Effect of Strain on Rodent Glaucoma Models: Magnetic Bead Injection Versus Hydrogel Injection Versus Circumlimbal Suture.

Purpose: To compare the inter-strain differences of three rodent glaucoma models as induced by magnetic bead injection, hydrogel injection, and circumlimbal suture. Methods: In Brown Norway (BN) and Sprague Dawley (SD) rat strains, intraocular pressure (IOP) was elevated by injection of magnetic beads or hydrogel to obstruct the aqueous humor outflow or by external compression of circumlimbal suture. Maximum and average IOP values were compared according to both procedure and rat strain over 1 month postoperatively. Retinal ganglion cell (RGC) density loss was evaluated using confocal microscopic images of the flat-mounted retina obtained at postoperative days 14 and 30. Results: The maximum IOPs were higher in the hydrogel injection or circumlimbal injection models than in the magnetic bead injection model (P < 0.001), whereas average IOP showed no difference between the two strains (both P ≥ 0.05). A generalized estimating equation regression model showed that the IOP increase was maintained better in the BN rats than in the SD rats (P < 0.001). Such inter-strain difference was smaller in the circumlimbal suture model. A significant decrease in RGC density was observed in all of the models for the BN rats and in the circumlimbal suture model for the SD rats at postoperative day 30. Conclusions: BN rats were advantageous for the magnetic bead or hydrogel injection model, but either rat strain could be used for the circumlimbal suture model. Strains should be considered cautiously when establishing rodent glaucoma models with different IOP profiles. Translational Relevance: This comparison offers the best strain for each rodent glaucoma model for assessment of glaucoma-relevant therapeutics.

Performance evaluation of immunoassay for infectious diseases on the Alinity i system.

BACKGROUND: Although a diagnosis of infectious diseases is essential for timely treatment, the performance of diagnostic tests has been hardly evaluated due to variable results that are influenced by multiple factors in different conditions. In the present study, the performance of the Alinity i system, which is a newly developed immunoassay to diagnose infectious diseases, was evaluated. METHODS: We evaluated the precision, linearity, correlation, and carryover of 16 analytes (HAV Ab IgG, HBsAg, HBeAg, anti-HBc, anti-HBe, anti-HBs, anti-HCV, HIV Ag/Ab, EBV VCA IgM, EBV VCA IgG, EBV EBNA IgG, CMV IgM, CMV IgG, Toxoplasma IgG, Rubella IgG, and Syphilis TP) of Alinity i by comparison with ARCHITECT i2000SR system following the rationale of the Clinical and Laboratory Standards Institute (CLSI). RESULTS: For quantitative tests, the coefficients of variation (CV) % of repeatability and intermediate precision were between 0% and 4.18%. The coefficients of the linearity (r2 ) over a widely tested analytical range were ≥ 0.990 and the correlation between Alinity i and the ARCHITECT i2000SR system was strong (r ≥ 0.994). For qualitative tests, the agreement between Alinity i and the ARCHITECT i2000SR system was excellent (kappa coefficient 1) with 100% sensitivity and specificity. Carryover rates for all analytes were less than 1.0% (-0.11% ~ 0.21%). CONCLUSION: The Alinity i system showed good analytical performance and favorable comparability with the ARCHITECT i2000SR. It could be suitable as a routine immunoassay analyzer for screening and diagnosis of infectious disease.

Activation of Factor XII and Kallikrein-Kinin System Combined with Neutrophil Extracellular Trap Formation in Diabetic Retinopathy.

