Multiple-Cycle Polymeric Extracellular Vesicle Precipitation and Its Evaluation by Targeted Mass Spectrometry.

The multiple roles of extracellular vesicles (EVs) in pathogenesis have received much attention, as they are valuable as diagnostic and prognostic biomarkers. We employed polymeric EV precipitation to isolate EVs from clinical specimens to overcome the limitations of ultracentrifugation (UC), such as low protein yields, a large volume of specimens used, and time requirements. Multiple-cycle polymeric EV precipitation was applied to enhance the purity of the EV fractions with a small sample volume. Then, the purity was assessed using a multiple reaction monitoring (MRM) panel consisting of alpha-2-macroglobulin (A2M), thrombospondin 1 (THBS 1), galectin 3 binding protein (LGALS3BP), and serum albumin (ALB). For purity evaluation, the EV fractions isolated from blood specimens were subjected to shotgun proteomics and MRM-based targeted proteomics analyses. We demonstrate that the modified method is an easy and convenient method compared with UC. In the shotgun proteomics analysis, the proteome profile of EV fraction contains 89% EV-related proteins by matching with the EVpedia database. In conclusion, we suggest that multiple-cycle polymeric EV precipitation is not only a more effective method for EV isolation for further proteomics-based experiments, but may also be useful for further clinical applications due to the higher EV yield and low sample requirements.

Effects of Electric Cortical Stimulation (ECS) and Transcranial Direct Current Stimulation (tDCS) on Rats With a Traumatic Brain Injury.

OBJECTIVE: To evaluate the effects of electric cortical stimulation (ECS) and transcranial direct current stimulation (tDCS) on motor and cognitive function recovery and brain plasticity in focal traumatic brain injury (TBI) of rats model. METHODS: Forty rats were pre-trained to perform a single pellet reaching task (SPRT), rotarod test (RRT), and Y-maze test for 14 days, then a focal TBI was induced by a weight drop model on the motor cortex. All rats were randomly assigned to one of the three groups: anodal ECS (50 Hz and 194 µs) (ECS group), tDCS (0.1 mA, 50 Hz and 200 µs) (tDCS group), and no stimulation as a control group. Four-week stimulation, including rehabilitation, was started 3 days after the operation. SPRT, RRT, and Y-maze were measured from day 1 to day 28 after the TBI was induced. Histopathological and immunohistochemistry staining evaluations were performed at 4 weeks. RESULTS: SPRT was improved from day 7 to day 26 in ECS, and from day 8 to day 26 in tDCS compared to the control group (p<0.05). SPRT of ECS group was significantly improved on days 3, 8, 9, and 17 compared to the tDCS group. Y-maze was improved from day 8 to day 16 in ECS, and on days 6, 12, and 16 in the tDCS group compared to the control group (p<0.05). Y-maze of the ECS group was significantly improved on day 9 to day 15 compared to the tDCS group. The c-Fos protein expression was better in the ECS group and the tDCS group compared to the control group. CONCLUSION: Electric stimulation in rats modified with a focal TBI is effective for motor recovery and brain plasticity. ECS induced faster behavioral and cognitive improvements compared to tDCS during the recovery period of rats with a focal TBI.

Development of elbow spasticity model for objective training of spasticity assessment of patients post stroke.

Reliable assessment is essential for the management of spasticity, one of the most frequent complication of various neurological diseases. For the spasticity assessment, several clinical tools have been developed and widely used in clinics. The most popular one is modified Ashworth scale (MAS). It has a simple protocol, but is subjective and qualitative. To improve its reliability, quantitative measurement and consistent training would be needed. This study presents an elbow spasticity simulator which mimics spastic response of adult post stroke survivors. First, spastic responses (i.e. resistance and joint motion) from patients with a stroke were measured during conventional MAS assessment. Each grade of MAS was quantified by using three parameters representing three characteristics of the spasticity. Based on the parameters, haptic models of MAS were developed for implementing repeatable and consistent haptic training of novice clinicians. Two experienced clinicians participated in preliminary evaluation of the models.

Urinary profiling of tryptophan and its related metabolites in patients with metabolic syndrome by liquid chromatography-electrospray ionization/mass spectrometry.

