Levels Utilizing Biotinylated Dideoxynucleotides and Mass Spectrometry

MicroRNAs (miRNAs) are important regulators of gene translation and have been suggested as potent biomarkers in various disease states. In this study, we established an efficient method for simultaneous determination of multiple miRNA levels, employing the previously developed SPC-SBE (solid phase capture-single base extension) approach and MALDI-TOF mass spectrometry (MS). In this approach, we first perform reverse transcription of miRNAs extracted using stem-loop primers. Then the cDNA is co-amplified with competitors, synthetic oligonucleotides whose sequences precisely match cDNA except for one base, and the amplicons serve as templates for a multiplexed SBE reaction. Extension products are isolated using SPC and quantitatively analyzed with MALDI-TOF MS to determine multiple miRNA levels. Here we demonstrated concurrent analysis of four miRNA levels utilizing the approach. Furthermore, we showed the presented method significantly facilitated MS analysis of peak area ratio owing to SPC. The SPC process allowed effective removal of irrelevant reaction components prior to MS and promoted MS sample purification. Data obtained in this study was verified with RT-qPCR and agreement was shown on one order of magnitude scale, suggesting the SPC-SBE and MS approach has strong potential as a viable tool for high throughput miRNA analysis.