Dynamic association of proteasomal machinery with the centrosome

Although the number of pathologies known to arise from the inappropriate folding of proteins continues to grow, mechanisms underlying the recognition and ultimate disposition of misfolded polypeptides remain obscure. For example, how and where such substrates are identified and processed is unknown. We report here the identification of a specific subcellular structure in which, under basal conditions, the 20S proteasome, the PA700 and PA28 (700- and 180-kD proteasome activator complexes, respectively), ubiquitin, Hsp70 and Hsp90 (70- and 90-kD heat shock protein, respectively) concentrate in HEK 293 and HeLa cells. The structure is perinuclear, surrounded by endoplasmic reticulum, adjacent to the Golgi, and colocalizes with gamma-tubulin, an established centrosomal marker. Density gradient fractions containing purified centrosomes are enriched in proteasomal components and cell stress chaperones. The centrosome-associated structure enlarges in response to inhibition of proteasome activity and the level of misfolded proteins. For example, folding mutants of CFTR form large inclusions which arise from the centrosome upon inhibition of proteasome activity. At high levels of misfolded protein, the structure not only expands but also extensively recruits the cytosolic pools of ubiquitin, Hsp70, PA700, PA28, and the 20S proteasome. Thus, the centrosome may act as a scaffold, which concentrates and recruits the systems which act as censors and modulators of the balance between folding, aggregation, and degradation.ope

Cystic fibrosis transmembrane conductance regulator regulates luminal Cl-/HCO3- exchange in mouse submandibular and pancreatic ducts

We have demonstrated previously the regulation of Cl-/HCO3- exchange activity by the cystic fibrosis transmembrane conductance regulator (CFTR) in model systems of cells stably or transiently transfected with CFTR (Lee, M. G., Wigley, W. C., Zeng, W., Noel, L. E., Marino, C. R., Thomas, P. J., and Muallem, S. (1999) J. Biol. Chem. 274, 3414-3421). In the present work we examine the significance of this regulation in cells naturally expressing CFTR. These include the human colonic T84 cell line and the mouse submandibular gland and pancreatic ducts, tissues that express high levels of CFTR in the luminal membrane. As in heterologous expression systems, stimulation of T84 cells with forskolin increased the Cl-/HCO3- exchange activity independently of CFTR Cl- channel activity. Freshly isolated submandibular gland ducts from wild type mice showed variable Cl-/HCO3- exchange activity. Measurement of [Cl-]i revealed that this was largely the result of variable steady-state [Cl-]i. Membrane depolarization with 5 mM Ba2+ or 100 mM K+ increased and stabilized [Cl-]i. Under depolarized conditions wild type and DeltaF/DeltaF mice had comparable basal Cl-/HCO3- exchange activity. Notably, stimulation with forskolin increased Cl-/HCO3- exchange activity in submandibular gland ducts from wild type but not DeltaF/DeltaF mice. Microperfusion of the main pancreatic duct showed Cl-/HCO3- exchange activity in both the basolateral and luminal membranes. Stimulation of ducts from wild type animals with forskolin had no effect on basolateral but markedly stimulated luminal Cl-/HCO3- exchange activity. By contrast, forskolin had no effect on either basolateral or luminal Cl-/HCO3- exchange activity of ducts from DeltaF/DeltaF animals. We conclude that CFTR regulates luminal Cl-/HCO3- exchange activity in CFTR-expressing cells, and we discuss the possible physiological significance of these findings regarding cystic fibrosis.ope