BACKGROUND: In diabetic retinopathy (DR), neutrophil extracellular traps (NET) and kallikrein-kinin system are considered as contributing factors. However, the detail activation mechanisms has not been fully understood. Since the NET could provide negative-charged surface for factor XII activation and the activated factor XII (XIIa) can initiate kallikrein-kinin system, this study investigated whether patients with DR show activation of NET, factor XII and kallikrein-kinin system. METHODS: The markers related to NET (DNA-histone complex) and kallikrein-kinin system (high-molecular-weight kininogen, prekallikrein, bradykinin) and factor XIIa were measured in 253 patients with diabetes. To access ex vivo effect of glucose, DNA-histone complex and factor XIIa were measured in whole blood stimulated by glucose. RESULTS: The circulating level of DNA-histone complex and factor XIIa were significantly higher in patients with DR than those without DR. In logistic regression analysis, DNA-histone complex, factor XIIa, and high-molecular-weight kininogen were the risk factors of DR. In recursive partitioning analysis, among patients with diabetes duration less than 10 years, patients with high level of DNA-histone complex (>426 AU) showed high risk of DR. In ex vivo experiment, glucose significantly elevated both DNA-histone complex and factor XIIa. CONCLUSION: Our findings suggest that activation of factor XII and kallikrein-kinin system combined with NET formation actively occur in patients with DR and circulating levels of DNA-histone complex, factor XIIa and HMWK can be potential biomarkers to estimate the risk of DR. Strategies against factor XII activation may be beneficial to inhibit DR.

Evaluation of analytical performance of Alinity i system on 31 measurands.

INTRODUCTION: Accurate, precise and reliable laboratory test results play a critical role in medical decision making. To satisfy the increasing needs in clinical laboratory tests, the analyzers have been advanced. In this study, authors aimed to evaluate the analytical performance of the Alinity i system (Abbott Laboratories, IL, USA) for diverse analytes measured by using immunoassay principle. MATERIALS AND METHODS: Analytical performance of recently launched Alinity i system has been evaluated for 31 assays in aspects of precision, linearity and analytical measurement range, correlation with the Architect i2000sr system (Abbott Laboratories), carry-over, and reference interval validation in accordance with CLSI guidelines. RESULTS: The within-laboratory CVs of the analytes tested in the study ranged between 1.00 and 7.84%, which met vendor claimed value in precision. In linearity test, most assays satisfied acceptable linearity criteria, best-fit first order regression or polynomial regression with nonlinearity smaller than ±10%, compared with linear regression. The recovery of each analyte distributed from 90.1 to 109.7%. The coefficient of determination (R2) for each test was larger than 0.95 except for folate when compared to the results obtained from existing routine analyzer and statistically or clinically equivalent. The carry-over rates were acceptable, and reference intervals were validated. CONCLUSION: Through this study, acceptable analytical performance of novel Alinity i system has been verified. It is expected to readily replace existing instrument and to be an option for laboratories considering introduction of automated immunoassay analyzer.

Urinary Sodium and Potassium Levels and Blood Pressure in Population with High Sodium Intake.

The purpose of this study was to examine the association of urinary sodium-to-creatinine ratio and potassium-to-creatinine ratio with blood pressure in a cross-sectional study comprising Korean adults who participated in the Healthy Twin Study. The participants consisted of 2653 men and women in the Healthy Twin Study aged ≥19 years. Participants' urinary excretion of sodium, potassium, and creatinine was measured from overnight half-day urine samples. Food intake was assessed using a validated food frequency questionnaire. We examined systolic and diastolic blood pressures according to sodium- or potassium-to-creatinine ratios using the generalized linear model. We determined food groups explaining high urinary sodium- or potassium-to-creatinine ratio using the reduced rank regression and calculated sodium- or potassium-contributing food score. We observed that systolic blood pressure was higher among men and women in the highest quintile of urinary sodium-to-creatinine ratio or sodium-to-potassium ratio than it was in the lowest quintile. Geometric means (95% CIs) of the lowest and the highest quintiles of systolic blood pressure (mmHg) were 113.4 (111.8-115.0) and 115.6 (114.1-117.2; P for trend = 0.02), respectively, for sodium-to-creatinine ratio. The association between urinary sodium-to-creatinine and systolic blood pressure was more pronounced among individuals whose body mass index (BMI) was less than 25 kg/m2 (P for interaction = 0.03). We found that vegetables, kimchi and seaweed intake contributed to high sodium intake and a sodium-contributing food score were associated with increased blood pressure. In our study, we identified the food groups contributing to high sodium intake and found that high urinary sodium levels were associated with increasing blood pressure among Korean adults.

High prevalence of increased sitosterol levels in hypercholesterolemic children suggest underestimation of sitosterolemia incidence.

