Heo, Hyun Young;Kim, Yong Tae;Chen, Yuchao;Choi, Jong Young;Seo, Tae Seok
Proceedings of the Korean Vacuum Society Conference
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2013.08a
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pp.273-273
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2013
Recently, Point-of-care (POC) testing microdevices enable to do the patient monitoring, drug screening, pathogen detection in the outside of hospital. Immunochromatographic strip (ICS) is one of the diagnostic technologies which are widely applied to POC detection. Relatively low cost, simplicity to use, easy interpretations of the diagnostic results and high stability under any circumstances are representative advantages of POC diagnosis. It would provide colorimetric results more conveniently, if the genetic analysis microsystem incorporates the ICS as a detector part. In this work, we develop a reverse transcriptase-polymerase chain reaction (RT-PCR) microfluidic device integrated with a ROSGENE strip for colorimetric influenza H1N1 virus detection. The integrated RT-PCR- ROSGENE device is consist of four functional units which are a pneumatic micropump for sample loading, 2 ${\mu}L$ volume RT-PCR chamber for target gene amplification, a resistance temperature detector (RTD) electrode for temperature control, and a ROSGENE strip for target gene detection. The device was fabricated by combining four layers: First wafer is for RTD microfabrication, the second wafer is for PCR chamber at the bottom and micropump channel on the top, the third is the monolithic PDMS, and the fourth is the manifold for micropump operation. The RT-PCR was performed with subtype specific forward and reverse primers which were labeled with Texas-red, serving as a fluorescent hapten. A biotin-dUTP was used to insert biotin moieties in the PCR amplicons, during the RT-PCR. The RT-PCR amplicons were loaded in the sample application area, and they were conjugated with Au NP-labeled hapten-antibody. The test band embedded with streptavidins captures the biotin labeled amplicons and we can see violet colorimetric signals if the target gene was amplified with the control line. The off-chip RT-PCR amplicons of the influenza H1N1 virus were analyzed with a ROSGENE strip in comparison with an agarose gel electrophoresis. The intensities of test line was proportional to the template quantity and the detection sensitivity of the strip was better than that of the agarose gel. The test band of the ROSGENE strip could be observed with only 10 copies of a RNA template by the naked eyes. For the on-chip RT-PCR-ROSGENE experiments, a RT-PCR cocktail was injected into the chamber from the inlet reservoir to the waste outlet by the micro-pump actuation. After filling without bubbles inside the chamber, a RT-PCR thermal cycling was executed for 2 hours with all the microvalves closed to isolate the PCR chamber. After thermal cycling, the RT-PCR product was delivered to the attached ROSGENE strip through the outlet reservoir. After dropping 40 ${\mu}L$ of an eluant buffer at the end of the strip, the violet test line was detected as a H1N1 virus indicator, while the negative experiment only revealed a control line and while the positive experiment a control and a test line was appeared.
This study was performed to investigate the effect of dietary protein and calcium levels on cadmium detoxication in rats. Seventy male Sprague-Dawley rats weighing 208 $\pm$ 19 g were blocked into 10 groups of 7 animals according to body weight. Five groups were fed 15% protein-0.6% calcium diet with 100ppm cadmium in drinking water for first 15days and the other 5groups fed same diet without cadmium in drinking water for same period and served as controls. After this 15-day intoxication period, each one of cadmium intoxication and control groups were fed each of 4 kinds of detoxifying diets different with protein(40%, 15%) and calcium(1.3%, 0.6%) levels without cadmimum in drinking water for following 15 days of detoxifying period. Results were summarized as follows: 1) Food intake, body weight gain, F.E.R. and weights of liver, kidney and femur were increased by detoxifying diets and high protein diet was most effective in weight gains of liver and kidney. 2) When cadmium and metallothionein contents of initial intoxication group and those of all detoxication groups were compared, cadmium and metallothionein contents in the liver were not changed, but those in kidney increased, and those in intestine decreased markedly. 3) Only dietary protein level affected cadmium and metallothionein distribution among organs, and cadmium contents of whole blood, liver, kidney and femur were lower in high protein diet, but metallothionein contents in liver and kidney were higher in high protein diet. 4) Gel filtration chromatogram showed that most of cadmium in the cytosol was bound to metallothionein fractions in high protein-high calcium group. Results obtained indicated that high protein diet was effective in cadmium detoxication by increasing the induction of metallothionein synthesis. But high calcium diet did not play a role in cadmium detoxication.
For overall system test, hidden-target test have been used using film which leads to inherent analysis error. The purpose of our study is to quantify this error and to propose gel dosimeter based verification technique for 3-dimensional target point error. The phantom was made for simulation of human head and this has ability to equip 10 gel-dosimeter. $BANGkit^{TM}$ which we are able to manufacture whenever it is needed as well as to easily change the container with different shapes was used as a gel dosimeter. The 10 targets were divided into two groups based on shapes of areas with a planned 50% isodose line. All treatment and analysis was performed three times using Novalis and $BrainSCAN^{TM}$. The target point error is $0.77{\pm}0.15mm$ for 10 targets and directional target point error in each direction is $0.54{\pm}0.23mm$, $0.37{\pm}0.08mm$, $0.33{\pm}0.10mm$ in AP (anterior-posterior), LAT (lateral), and VERT (vertical) direction, respectively. The result of less than 1 mm shows that the treatment was performed through each precise step in treatment procedure. In conclusion, the 3-dimensional target point verification technique can be one of the techniques for overall system test.