자동차 사양 변경을 실시간 반영하는 데이터 기반 디자인 접근 방법

학위논문 (박사) -- 서울대학교 대학원 : 융합과학기술대학원 융합과학부(지능형융합시스템전공), 2020. 8. 곽노준.The automotive industry is entering a new phase in response to changes in the external environment through the expansion of eco-friendly electric/hydrogen vehicles and the simplification of modules during the manufacturing process. However, in the existing automotive industry, conflicts between structured production guidelines and various stake-holders, who are aligned with periodic production plans, can be problematic. For example, if there is a sudden need to change either production parts or situation-specific designs, it is often difficult for designers to reflect those requirements within the preexisting guidelines. Automotive design includes comprehensive processes that represent the philosophy and ideology of a vehicle, and seeks to derive maximum value from the vehicle specifications. In this study, a system that displays information on parts/module components necessary for real-time design was proposed. Designers will be able to use this system in automotive design processes, based on data from various sources. By applying the system, three channels of information provision were established. These channels will aid in the replacement of specific component parts if an unexpected external problem occurs during the design process, and will help in understanding and using the components in advance. The first approach is to visualize real-time data aggregation in automobile factories using Google Analytics, and to reflect these in self-growing characters to be provided to designers. Through this, it is possible to check production and quality status data in real time without the use of complicated labor resources such as command centers. The second approach is to configure the data flow to be able to recognize and analyze the surrounding situation. This is done by applying the vehicles camera to the CCTV in the inventory and distribution center, as well as the direction inside the vehicle. Therefore, it is possible to identify and record the parts resources and real-time delivery status from the internal camera function without hesitation from existing stakeholders. The final approach is to supply real-time databases of vehicle parts at the site of an accident for on-site repair, using a public API and sensor-based IoT. This allows the designer to obtain information on the behavior of parts to be replaced after accidents involving light contact, so that it can be reflected in the design of the vehicle. The advantage of using these three information channels is that designers can accurately understand and reflect the modules and components that are brought in during the automotive design process. In order to easily compose the interface for the purpose of providing information, the information coming from the three channels is displayed in their respective, case-specific color in the CAD software that designers use in the automobile development process. Its eye tracking usability evaluation makes it easy for business designers to use as well. The improved evaluation process including usability test is also included in this study. The impact of the research is both dashboard application and CAD system as well as data systems from case studies are currently reflected to the design ecosystem of the motors group.자동차 산업은 친환경 전기/수소 자동차의 확대와 제조 공정에서의 모듈 단순화를 통해서 외부 환경의 변화에 따른 새로운 국면을 맞이하고 있다. 하지만 기존의 자동차 산업에서 구조화된 생산 가이드라인과 기간 단위 생산 계획에 맞춰진 여러 이해관계자들과의 갈등은 변화에 대응하는 방안이 관성과 부딪히는 문제로 나타날 수 있다. 예를 들어, 갑작스럽게 생산에 필요한 부품을 변경해야 하거나 특정 상황에 적용되는 디자인을 변경할 경우, 주어진 가이드라인에 따라 디자이너가 직접 의견을 반영하기 어려운 경우가 많다. 자동차 디자인은 차종의 철학과 이념을 나타내고 해당 차량제원으로 최대의 가치를 끌어내고자 하는 종합적인 과정이다. 본 연구에서는 여러 원천의 데이터를 기반으로 자동차 디자인 과정에서 활용할 수 있도록 디자인에 필요한 부품/모듈 구성요소들에 대한 정보를 실시간으로 표시해주는 시스템을 고안하였다. 이를 적용하여 자동차 디자인 과정에서 예상 못한 외부 문제가 발생했을 때 선택할 구성 부품을 대체하거나 사전에 해당 부품을 이해하고 디자인에 활용할 수 있도록 세 가지 정보 제공 채널을 구성하였다. 첫 번째는 자동차 공장 내 실시간 데이터 집계를 Google Analytics를 활용하여 시각화하고, 이를 공장 자체의 자가 성장 캐릭터에 반영하여 디자이너에게 제공하는 방식이다. 이를 통해 종합상황실 등의 복잡한 인력 체계 없이도 생산 및 품질 현황 데이터를 실시간으로 확인 가능하도록 하였다. 두 번째는 차량용 주차보조 센서 카메라를 차량 부착 뿐만 아니라 인벤토리와 물류센터의 CCTV에도 적용하여 주변상황을 인식하고 분석할 수 있도록 구성하였다. 차량의 조립 생산 단계에서 부품 단위의 이동, 운송, 출하를 거쳐 완성차의 주행 단계에 이르기까지 데이터 흐름을 파악하는 것이 디자인 부문에 필요한 정보를 제공할 수 있는 방법으로 활용되었다. 이를 통해 기존 이해관계자들의 큰 반발 없이 내부의 카메라 기능으로부터 부품 리소스와 운송 상태를 실시간 파악 및 기록 가능하도록 하였다. 마지막으로 공공 API와 센서 기반의 사물인터넷을 활용해서 도로 위 차량 사고가 발생한 위치에서의 현장 수리를 위한 차량 부품 즉시 수급 및 데이터베이스화 방법도 개발 되었다. 이는 디자이너로 하여금 가벼운 접촉 사고에서의 부품 교체 행태에 대한 정보를 얻게 하여 차량의 디자인에 반영 가능하도록 하였다. 시나리오를 바탕으로 이 세 가지 정보 제공 채널을 활용할 경우, 자동차 디자인 과정에서 불러들여오는 부품 및 모듈의 구성 요소들을 디자이너가 정확히 알고 반영할 수 있다는 장점이 부각되었다. 정보 제공의 인터페이스를 쉽게 구성하기 위해서, 실제로 디자이너들이 자동차 개발 과정에서 디자인 프로세스 상에서 활용하는 CAD software에 세 가지 채널들로부터 들어오는 정보를 사례별 컬러로 표시하고, 이를 시선추적 사용성 평가를 통해 현업 디자이너들이 사용하기 쉽게 개선한 과정도 본 연구에 포함시켜 설명하였다.1 Introduction 1 1.1 Research Background 1 1.2 Objective and Scope 2 1.3 Environmental Changes 3 1.4 Research Method 3 1.4.1 Causal Inference with Graphical Model 3 1.4.2 Design Thinking Methodology with Co-Evolution 4 1.4.3 Required Resources 4 1.5 Research Flow 4 2 Data-driven Design 7 2.1 Big Data and Data Management 6 2.1.1 Artificial Intelligence and Data Economy 6 2.1.2 API (Application Programming Interface) 7 2.1.3 AI driven Data Management for Designer 7 2.2 Datatype from Automotive Industry 8 2.2.1 Data-driven Management in Automotive Industry 8 2.2.2 Automotive Parts Case Studies 8 2.2.3 Parameter for Generative Design 9 2.3 Examples of Data-driven Design 9 2.3.1 Responsive-reactive 9 2.3.2 Dynamic Document Design 9 2.3.3 Insignts from Data-driven Design 10 3 Benchmark of Data-driven Automotive Design 12 3.1 Method of Global Benchmarking 11 3.2 Automotive Design 11 3.2.1 HMI Design and UI/UX 11 3.2.2 Hardware Design 12 3.2.3 Software Design 12 3.2.4 Convergence Design Process Model 13 3.3 Component Design Management 14 4 Vehicle Specification Design in Mobility Industry 16 4.1 Definition of Vehicle Specification 16 4.2 Field Study 17 4.3 Hypothesis 18 5 Three Preliminary Practical Case Studies for Vehicle Specification to Datadriven 21 5.1 Production Level 31 5.1.1 Background and Input 31 5.1.2 Data Process from Inventory to Designer 41 5.1.3 Output to Designer 51 5.2 Delivery Level 61 5.2.1 Background and Input 61 5.2.2 Data Process from Inventory to Designer 71 5.2.3 Output to Designer 81 5.3 Consumer Level 91 5.3.1 Background and Input 91 5.3.2 Data Process from Inventory to Designer 101 5.3.3 Output to Designer 111 6 Two Applications for Vehicle Designer 86 6.1 Real-time Dashboard DB for Decision Making 123 6.1.1 Searchable Infographic as a Designer's Tool 123 6.1.2 Scope and Method 123 6.1.3 Implementation 123 6.1.4 Result 124 6.1.5 Evaluation 124 6.1.6 Summary 124 6.2 Application to CAD for vehicle designer 124 6.2.1 CAD as a Designer's Tool 124 6.2.2 Scope and Method 125 6.2.3 Implementation and the Display of the CAD Software 125 6.2.4 Result 125 6.2.5 Evaluation: Usability Test with Eyetracking 126 6.2.6 Summary 128 7 Conclusion 96 7.1 Summary of Case Studies and Application Release 129 7.2 Impact of the Research 130 7.3 Further Study 131Docto