BACKGROUND: Sitosterolemia is an inherited lipid disorder which presents with elevated serum sitosterol and can result in an increased risk of premature cardiovascular disease. However, sitosterol cannot be accurately measured by routine diagnostic assays, meaning that sitosterolemia diagnosis can often be difficult, especially with many clinical features overlapping with familial hypercholesterolemia. With such complications resulting in increasing reports of misdiagnosis, the prevalence of sitosterolemia is predicted to be much higher than previously reported. METHODS: Gas chromatography-mass spectrometry was utilized to measure sitosterol levels of normocholesterolemic and hypercholesterolemic children. Subsequently, an epidemiologically determined cutoff level of sitosterol was calculated and applied to estimate the prevalence of children with increased sitosterol and identify potential sitosterolemia patients. Massively parallel sequencing was used to confirm the diagnosis in suspected patients. RESULTS: Samples from 109 normocholesterolemic and 220 hypercholesterolemic were tested for phytosterols. Sitosterol and campesterol levels were significantly increased in hypercholesterolemic children (mean 22.0±45.9 µmol/L for sitosterol and 26.0±32.8 µmol/L for campesterol) compared to normocholesterolemic children (mean 12.1±4.9 µmol/L for sistosterol and 14.8±6.7 µmol/L for campesterol). Via application of a cutoff of 35.9 µmol/L, the prevalence rates for increased and overtly increased sitosterol in hypercholesterolemic children were 6.4% and 1.4% respectively. Furthermore, 3 suspected sitosterolemia patients were identified, with 2 patients receiving molecular confirmation for sitosterolemia diagnosis. CONCLUSIONS: Our findings reaffirm that the prevalence of sitosterolemia is probably much higher than previously reported, which also indicates the significant risk of misdiagnosis of sitosterolemia with familial hypercholesterolemia. Special lipid testing including sitosterol, especially in children with uncontrolled hypercholesterolemia, is recommended in children in order to identify potential sitosterolemia patients that would otherwise be neglected.

Poly-(γ-glutamic acid) Production and Optimization from Agro-Industrial Bioresources as Renewable Substrates by Bacillus sp. FBL-2 through Response Surface Methodology.

We optimized culture conditions using Bacillus sp. FBL-2 as a poly-(γ-glutamic acid) (PGA) producing strain isolated from cheonggukjang. All experiments were performed under aerobic conditions using a laboratory scale 2.5 L fermentor. We investigated the effects of fermentation parameters (temperature, pH, agitation, and aeration) and medium components (glutamic acid, citric acid, and yeast extract) on poly-(γ-glutamic acid) production, viscosity, and dry cell mass. A non-optimized fermentation method (1.5 vvm, 350 rpm, and 37 °C) yielded PGA, viscosity, and dry cell mass at levels of 100.7 g/L, 483.2 cP, and 3.4 g/L, respectively. L-glutamic acid, citric acid, and yeast extract supplementation enhanced poly-(γ-glutamic acid) production to 175.9 g/L. Additionally, the production of poly-(γ-glutamic acid) from rice bran and wheat bran was assessed using response surface methodology (central composite rotatable design). Agricultural byproducts (rice bran and wheat bran) and H2SO4 were selected as factors, and experiments were performed by combining various component concentrations to determine optimal component concentrations. Our experimentally-derived optimal parameters included 38.6 g/L of rice bran, 0.42% of H2SO4, 28.0 g/L of wheat bran, and 0.32% of H2SO4. Under optimum conditions, rice bran medium facilitated poly-(γ-glutamic acid) production of up to 22.64 g/L, and the use of wheat bran medium yielded up to 14.6 g/L. Based on a validity test using the optimized culture conditions, poly-(γ-glutamic acid) was produced at 47.6 g/L and 36.4 g/L from these respective mediums, and both results were higher than statistically predicted. This study suggests that rice bran can be used as a potential alternative substrate for poly-(γ-glutamic acid) production.

Telomere length and its correlation with gene mutations in chronic lymphocytic leukemia in a Korean population.

