Label-free Detection for a DNA Methylation Assay Using Raman Spectroscopy / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>DNA methylation has been suggested as a biomarker for early cancer detection and treatment. Varieties of technologies for detecting DNA methylation have been developed, but they are not sufficiently sensitive for use in diagnostic devices. The aim of this study was to determine the suitability of Raman spectroscopy for label-free detection of methylated DNA.</p><p><b>METHODS</b>The methylated promoter regions of cancer-related genes cadherin 1 (CDH1) and retinoic acid receptor beta (RARB) served as target DNA sequences. Based on bisulfite conversion, oligonucleotides of methylated or nonmethylated probes and targets were synthesized for the DNA methylation assay. Principal component analysis with linear discriminant analysis (PCA-DA) was used to discriminate the hybridization between probes and targets (methylated probe and methylated target or nonmethylated probe and nonmethylated target) of CDH1 and RARB from nonhybridization between the probe and targets (methylated probe and nonmethylated target or nonmethylated probe and methylated target).</p><p><b>RESULTS</b>This study revealed that the CDH1 and RARB oligo sets and their hybridization data could be classified using PCA-DA. The classification results for CDH1 methylated probe + CDH1 methylated target versus CDH1 methylated probe + CDH1 unmethylated target showed sensitivity, specificity, and error rates of 92%, 100%, and 8%, respectively. The classification results for the RARB methylated probe + RARB methylated target versus RARB methylated probe + RARB unmethylated target showed sensitivity, specificity, and error rates of 92%, 93%, and 11%, respectively.</p><p><b>CONCLUSIONS</b>Label-free detection of DNA methylation could be achieved using Raman spectroscopy with discriminant analysis.</p>

Applications of Microfluidic Devices for Urology / 대한배뇨장애요실금학회지

Microfluidics is considered an important technology that is suitable for numerous biomedical applications, including cancer diagnosis, metastasis, drug delivery, and tissue engineering. Although microfluidics is still considered to be a new approach in urological research, several pioneering studies have been reported in recent years. In this paper, we reviewed urological research works using microfluidic devices. Microfluidic devices were used for the detection of prostate and bladder cancer and the characterization of cancer microenvironments. The potential applications of microfluidics in urinary analysis and sperm sorting were demonstrated. The use of microfluidic devices in urology research can provide high-throughput, high-precision, and low-cost analyzing platforms.

Differentiation Potential of Mesenchymal Stem Cells Is Related to Their Intrinsic Mechanical Properties / 대한배뇨장애요실금학회지

PURPOSE: The differentiation properties of stem cells are not yet fully understood due to their close association with multiple environmental and extrinsic factors. This study investigates the differentiation properties of mesenchymal stem cells (MSCs) and correlates them with their intrinsic mechanical properties. METHODS: A total of 3 different types of MSCs, namely bone marrow-derived MSCs (BMSCs), umbilical cord-derived MSCs (UCSCs), and adipose-derived MSCs (ADSCs) were evaluated. These 3 MSCs were individually differentiated into adipocytes and osteoblasts for 3 weeks. The mechanical properties of the MSCs and differentiated cells were determined by atomic force microscopy. RESULTS: ADSCs showed the greatest ability to differentiate into adipocytes, followed by BMSCs and UCSCs. While UCSCs differentiated readily into osteoblasts, BMSCs and ADSCs were less likely to undergo this differentiation. UCSCs were the “hardest” cells, while ADSCs were the “softest.” The cells differentiated from “hard” MSCs were stiffer than the cells differentiated from “soft” MSCs, irrespective of lineage specification. CONCLUSIONS: The differentiation ability of MSCs and the mechanical properties of the differentiated cells were closely linked. However, there were no significant correlations regarding changes in the mechanical properties between the nuclear region and the cytoplasm during differentiation.

Effects of Mitomycin C on Scleral Collagen Fibrils According to Atomic Force Microscopy

PURPOSE: To investigate the effects of mitomycin C on the scleral collagen surfaces using atomic force microscopy (AFM). METHODS: Two non-contact mode AFM machines were used to observe changes in the morphological characteristics of human scleral surfaces before and after one, three, and five minutes of 0.02% mitomycin C application. Based on AFM topography and deflection images of the collagen fibril, the morphological characteristics of scleral fibrils including the fibril diameter and D-period were measured using the line profile. RESULTS: The sclera collagen fibril treated with 0.02% mitomycin C for one minute did not show any significant increases in mean fibril diameter (155.04 +/- 17.46 nm) or mean D-periodicity (70.02 +/- 3.33 nm), compared to those of the control group. However, the scleral collagen fibrils treated with 0.02% mitomycin C for three and five minutes showed significant increases in mean fibril diameter (182.33 +/- 16.33 nm, 199.20 +/- 12.40 nm, respectively) and mean D-periodicity (70.27 +/- 13.66 nm, 72.75 +/- 19.32 nm, respectively), compared to those of the control group. CONCLUSIONS: The present study examined the structural changes in the scleral collagen fibrils before and after mitomycin C application according to atomic force microscopy. The results indirectly suggest that three or more minutes of 0.02% mitomycin C application affects the morphology of scleral collagen.