Multicenter Surveillance of Cystic Fibrosis in Korean Children

Purpose: Cystic fibrosis (CF), caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, is rare among non-Caucasians. We aimed to identify the clinical features and CFTR mutations in Korean children. Methods: We included 18 pediatric patients with CF diagnosed using sweat chloride test or genetic analysis for 30 years. HEK293 cells were transfected with wild-type CFTR, ΔF508-CFTR, and L441P-CFTR mutant plasmids for 24 hours and treated with CFTR correctors (VX809 and VX661). Results: The median age at diagnosis was 9.2 years. Eleven patients had growth retardation, and 6 had a respiratory failure at diagnosis. Genetic analysis was used for all patients, while sweat testing was for 8 patients. At diagnosis, the median z scores of forced expiratory volume in one second (FEV1), FEV1/forced vital capacity, and forced expiratory flow at 25%-75% of forced vital capacity were -3.61 (-5.78, 1.78), -3.38 (-4.40, -0.60), and -4.45 (-5.78, 0.54), respectively. Two patients were treated with dornase alfa and only one with CFTR modulator. Patients were followed up for 3.7 years as a median. Four patients died at 10.6 years, with 4.2 years of post-diagnosis survival. The most common mutation was exon 16-17b deletion (19.4%). Among 11 single nucleotide variants, c.1322T>C (p.Leu441Pro, L441P) was detected in 4 patients. In the functional assay, L441P-CFTR correction was well restored by CFTR correctors compared with ΔF508. Conclusions: CF is extremely rare in Korean children and is caused by different mutations from those commonly observed in Caucasians. Early diagnosis and treatment availability may improve outcomes. CFTR modulators may be effective for Asian patients with rare CFTR mutations, c.1322T>C (p.Leu441Pro).ope