Tryptophan (Trp) is an essential amino acid that plays an important role in protein synthesis and is a precursor of various substances related to diverse biological functions. An imbalance in Trp metabolites is associated with inflammatory diseases. The accurate and precise measurement of these compounds in biological specimens would provide meaningful information for understanding the biochemical states of various metabolic syndrome-related diseases, such as hyperlipidemia, hypertension, diabetes, and obesity. In this study, we developed a rapid, accurate, and sensitive liquid chromatography-tandem mass spectrometry-based method for the simultaneous targeted analysis of Trp and its related metabolites of the kynurenine (Kyn), serotonin, and tryptamine pathways in urine. The application of the developed method was tested using urine samples after protein precipitation. The detection limits of Trp and its metabolites were in the range of 0.01 to 0.1 µg/mL. The method was successfully validated and applied to urine samples from controls and patients with metabolic syndrome. Our results revealed high concentrations of Kyn, kynurenic acid, xanthurenic acid, and quinolinic acid as well as a high Kyn-to-Trp ratio (KTR) in patients with metabolic syndromes. The levels of urine Kyn and KTR were significantly increased in patients under 60 years old. The profiling of urinary Trp metabolites could be a useful indicator for age-related diseases including metabolic syndrome. ᅟ.

Pheophorbide a from Capsosiphon fulvescens Inhibits Advanced Glycation End Products Mediated Endothelial Dysfunction.

During hyperglycemia, the first step toward the formation of advanced glycation end products is the nonenzymatic glycation between the carbonyl group of a sugar and the primary amino group of a protein. Advanced glycation end products are then produced through more complex reactions. Reactive oxygen species derived from advanced glycation end products may play a key role in inflammation of the endothelium, leading to the complications seen in diabetes. Glycolaldehyde-induced advanced glycation end products have been reported to express proinflammatory cytokines, such as tumor necrosis factor-α and interleukin-1ß. This study focused on Capsosiphon fulvescens, a Capsosiphonaceae type of green algae that has shown potential as a functional food material. Pheophorbide a, an anti-glycation compound, was isolated from C. fulvescens by extraction using a mixture of ethanol and water, followed by column fractionation of the resulting extract. The compound separated from C. fulvescens was identified by means of high-performance liquid chromatography combined with mass spectrometry. Pheophorbide a showed scavenging activity of the intracellular reactive oxygen species as well as monocyte adhesiveness inhibitory activity on the human myelomonocytic cell line (THP-1) and human umbilical vein endothelial cells cocultivation system. The mRNA levels of inflammation-related genes such as monocyte chemoattractant protein-1 and interleukin-6 were significantly decreased by pheophorbide a, and advanced glycation end products-stimulated tumor necrosis factor-α and interleukin-1ß were downregulated as well. These results indicate that pheophorbide a has significant reactive oxygen species-scavenging activity, monocyte adhesive inhibitory activity, and downregulatory activity of cytokines related to inflammation affecting the endothelium. Pheophorbide a could therefore be a promising candidate for modulating endothelial cell dysfunction.

Effect of pneumatic compressing powered orthosis in stroke patients: preliminary study.

OBJECTIVE: To evaluate the feasibility and effectiveness of a knee-ankle-foot orthosis powered by artificial pneumatic muscles (PKAFO). METHODS: Twenty-three hemiplegic patients (age, 59.6±13.7 years) were assessed 19.7±36.6 months after brain lesion. The 10-m walking time was measured as a gait parameter while the individual walked on a treadmill. Walking speed (m/s), step cycle (cycle/s), and step length (m) were also measured on a treadmill with and without PKAFO, and before and after gait training. Clinical parameters measured before and after gait training included Korean version of Modified Bathel Index (K-MBI), manual muscle test (MMT), and Modified Ashworth Scale (MAS) of hemiplegic ankle. Gait training comprised treadmill walking for 20 minutes, 5 days a week for 3 weeks at a comfortable speed. RESULTS: The 10-m walking time, walking speed, step length, and step cycle were significantly greater with PKAFO than without PKAFO, and after gait training (both p<0.05). K-MBI was improved after gait training (p<0.05), but MMT and MAS were not. CONCLUSION: PKAFO may improve gait function in hemiplegic patients. It can be a useful orthosis for gait training in hemiplegic patients.