Neuroprotective Effects by Nimodipine Treatment in the Experimental Global Ischemic Rat Model : Real Time Estimation of Glutamate

OBJECTIVE: Glutamate is a key excitatory neurotransmitter in the brain, and its excessive release plays a key role in the development of neuronal injury. In order to define the effect of nimodipine on glutamate release, we monitored extracellular glutamate release in real-time in a global ischemia rat model with eleven vessel occlusion. METHODS: Twelve rats were randomly divided into two groups: the ischemia group and the nimodipine treatment group. The changes of extracellular glutamate level were measured using microdialysis amperometric biosensor, in coincident with cerebral blood flow (CBF) and electroencephalogram. Nimodipine (0.025 microg/100 gm/min) was infused into lateral to the CBF probe, during the ischemic period. Also, we performed Nissl staining method to assess the neuroprotective effect of nimodipine. RESULTS: During the ischemic period, the mean maximum change in glutamate concentration was 133.22+/-2.57 microM in the ischemia group and 75.42+/-4.22 microM (p<0.001) in the group treated with nimodipine. The total amount of glutamate released was significantly different (p<0.001) between groups during the ischemic period. The %cell viability in hippocampus was 47.50+/-5.64 (p<0.005) in ischemia group, compared with sham group. But, the %cell viability in nimodipine treatment group was 95.46+/-6.60 in hippocampus (p<0.005). CONCLUSION: From the real-time monitoring and Nissl staining results, we suggest that the nimodipine treatment is responsible for the protection of the neuronal cell death through the suppression of extracellular glutamate release in the 11-VO global ischemia model of rat.

Changes in surface roughness of bracket and wire after experimental sliding - preliminary study using an atomic force microscopy / 대한치과교정학회지

OBJECTIVE: The surface roughness of orthodontic materials is an essential factor that determines the coefficient of friction and the effectiveness of tooth movement. The aim of this study is to evaluate the surface roughness change of the brackets and wires after experimental sliding quantitatively. METHODS: Before and after experimental sliding tests, the surface roughness of stainless steel brackets, ceramic brackets, stainless steel wires, and beta-titanium (TMA) wires were investigated and compared using atomic force microscopy (AFM). RESULTS: After sliding tests, changes in the surface of the wire were greater than changes in the bracket slot surface. The surface roughness of the stainless steel bracket was not significantly increased after sliding test, whereas the roughness of ceramic brackets was decreased. Both the surface roughness of stainless steel and TMA wires were increased after sliding test. More changes were observed on the ceramic bracket than the stainless steel bracket. CONCLUSIONS: AFM is a valuable research tool when analyzing the surface roughness of the brackets and wires quantitatively.

Surface roughness analysis of ceramic bracket slots using atomic force microscope / 대한치과교정학회지

OBJECTIVE: This study was designed to measure the surface roughness at the slot floor of various ceramic brackets. METHODS: One kind of stainless steel bracket (Succes(R)), two kinds of monocrystalline brackets (Inspire Ice(R), Perfect(R)) and two kinds of polycrystalline brackets (Crystalline V(R), Invu(R)) were examined. Atomic force microscopy (AFM) was used to measure the surface roughness of each bracket. Data acquisition and processing were performed using SPIP(TM). RESULTS: The differences in values of Sa, Sq, and Sz in Invu(R) and Inspire Ice(R) were not statistically different from the control group Succes(R). The values of Sa, Sq, and Sz of Perfect(R) and Crystalline V(R) were greater than those of Succes(R). Differences of all the Sa, Sq, and Sz values between Perfect(R) and Crystalline V(R) were not statistically significant. CONCLUSIONS: It is concluded that the slot surfaces of Succes(R), Inspire Ice(R), and Invu(R) were smooth compared to those of Crystalline V(R) and Perfect(R).

