Large-Scale Proteomics and Phosphoproteomics of Urinary Exosomes

Normal human urine contains large numbers of exosomes, which are 40- to 100-nm vesicles that originate as the internal vesicles in multivesicular bodies from every renal epithelial cell type facing the urinary space. Here, we used LC-MS/MS to profile the proteome of human urinary exosomes. Overall, the analysis identified 1132 proteins unambiguously, including 177 that are represented on the Online Mendelian Inheritance in Man database of disease-related genes, suggesting that exosome analysis is a potential approach to discover urinary biomarkers. We extended the proteomic analysis to phosphoproteomic profiling using neutral loss scanning, and this yielded multiple novel phosphorylation sites, including serine-811 in the thiazide-sensitive Na-Cl co-transporter, NCC. To demonstrate the potential use of exosome analysis to identify a genetic renal disease, we carried out immunoblotting of exosomes from urine samples of patients with a clinical diagnosis of Bartter syndrome type I, showing an absence of the sodium-potassium-chloride co-transporter 2, NKCC2. The proteomic data are publicly accessible at http://dir.nhlbi.nih.gov/papers/lkem/exosome/.Urinary exosomes are small extracellular vesicles (<100 nm in diameter) that originate from the internal vesicles of multivesicular bodies (MVB) in renal epithelial cells, including glomerular podocytes, renal tubule cells, and the cells lining the urinary drainage system.¹ Exosomes are released into the urine when the outer membrane of the MVB fuses with the apical plasma membrane of the epithelial cell.Exosomes can be recovered from the urine by differential centrifugation as a low-density membrane fraction. Exosome isolation can result in marked enrichment of low-abundance urinary proteins that have potential pathophysiologic significance. As a consequence, we and others have been working to define optimal conditions for their isolation and purification as a prelude to their use in biomarker discovery studies.^1–3In this study, we thoroughly expanded the known proteome of human urinary exosomes by using a highly sensitive LC-MS/MS system, improved software for identification of peptide ions and a more elaborate data analysis strategy than in our previous study. In addition, we used a neutral loss scanning approach⁴ to investigate the phosphoproteome of human urinary exosomes. The study identified 1412 proteins including 14 phosphoproteins in human urinary exosomes. Overall, there are 177 proteins that are associated with diseases as judged by their presence on the Online Mendelian Inheritance in Man (OMIM) database, 34 of which are known to be associated with renal diseases. The potential clinical usefulness of urinary exosomes was demonstrated using the well-defined renal tubulopathy, Bartter syndrome type I, as an example. The rich information from the proteomic analysis also provides further insight into the biogenesis of urinary exosomes.     

Two-stage total knee arthroplasty for non-salvageable septic arthritis in diabetes mellitus patients

Pyogenic knee arthritis in a patient with advanced osteoarthritis is a serious medical problem. We have performed arthroscopic debridement in 136 patients with pyogenic knee arthritis from January 1999 to December 2001. Five of these patients were diabetic, they did not respond to the standard treatment protocol and they continued to have infection. For these patients, we performed open arthrotomy, with implantation of antibiotic cement as a spacer, and staged total knee arthroplasty. The clinical results were evaluated using the Hospital for Special Surgery (HSS) scoring system. At an average follow-up of 38 months (range: 29 to 46), the average pain score was 83 and the functional score was 73 with no patient having recurrence of the infection. This study shows that just as a 2-stage revision is now done for infected total knee arthroplasty, primary uncontrolled infected knees may be treated by a 2-stage arthroplasty as well.     

SPECIFIC IDENTIFICATION OF HERPES SIMPLEX VIRUS IN HUMAN ESOPHAGUS WITH RAPID IN SITU HYBRIDIZATION IN 5 CASES

HUMAN herpes simplex virus esophagitis （HSVE） was first reported in 1940 by Johnson. ^1HSVE usually occurs in immunocompromised patients,such as those with acquired immunodeficiency syndrome （AIDS）, 2-4 malignancies, cutaneous burns, connective tissue diseases, inflammatory bowel disease, those taking immuno-suppressive therapy, and those undergoing organ transplantation,5 etc. In the immunocompetent individuals, HSVE is rare, having been reported in 39 cases and mainly affecting young males^6,7 The aim of this study was to delineate the clinical experience in the diagnosis of HSVE using rapid in situ hybridization and assess the various detection methods.     