Aerobic exercise improves gastrointestinal motility in psychiatric inpatients.

AIM: To evaluate the benefit of aerobic exercise on colonic transit time (CTT) for psychiatric inpatients in a closed ward. METHODS: Sixty consecutive adult inpatients of the Somang Hospital Psychiatry Unit (Eumsung-gun, South Korea), without CTT-related diseases or drug therapies, were recruited for study from March to June of 2012. Upon enrollment, the patients were randomly assigned to partake in a 12-wk instructor-led group aerobic exercise program (exercise group; n = 30) or to maintain their ordinary daily activities (control group; n = 30). The exercise program was structured as 10 min warm-up (stretching), 40 min exercise, and 10 min cool-down (stretching) for three days each week. The exercise sessions consisted of walking only in week one and aerobics from weeks two to 12, with increasing intensity (50% heart rate reserve (HRR) for weeks one to four, 60% HRR for weeks five to eight, and 70% HRR for weeks nine to 12). CTT was measured before (baseline) and after (week 12) the exercise program, in duplicate (on days four and seven), using abdominal radiography and the multiple radio-opaque marker technique. Changes in the exercising patients' CTT and weight-, cardiovascular- and fitness-related parameters were statistically assessed. RESULTS: The study dropout rate was 30.0%, with 23 patients in the exercise group and 19 patients in the control group completing the study. At week 12, the exercise group showed decreases in body weight (mean ± SE) baseline: 69.4 ± 2.8 vs study-end: 67.6 ± 2.7; P < 0.635) and body mass index (BMI) (25.2 ± 1.1 vs 24.9 ± 0.8; P < 0.810), but the extent of change was not significantly different from that experienced by the control group (body weight: 68.8 ± 4.0 vs 68.8 ± 3.9; BMI: 24.3 ± 1.1 vs 24.4 ± 1.2). However, the exercise group showed significant improvements in leg muscle strength (baseline: 41.7 ± 4.3 vs study-end: 64.1 ± 5.0; P < 0.001), cardio-respiratory endurance (120.5 ± 4.5 vs 105.4 ± 2.8; P < 0.004), and leg muscle elasticity and power output (21.5 ± 2.6 vs 30.6 ± 2.8; P < 0.001). The exercise group showed an exercise-induced reduction in total CTT (baseline: 54.2 ± 8.0 vs 30.3 ± 6.1), which was significantly different from that experienced by the control group over the 12-wk period (48.6 ± 9.3 vs 48.3 ± 12.3; P = 0.027); however, the exercise-induced decreases in CTT involving the three colonic segments examined (right, left and recto-sigmoid) showed no significant differences from the control group. CONCLUSION: A 12-wk aerobic exercise program can benefit psychiatric inpatients by increasing intestinal motility, possibly decreasing risk of metabolic- and cardiovascular-related disease.

Development of microRNA-145 for therapeutic application in breast cancer.

MicroRNAs, small non-coding RNAs, are key regulators of tumorigenesis and cancer metastasis through inhibition of gene expression. Therefore, there is increasing interest in developing anti-cancer therapies using microRNAs. In this study, we determined the therapeutic potency of microRNA-145(miR-145) against breast cancer. We found a reverse-correlation between the expression of miR-145 and its target genes, such as fascin-1, c-myc, SMAD2/3 and IGF-1R in breast cancer cell lines and breast cancer patient tissues. Transfected miR-145 mimicking double-stranded oligonucleotides was directly reduced cell proliferation and motility via interaction with 3'UTR of target gene and also indirectly regulates Wnt signaling. An inhibitor of miR-145 nullified this decreasing effect of miR-145 on cell proliferation and motility. We prepared an adenoviral constructed miR-145(Ad-miR-145) and subjected it to breast cancer cells in vitro and orthotopic breast cancer mice in vivo. Ad-miR-145 suppressed cell growth and motility in both the in vitro and in vivo systems. Furthermore, a treatment combining Ad-miR-145 with 5-FU significantly showed anti-tumor effects, compared to treating alone. In conclusion, this study demonstrated that miR-145 suppresses tumor growth by inhibition of multiple tumor survival effectors, and more we suppose that miR-145 is potentially useful in the therapy of breast cancers.