Proximal caries detection using digital subtraction radiography in the artificial caries activity model / 대한구강악안면방사선학회지

PURPOSE: The purpose of the experiment was to evaluating the diagnostic ability of dental caries detection using digital subtraction in the artificial caries activity model. MATERIALS AND METHODS: Digital radiographies of five teeth with 8 proximal surfaces were obtained by CCD sensor (Kodak RVG 6100 using a size #2). The digital radiographic images and subtraction images from artificial proximal caries were examined and interpreted. In this study, we proposed novel caries detection method which could diagnose the dental proximal caries from single digital radiographic image. RESULTS: In artificial caries activity model, the range of lesional depth was 572-1,374 micrometer and the range of lesional area was 36.95-138.52mm2. The lesional depth and the area were significantly increased with demineralization time (p<0.001). Furthermore, the proximal caries detection using digital subtraction radiography showed high detection rate compared to the proximal caries examination using simple digital radiograph. CONCLUSION: The results demonstrated that the digital subtraction radiography from single radiographic image of artificial caries was highly efficient in the detection of dental caries compared to the data from simple digital radiograph.

Detection for demineralization of dental hard tissues using index-sequenced radiographs / 대한구강악안면방사선학회지

PURPOSE : The purpose of this study was to develop the radiographic technique for detecting the demineralization which is known as indication of dental caries MATERIALS AND METHODS : This technique was based on the comparing of multiple radiographs which was irradiated by multiple X-ray spectra. For the meaningful comparing, the multiple radiographs were reconstructed to the dosimetrically consistent images using a standard material. The difference of resulting images of same target with multiple spectra represents the difference of response of material as regards the spectra. RESULTS : We have found about 10% of demineralization of dental hard tissues particularly in the proximal region through the analyzing of differences. CONCLUSION : Most intriguing thing in this investigation was that the method to analyze difference shows us to an anatomic structure of dental hard tissues even if absolute values of optical density were excluded during the procedures.

Detection method of proximal caries using line profile in digital intra-oral radiography / 대한구강악안면방사선학회지

PURPOSE: The purpose of this study was to investigate how to detect proximal caries using line profile and validate linear measurements of proximal caries lesions by basic digital manipulation of radiographic images. MATERIALS AND METHODS: The X-ray images of control group (15) and caries teeth (15) from patients were used. For each image, the line profile at the proximal caries-susceptible zone was calculated. To evaluate the contrast as a function of line profile to detect proximal caries, a difference coefficient (D) that indicates the relative difference between caries and sound dentin or intact enamel was measured. RESULTS: Mean values of D were 0.0354+/-0.0155 in non-caries and 0.2632+/-0.0982 in caries (p<0.001). CONCLUSION: The mean values of caries group were higher than non-caries group and there was correlation between proximal dental caries and D. It is demonstrated that the mean value of D from caries group was higher than that of control group. From the result, values of D possess great potentiality as a new detection parameter for proximal dental caries.

Identification of Genetic Variations in CBL, SORBS1, CRK, and RHOQ,Key Modulators in the CAP/TC10 Pathway of Insulin Signal Transduction and Their Association with Type 2 Diabetes Mellitus in the Korean Population

Recent evidence has strongly suggested that the CAP/TC10 pathway is involved in the trafficking, docking,and fusion of vesicles containing the insulin- responsive glucose transporter Glut4 to the plasma membrane. However, little is known about how the genes employed in the CAP/TC10 pathway are associated with the development of type 2 diabetes mellitus. In this study, we sequenced 4 genes of the CAP/TC10 pathway [SORBS1, CBL, CRK, and RHOQ] in 24 individuals to identify genetic variations in these loci. A total of 48 sequence variants were identified, including 23 novel variations. To investigate the possible association with type 2 diabetes mellitus, 3 single nucleotide polymorphisms from SORBS1, 3 from CBL , and 4 from RHOQ were genotyped in 1122 Korean type 2 diabetic patients and 1138 nondiabetic controls. Using logistic regression analysis, 1 significant association between SNP rs1376405 in RHOQ and type 2 diabetes mellitus [OR = 8.714 (C.I. 1.714-44.29), p = 0.009] was found in the recessive model. Our data demonstrate a positive association of the RHOQ gene in the CAP/TC10 pathway with T2DM in the Korean population.

The Effect of Extracellular Glutamate Release on Repetitive Transient Ischemic Injury in Global Ischemia Model

During operations, neurosurgeons usually perform multiple temporary occlusions of parental artery, possibly resulting in the neuronal damage. It is generally thought that neuronal damage by cerebral ischemia is associated with extracellular concentrations of the excitatory amino acids. In this study, we measured the dynamics of extracellular glutamate release in 11 vessel occlusion (VO) model to compare between single occlusion and repeated transient occlusions within short interval. Changes in cerebral blood flow were monitored by laser-Doppler flowmetry simultaneously with cortical glutamate level measured by amperometric biosensor. From real time monitoring of glutamate release in 11 VO model, the change of extracellular glutamate level in repeated transient occlusion group was smaller than that of single occlusion group, and the onset time of glutamate release in the second ischemic episode of repeated occlusion group was delayed compared to the first ischemic episode which was similar to that of single 10 min ischemic episode. These results suggested that repeated transient occlusion induces less glutamate release from neuronal cell than single occlusion, and the delayed onset time of glutamate release is attributed to endogeneous protective mechanism of ischemic tolerance.