Comparison of the skin biophysical parameters of Southeast Asia females: forehead–cheek and ethnic groups

Background Regional and ethnic (racial) differences in skin properties are well known. However, regional and racial studies are limited and have studied skin properties using an insufficient number of subjects and limited ethnic groups, except in the case of some recent studies. Objective The aim of this study was (1) to compare the skin biophysical parameter among the large scale of Southeast Asia females group and (2) to compare skin properties of the forehead and cheek. Methods We measured and compared seven skin biophysical parameters, such as skin hydration, sebum, skin pH, melanin index, erythema index, skin elasticity and transepidermal water loss (TEWL), of the forehead and cheek of a large population of Indonesian (n = 200), Vietnamese (n = 100) and Singaporean females (n = 97). Results At the point of site difference, there were significant differences in five biophysical parameters (skin hydration, skin pH, melanin index, erythema index and skin elasticity) in Indonesian females. In Vietnamese and Singaporean women, they were significant differences in five biophysical parameters (skin hydration, skin pH, melanin index, erythema index and TEWL). At the point of ethnic difference, the Indonesian women had the highest skin pH and melanin index between the different ethnic groups. Vietnamese women had the highest skin hydration and TEWL in the forehead, whereas Singaporean women had the highest skin elasticity. Conclusion The skin biophysical parameters are different between the forehead and cheek among Southeast Asian females. It also reveals that the biophysical parameters are different in same racial group.     

Association of CD26 with CD45RA outside lipid rafts attenuates cord blood T-cell activation

CD26 is a T-cell activation antigen that contains dipeptidyl peptidase IV activity and binds adenosine deaminase. Recent work showed that specialized membrane microdomains, also known as lipid rafts, play a key role in T-cell signaling. In this study, we investigate the role of CD26 in cord blood T-cell activation and signal transduction. We demonstrated that different expression levels of CD26 were observed between cord blood T cells (CBTCs) and peripheral blood T cells (PBTCs) and that CD26(+)CD45RA(+) CBTCs were different compared with CD26(+)CD45RA(+) PBTCs. Moreover, the comitogenic effect of CD26 was not as pronounced in CBTCs as in PBTCs. We also showed that CD26 cross-linking induced less phosphorylation of T-cell receptor-signaling molecules, lymphoid T-cell protein tyrosine kinase (Lck), zeta-associated protein 70 (ZAP-70), T-cell receptor zeta (TCRzeta), and linker for activator of T cells (LAT) in CBTCs than in PBTCs. Furthermore, CD26 molecules associated with CD45RA molecules outside lipid rafts in CBTCs. Our results suggest that strong physical linkage of CD26 with CD45RA outside lipid rafts may be responsible for the attenuation of T-cell activation signaling through CD26, which may be responsible for immature immune response and the low incidence of severe graft-versus-host disease in cord blood transplantation.     

Photodynamic therapy with verteporfin for choroidal neovascularization in patients with angioid streaks

PURPOSE: To evaluate the functional and anatomic outcomes of photodynamic therapy (PDT) for choroidal neovascularization (CNV) in patients with angioid streaks. METHODS: The authors retrospectively evaluated 6 consecutive patients (6 eyes) with CNV secondary to angioid streaks. All patients were treated with standard PDT with verteporfin protocol. Standardized protocol refraction, visual acuity testing, ophthalmologic examination, color photographs, fluorescein angiograms and indocyanin angiograms were used to evaluate the results of PDT with verteporfin. Main outcome measures were visual acuity and CNV size. RESULTS: Their mean age was 61.3+/-5.50 years (range, 53-68 years). Follow-up time ranged from 12 to 38 months with mean of 20.5+/-10.91 months. The mean visual acuity at baseline was 20/100 (range 20/25-20/500), and the mean visual acuity at the last examination was 20/320(range 20/125-counting finger). The mean greatest linear dimension (GLD) at baseline was 2400+/-766.81 micrometer, and the mean GLD at the last examination was 3483+/-444.59 micrometer. CONCLUSIONS: PDT for CNV associated with angioid streaks seemed to slow down but not prevent the progression of the disease and associated visual loss.     