Telomere length (TL) is a prognostic indicator in Caucasian chronic lymphocytic leukemia (CLL), but its significance in Asian CLL remains unknown. To investigate the prognostic significance of TL and its correlation with cytogenetic aberrations and somatic mutations, we analyzed TL measurements at the cellular level by interphase fluorescence in situ hybridization in patients with CLL in Korea. The present study enrolled 110 patients (41 females and 69 males) diagnosed with CLL according to the World Health Organization criteria (2001-2017). TLs of bone marrow nucleated cells at the single-cell level were measured by quantitative fluorescence in situ hybridization (Q-FISH) in 71 patients. The correlations of TL with clinical characteristics, cytogenetic aberrations, genetic mutations, and overall survival were assessed. The median value of mean TL in CLL patients (T/C ratio 7.46 (range 1.19-18.14) was significantly shorter than that in the normal controls (T/C ratio 15.28 (range 8.59-24.93) (p < 0.001). Shorter TLs were associated with complex karyotypes (p = 0.030), del(11q22) (p = 0.023), presence of deletion and/or mutation in ATM and/or TP53 (p = 0.019), and SH2B3 mutation (p = 0.015). A shorter TL was correlated with lower hemoglobin levels and adverse survival (mean TL < 9.35, p = 0.021). When the proportion of cells with extremely short TLs (< 7.61) was greater than 90%, CLL patients showed poor survival (p = 0.002). Complex karyotypes, TP53 mutation, and the number of mutated genes were determined to be significant adverse variables by multivariable Cox analysis (p = 0.011, p = 0.002, and p = 0.002, respectively). TL was attrited in CLL, and attrited telomeres were correlated with adverse survival and other well-known adverse prognostic factors. We infer that TL is an independent adverse prognostic predictor in Korean CLL.

Proliferation Profile of Uterine Endometrial Stromal Cells during In Vitro Culture with Gonadotropins: Recombinant versus Urinary Follicle Stimulating Hormone.

BACKGROUND: Provision of optimal endometrial stromal cells is essential in uterine tissue engineering. Culture of these cells is significantly influenced by gonadotropin hormones. This investigation attempted to define the proliferation profiles of murine uterine endometrial stromal cells during in vitro culture with recombinant follicle stimulating hormone (rFSH), urinary follicle stimulating hormone (uFSH), and human chorionic gonadotropin (hCG). METHODS: Murine uterine endometrial stromal cells were collected from 8-week-old mice and cultured in vitro up to 72 h, with rFSH, uFSH, or hCG. Cell cycles were analyzed by BrdU assay, and cyclin D1 expression was evaluated according to dose and duration of gonadotropin treatment. RESULTS: BrdU assay showed a further inhibitory effect on murine uterine endometrial stromal cell proliferation when cultured with rFSH compared to uFSH, and a similar inhibitory proliferation profile when cultured with hCG at a specific range of concentrations. The expression of cyclin D1 of murine uterine endometrial stromal cells was down-regulated when cultured with rFSH, uFSH, or hCG, compared to control. CONCLUSIONS: FSH may inhibit the proliferation of murine uterine endometrial stromal cells during in vitro culture. rFSH may have more significant inhibitory effects on the proliferation of endometrial stromal cells than uFSH. Establishing an optimal endocrine milieu is necessary using more advanced combination of female hormones for in vitro culture of this type of cells.

Calf Circumference as a Screening Tool for Cognitive Frailty in Community-Dwelling Older Adults: The Korean Frailty and Aging Cohort Study (KFACS).

The aim of this study was to examine calf circumference in relation to cognitive frailty in community-dwelling older adults. Cross-sectional analysis was performed on the first-year baseline data of 1559 adults aged 70⁻84 years enrolled in the Korean Frailty and Aging Cohort Study. The final analysis included 1221 adults who were non-dependent in terms of instrumental activities of daily living, who underwent frailty and cognitive function assessments. Physical frailty was defined using the Fried Frailty Index. Cognitive impairment was defined as a score 1.5 standard deviations below the age-, sex- and education-matched norms on any of four cognitive-function tests. The prevalence of cognitive frailty was 2.8% for men and 3.8% for women. After adjusting for potential confounders, in comparison to the "physically robust without cognitive impairment" group, the estimates of increased odds ratios (ORs) for low calf circumference (<32 cm) were much greater in the prefrail with cognitive impairment (OR 4.62, 95% confidence interval (CI): 2.02⁻10.61) and frail with cognitive impairment (OR 10.94, 95% CI: 2.87⁻41.68) groups in men but not in women. Low calf circumference was strongly related to cognitive frailty in men only, suggesting calf circumference can be used as an indicator of these outcomes.