Dysfunctional cerebellar Purkinje cells contribute to autism-like behaviour in Shank2-deficient mice

Loss-of-function mutations in the gene encoding the postsynaptic scaffolding protein SHANK2 are a highly penetrant cause of autism spectrum disorders (ASD) involving cerebellum-related motor problems. Recent studies have implicated cerebellar pathology in the aetiology of ASD. Here we evaluate the possibility that cerebellar Purkinje cells (PCs) represent a critical locus of ASD-like pathophysiology in mice lacking Shank2. Absence of Shank2 impairs both PC intrinsic plasticity and induction of long-term potentiation at the parallel fibre to PC synapse. Moreover, inhibitory input onto PCs is significantly enhanced, most prominently in the posterior lobe where simple spike (SS) regularity is most affected. Using PC-specific Shank2 knockouts, we replicate alterations of SS regularity in vivo and establish cerebellar dependence of ASD-like behavioural phenotypes in motor learning and social interaction. These data highlight the importance of Shank2 for PC function, and support a model by which cerebellar pathology is prominent in certain forms of ASD.ope

Novel amiloride-sensitive sodium-dependent proton secretion in the mouse proximal convoluted tubule.

The proximal convoluted tubule (PCT) reabsorbs most of the filtered bicarbonate. Proton secretion is believed to be mediated predominantly by an apical membrane Na(+)/H(+) exchanger (NHE). Several NHE isoforms have been cloned, but only NHE3 and NHE2 are known to be present on the apical membrane of the PCT. Here we examined apical membrane PCT sodium-dependent proton secretion of wild-type (NHE3(+/+)/NHE2(+/+)), NHE3(-/-), NHE2(-/-), and double-knockout NHE3(-/-)/NHE2(-/-) mice to determine their relative contribution to luminal proton secretion. NHE2(-/-) and wild-type mice had comparable rates of sodium-dependent proton secretion. Sodium-dependent proton secretion in NHE3(-/-) mice was approximately 50% that of wild-type mice. The residual sodium-dependent proton secretion was inhibited by 100 microM 5-(N-ethyl-N-isopropyl) amiloride (EIPA). Luminal sodium-dependent proton secretion was the same in NHE3(-/-)/NHE2(-/-) as in NHE3(-/-) mice. These data point to a previously unrecognized Na(+)-dependent EIPA-sensitive proton secretory mechanism in the proximal tubule that may play an important role in acid-base homeostasis.ope

Na+-dependent transporters mediate HCO3-salvage across the luminal membrane of the main pancreatic duct

To study the roles of Na+-dependent H+ transporters, we characterized H+ efflux mechanisms in the pancreatic duct in wild-type, NHE2–/–, and NHE3–/– mice. The pancreatic duct expresses NHE1 in the basolateral membrane, and NHE2 and NHE3 in the luminal membrane, but does not contain NHE4 or NHE5. Basolateral Na+-dependent H+ efflux in the microperfused duct was inhibited by 1.5 μM of the amiloride analogue HOE 694, consistent with expression of NHE1, whereas the luminal activity required 50 μM HOE 694 for effective inhibition, suggesting that the efflux might be mediated by NHE2. However, disruption of NHE2 had no effect on luminal transport, while disruption of the NHE3 gene reduced luminal Na+-dependent H+ efflux by ∼45%. Notably, the remaining luminal Na+-dependent H+ efflux in ducts from NHE3–/– mice was inhibited by 50 μM HOE 694. Hence, ∼55% of luminal H+ efflux (or HCO3– influx) in the pancreatic duct is mediated by a novel, HOE 694–sensitive, Na+-dependent mechanism. H+ transport by NHE3 and the novel transporter is inhibited by cAMP, albeit to different extents. We propose that multiple Na+-dependent mechanisms in the luminal membrane of the pancreatic duct absorb Na+ and HCO3– to produce a pancreatic juice that is poor in HCO3– and rich in Cl– during basal secretion. Inhibition of the transporters during stimulated secretion aids in producing the HCO3–-rich pancreatic juice.ope