Evaluation of a deep learning-based computer-aided detection algorithm on chest radiographs: Case–control study

Along with recent developments in deep learning techniques, computer-aided diagnosis (CAD) has been growing rapidly in the medical imaging field. In this work, we evaluate the deep learning-based CAD algorithm (DCAD) for detecting and localizing 3 major thoracic abnormalities visible on chest radiographs (CR) and to compare the performance of physicians with and without the assistance of the algorithm. A subset of 244 subjects (60% abnormal CRs) was evaluated. Abnormal findings included mass/nodules (55%), consolidation (21%), and pneumothorax (24%). Observer performance tests were conducted to assess whether the performance of physicians could be enhanced with the algorithm. The area under the receiver operating characteristic (ROC) curve (AUC) and the area under the jackknife alternative free-response ROC (JAFROC) were measured to evaluate the performance of the algorithm and physicians in image classification and lesion detection, respectively. The AUCs for nodule/mass, consolidation, and pneumothorax were 0.9883, 1.000, and 0.9997, respectively. For the image classification, the overall AUC of the pooled physicians was 0.8679 without DCAD and 0.9112 with DCAD. Regarding lesion detection, the pooled observers exhibited a weighted JAFROC figure of merit (FOM) of 0.8426 without DCAD and 0.9112 with DCAD. DCAD for CRs could enhance physicians’ performance in the detection of 3 major thoracic abnormalities.     

Development of Diagnostic Reference Levels Using a Real-Time Radiation Dose Monitoring System at a Cardiovascular Center in Korea

Digital cardiovascular angiography accounts for a major portion of the radiation dose among the examinations performed at cardiovascular centres. However, dose-related information is neither monitored nor recorded systemically. This report concerns the construction of a radiation dose monitoring system based on digital imaging and communications in medicine (DICOM) data and its use at the cardiovascular centre of the University Hospitals in Korea. The dose information was analysed according to DICOM standards for a series of procedures, and the formulation of diagnostic reference levels (DRLs) at our cardiovascular centre represents the first of its kind in Korea. We determined a dose area product (DAP) DRL for coronary angiography of 75.6 Gy cm² and a fluoroscopic time DRL of 318.0 s. The DAP DRL for percutaneous transluminal coronary intervention was 213.3 Gy cm², and the DRL for fluoroscopic time was 1207.5 s.     

Randomized Comparison of Actual and Ideal Body Weight for Size Selection of the Laryngeal Mask Airway Classic in Overweight Patients

Size selection of the laryngeal mask airway (LMA) Classic based on actual body weight remains a common practice. However, ideal body weight might allow for a better size selection in obese patients. The purpose of our study was to compare the utility of ideal body weight and actual body weight when choosing the appropriate size of the LMA Classic by a randomized clinical trial. One hundred patients with age 20 to 70 yr, body mass index ≥25 kg/m², and the difference between LMA sizes based on actual weight and ideal weight were allocated to insert the LMA Classic using either actual body weight or ideal body weight in a weight-based formula for size selection. After insertion of the device, several variables including insertion parameters, sealing function, fiberoptic imaging, and complications were investigated. The insertion success rate at the first attempt was lower in the actual weight group (82%) than in the ideal weight group (96%), even it did not show significant difference. The ideal weight group had significantly shorter insertion time and easier placement. However, fiberoptic views were significantly better in the actual weight group. Intraoperative complications, sore throat in the recovery room, and dysphonia at postoperative 24 hr occurred significantly less often in the ideal weight group than in the actual weight group. It is suggested that the ideal body weight may be beneficial to the size selection of the LMA Classic in overweight patients (Clinical Trial Registry, {"type":"clinical-trial","attrs":{"text":"NCT 01843270","term_id":"NCT01843270"}}NCT 01843270).

Graphical Abstract

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