Optimization of Wet Fixation Methods for AFM Imaging of Human Fibroblast Cells

We investigated the effect by the chemical fixative on human fibroblast cells (HFCs) in order to make nano-scale images using by the atomic force microscopy (AFM). The cell fixation needed to be optimized as prerequisite step for the preparation before analysis. AFM imaging after optimal wet fixation can provide practical, simple and fast technique for scanning living cells. In this study, AFM images - topography and amplitude - and the optic images of HFCs which were fixed with phosphate buffered saline (PBS), 2:1 ethanol:acetic acid, 4% glutaraldehyde and 37% formaldehyde were compared respectively. The final effect by washing with PBS or distilled water (D.W.) was examined after 4% glutaraldehyde fixation. To determine the optimal fixation method for HFCs, we performed quantitative and qualitative analysis by the height profile, the presence of artifacts and the morphology of well-conserved fibroblastic topography image by AFM. From AFM image which showed fibroblastic cellular morphology and differential height value of cytoplasm (670+/-47 nm, n=10) and nucleus (847+/-32 nm, n=10) in HFCs, we proposed that wet fixation by 4% glutaraldehyde, followed by final washing with PBS, could be the most suitable preparation for AFM imaging of HFCs, which enable us to approach easily on living cells with the least shrinkage.

Protein Tyrosine Phosphatase N1 Gene Variants Associated with Type 2 Diabetes Mellitus and Its Related Phenotypes in the Korean Population

Protein phosphorylation at tyrosine residues is a key regulatory event that modulates insulin signal transduction. We studied the PTPN1 gene with regard to susceptibility to Korean type 2 diabetes mellitus (T2DM) and its related quantitative traits. A total of seven SNPs [g.36171G>A (rs941798), g.58166G>A (rs3787343), g.58208A>G (rs2909270), g.64840C>T (rs754118), g.69560C>G (rs6020612), g.69866G>A (rs718050), and g.69934T>G (rs3787343)] were selected based on frequency (>0.05), linkage disequilibrium (LD) status, and haplotype tagging status. We studied the seven SNPs in 483 unrelated patients with type 2 diabetes (age: 64+/-2.8 years, onset age: 56+/-8.1 years; 206 men, 277 women) and 1138 nondiabetic control subjects (age: 64+/-2.9; 516 men, 622 women). The SNP rs941798 had protective effects against T2DM with an odds ratio of 0.726 (C.I. 0.541~0.975) and p-value=0.034, but none of the remaining six SNPs was associated with T2DM. Also, rs941798 was associated with blood pressure, HDL cholesterol, insulin sensitivity. rs941798 also has been associated with T2DM in previous reports of Caucasian-American and Hispanic-American populations. This is the first report that shows an association between PTPN1 and T2DM in the Korean as well as Asian population.

Association between Prostaglandin-endoperoxide Synthase 2 (PTGS2) Polymorphisms and Blood Pressure in Korean Population

Blood pressure refers to the force exerted by circulating blood on the walls of blood vessels, and chronical elevation of blood pressure is known as hypertension. Although hypertension is affected by genetic and environmental factors, the genetic background of hypertension is not fully understood. One of the candidate genetic factors, Prostaglandin-endoperoxide synthase 2 (PTGS2), is a membrane-bound enzyme, catalyzing the conversion of arachidonic acid to prostaglandin, and recently SNPs of PTGS2 gene was associated with hypertension in Japanese population. Therefore the association of PTGS2 polymorphisms was investigated with blood pressure in healthy Korean subjects, 470 unrelated individuals randomly selected from Ansung and Ansan cohorts. The 25 SNPs of PTGS2 gene were identified by the sequencing analysis of 24 Korean samples. Among identified polymorphisms, three SNPs (rs689466, -1329A>G; rs5275, +6365T>C; rs4648308, +8806G> A) were selected for further association analysis, and rs689466 located in promoter region was associated with blood pressure as well as triglyceride level in the blood. By in silico analysis, rs689466 locates in v-Myb transcription factor binding site, and the v-Myb site disappears when the SNP is changed from A to G nucleotide. Individuals with A/G and G/G genotype in rs689466 have higher blood pressure than those with A/A genotype, and the regression p-value is 0.008 for systolic and 0.004 for diastolic blood pressure. In summary, the PTGS2 polymorphism (rs689466) is associated with blood pressure in Asian populations based on this and Japanese studies, shedding light on it as a genetic risk marker of hypertension.