Application of linear gradient elution in countercurrent chromatography for the separation of triterpenoid saponins from the roots of Pulsatilla koreana Nakai.

A linear gradient elution method using countercurrent chromatography was developed for the separation of four triterpenoid saponins from the roots of Pulsatilla koreana Nakai, including hederacolchiside E, which is responsible for the neuroprotective activity of this plant. The target fraction was obtained by 80% methanol elution of solid phase column chromatography. The partition coefficients of the target compounds were very different, which means they are difficult to separate with a single biphasic solvent system. Several important parameters for gradient elution, such as addition of alcohol content to the solvent system, starting point of the second mobile phase, and the time for the gradient change were logically determined and optimized. Four triterpenoid saponins could ultimately be separated, analyzed by high-performance liquid chromatography, and their structures were identified by comparing the mass spectra and NMR spectra with the literature data. The compounds and yields were: hederasaponin B (1; 21.3 mg/100 mg), hederacolchiside E (2; 19.8 mg/100 mg), cernuoside A (3; 18.4 mg/100 mg), and cernuoside B (4; 17.3 mg/100 mg). Gradient-elution countercurrent chromatography allows the effective separation of compounds with a wide polarity range.

Performance of urine cotinine and hypomethylation of AHRR and F2RL3 as biomarkers for smoking exposure in a population-based cohort.

There is a growing body of evidence demonstrating an association between smoking and DNA methylation. Accordingly, DNA methylation is now considered a promising biomarker of smoking exposure. We evaluated the relationship between methylation markers (AHRR and F2RL3) and urine cotinine as well as self-reported smoking status. DNA methylation levels of AHRR and F2RL3 in blood as well as urine cotinine were measured in 330 adults (46 to 87 years of age). Pyrosequencing was performed to measure DNA methylation of AHRR and F2RL3 associated with smoking exposure. The lung cancer risk associated with DNA methylation and urine cotinine was analyzed using logistic regression analysis. The AHRR and F2RL3 genes were significantly hypomethylated in current smokers compared to in individuals who have never smoked. An inverse relationship was observed between urine cotinine and methylation levels. Methylation of AHRR and F2RL3 distinguished current smokers from never-smokers with high accuracy. Logistic multivariate analysis showed that AHRR methylation is significantly associated with the risk of lung cancer (OR = 0.96, P = 0.011). Our study validated the smoking-associated DNA methylation markers reported in a Korean population-based cohort. In conclusion, DNA methylation of AHRR and F2RL3 provided accurate measures for smoking exposure. Methylation markers reflecting the long-term effect of smoking on the risk of lung cancer showed better performance in distinguishing former smokers from never-smokers.

Body mass index and biliary tract disease: a systematic review and meta-analysis of prospective studies.

OBJECTIVE: To evaluate the association between body mass index (BMI, kg/m(2)) and incidence of biliary tract disease. METHODS: We performed a systematic review and a meta-analysis of prospective studies by searching the database of PubMed and EMBASE published up to December 31, 2013. Outcome of interest was disease of biliary tract system (gallbladder, extrahepatic bile duct and Ampullar of Vater). We used a random-effects model to combine the study-specific relative risks (RRs) and 95% confidence intervals (95% CIs) from 22 prospective studies. We examined whether BMI was associated with a higher risk of biliary tract disease in a combined analysis. RESULTS: The positive association was stronger for non-cancer biliary tract disease than biliary tract cancer; combined RRs (95% CIs) comparing the top with bottom categories were 1.40 (1.15-1.65) for biliary tract cancer and 2.75 (2.35-3.15) for non-cancer biliary tract disease (P for difference<0.001). For non-cancer biliary tract disease, combined RRs (95% CIs) comparing the top with bottom categories were 3.21 (2.48-3.93) for women and 2.01 (1.66-2.37) for men (P for difference=0.04). CONCLUSION: Obesity is associated with higher risks of biliary tract cancer and, to a greater extent, non-cancer biliary tract disease.