Effect of Slc26a6 deletion on apical Cl−/HCO3− exchanger activity and cAMP-stimulated bicarbonate secretion in pancreatic duct

The role of Slc26a6 (PAT1) on apical Cl-/HCO3- exchange and bicarbonate secretion in pancreatic duct cells was investigated using Slc26a6 null and wild-type (WT) mice. Apical Cl-/HCO3- exchange activity was measured with the pH-sensitive dye BCECF in microperfused interlobular ducts. The HCO3(-)-influx mode of apical [Cl-]i/[HCO3-]o exchange (where brackets denote concentration and subscripts i and o denote intra- and extracellular, respectively) was dramatically upregulated in Slc26a6 null mice (P 0.05). In addition, when pancreatic juice was collected from whole animal in basal and secretin-stimulated conditions, neither juice volume nor its pH showed differences between WT and Slc26a6 null mice. Semiquantitative RT-PCR demonstrated more than fivefold upregulation in Slc26a3 (DRA) expression in Slc26a6 knockout pancreas. In conclusion, these results point to the role of Slc26a6 in HCO3- efflux at the apical membrane and also suggest the presence of a robust Slc26a3 compensatory upregulation, which can replace the function of Slc26a6 in pancreatic ducts.ope

Hippocampus-Dependent Goal Localization by Head-Fixed Mice in Virtual Reality

The demonstration of the ability of rodents to navigate in virtual reality (VR) has made it an important behavioral paradigm for studying spatially modulated neuronal activity in these animals. However, their behavior in such simulated environments remains poorly understood. Here, we show that encoding and retrieval of goal location memory in mice head-fixed in VR depends on the postsynaptic scaffolding protein Shank2 and the dorsal hippocampus. In our newly developed virtual cued goal location task, a head-fixed mouse moves from one end of a virtual linear track to seek rewards given at a target location along the track. The mouse needs to visually recognize the target location and stay there for a short period of time to receive the reward. Transient pharmacological blockade of fast glutamatergic synaptic transmission in the dorsal hippocampus dramatically and reversibly impaired performance of this task. Encoding and updating of virtual cued goal location memory was impaired in mice deficient in the postsynaptic scaffolding protein Shank2, a mouse model of autism that exhibits impaired spatial learning in a real environment. These results highlight the crucial roles of the dorsal hippocampus and postsynaptic protein complexes in spatial learning and navigation in VRope

An Investigation of Causal Relationships among Skin SCUBA Participation Motivation, Enjoyment and Life Satisfaction in College Students

This study is to investigate the effects of participation motivation and enjoyment factors on life satisfaction in college students participating in skin-scuba classes and to provide the practical research data for the development of the proper operation of skin-scuba classes for the leisure life which the university students may lack. For these purposes, technical statistics between questions were disposed in order to grasp the average and standard deviation on each variables, exploratory factor analysis and reliability analysis were conducted for the reliability and validity of the scale, and correlation analysis and multiple regression analysis were conducted to test the casuality of amusement factor, the motive of participation in skin-scuba class and the satisfaction with university life as the research methods. Research findings showed that the amusement factor and the motive of participation in skin-scuba class act as the important variables in the satisfaction with university life and it was predicted that the motive of participation in skin-scuba class and the amusement factor had interrelation with parameter. Therefore, a variety of programs reflecting the amusement factors should motivate the university students to participate in the skin-scuba class to increase the satisfaction with university life and to encourage the re-participation of students.1. 서 론 1.1 연구의 필요성 및 목적 1 1.2 연구가설 4 1.3 연구의 제한점 4 1.4 용어의 정의 5 2. 이론적 배경 2.1 스킨스쿠버 6 2.2 참여동기 8 2.3 재미요인 12 2.4 생활만족도 15 2.5 선행연구의 고찰 20 3. 연구방법 3.1 연구모형 21 3.2 연구대상 22 3.3 연구도구 24 3.4 연구절차 29 3.5 자료 분석 30 4. 연구결과 4.1 대학생의 기초배경변인에 따른 참여동기와 재미요인 및 대학생활 만족도의 비교 31 4.2 대학생의 스킨스쿠버 참여동기와 재미요인 및 대학생활 만족도의 인과관계 검증 36 5. 논 의 5.1 기초배경 특성(성별, 인증증 레벨, 다이빙 활동참여 빈도)에 따른 참여동기, 재미요인 및 대학생활 만족도의 차이 54 5.2 대학생의 스킨스쿠버 참여동기와 재미요인 및 대학생활 만족도의 인과관계 58 6. 결 론 6.1 결론 62 6.2 제언 63 참고문헌 64 부록 A 7