0.5 mg of natural ginsenoside mixture and 0.8 $\mu$Ci of synthesized 14C-ginsenosides were administered orally to a rat and killed at one hour after the ginsenoside administration and the liver was fractionated into nuclear fraction, mitrochondria microsomes and cytosol fraction. Radioactivity distribu lion in subcellular fractions of the liver showed that 32o1c of total radioactivity absorbed in the liver was in cytosol fraction but a significant portion of the radioactivity was also found in mitochondria (26.6%) and microsomal fraction (18.l%). 5.8% of the total radioactivity was recovered from the nuclear fraction as well. This suggested that ginsenosides might be distributed into all subcellular fractions. Activities of mitochondrial aldehyde dehydrogenase, lactate dehydrogenase and malate dehydrogenase of the liver of rat at two hours after the ginsenoside administraion were found appreciably stimulated, suggesting that the ginsenoside concentration in the liver might be around 10-5%, since optimum concentrations for most enzyme catalyzed reactions in vitro were known to be 10-6% 10-4%. A significant portion of the radioactivity recovered from subcellular fractions of the liver was found in protein fractions, suggesting that proteins might interact with ginsenosides. Examination of protein-ginsenoside interation by gel filtration, equilibrium dialysis and amonium sulfate precipitation technique suggesting that proteins and ginsenosides do not bound covalently but weakl\ulcorner combined. When purified ginsenoside Rbl and Rgl were incubated with rat liver cytosolic enzymes for 20 min, the above ginsenosides were hydrolyzed quickly, suggesting that ginsenosides might be rapidly hydrolyzed and metabolized in the liver. It was also observed in vitro that the ginsenosides such as Rbl and Rgl were easily hydrolyzed by rat liver cytosol preparation suggesting that absorbed ginsenosides might be quickly hydrolyzed and metabolized in the liver.
Background: Promoter hypermethylation mediated gene silencing of tumor suppressor genes is considered as most frequent mechanism than genetic aberrations such as mutations in the development of cancers. BRD7 is a single bromodomain containing protein that functions as a subunit of SWI/SNF chromatin-remodeling complex to regulate transcription. It also interacts with the well know tumor suppressor protein p53 to trans-activate genes involved in cell cycle arrest. Loss of expression of BRD7 has been observed in breast cancers and nasopharyngeal carcinomas due to promoter hypermethylation. However, the genetic status of BRD7 in oral squamous cell carcinomas (OSCCs) is not known, although OSCC is one of the most common among all reported cancers in the Indian population. Hence, in the present study we investigated OSCC samples to determine the occurrence of hypermethylation in the promoter region of BRD7 and understand its prevalence. Materials and Methods: Genomic DNA extracted from biopsy tissues of twenty three oral squamous cell carcinomas were digested with methylation sensitive HpaII type2 restriction enzyme that recognizes and cuts unmethylated CCGG motifs. The digested DNA samples were amplified with primers flanking the CCGG motifs in promoter region of BRD7 gene. The PCR amplified products were analyzed by agarose gel electrophoresis along with undigested amplification control. Results: Methylation sensitive enzyme technique identified methylation of BRD7 promoter region seventeen out of twenty three (74%) well differentiated oral squamous cell carcinoma samples. Conclusions: The identification of BRD7 promoter hypermethylation in 74% of well differentiated oral squamous cell carcinomas indicates that the methylation dependent silencing of BRD7 gene is a frequent event in carcinogenesis. To the best of our knowledge, the present study is the first to report the occurrence of BRD7and its high prevalence in oral squamous cell carcinomas.
Background: Sequential use of circulating prostate cell (CPC) detection has been reported to potentially decrease the number of unnecessary prostate biopsies in men suspected of prostate cancer. In order to determine the real world effectiveness of the test, we present a prospective study of men referred to two hospitals from primary care physicians, one using CPC detection to determine the necessity of prostate biopsy the other not doing so. Materials and Methods: Men with a suspicion of prostate cancer because of elevated PSA >4.0ng/ml or abnormal DRE were referred to Hospitals A or B. In Hospital A all underwent 12 core TRUS biopsy, in Hospital B only men CPC (+), with mononuclear cells obtained by differential gel centrifugation identified using double immunomarking with anti-PSA and anti-P504S, were recommended to undergo TRUS biopsy. Biopsies were classifed as cancer or no-cancer. Diagnostic yields were calculated, including the number of posible biopsies that could be avoided and the number of clinically significant cancers that would be missed. Results: Totals of 649 men attended Hospital A, and 552 men attended Hospital B; there were no significant differences in age or serum PSA levels. In Hospital A, 228 (35.1%) men had prostate cancer detected, CPC detection had a sensitivity of 80.7%, a specificity of 88.6%, and a negative predictive value of 89.5%. Some 39/44 men CPC negative with a positive biopsy had low grade small volume tumors. In Hospital B, 316 (57.2%) underwent biopsy. There were no significant differences between populations in terms of CPC and biopsy results. The reduction in the number of biopsies was 40%. Conclusions: The use of sequential CPC testing in the real world gives a clear decision structure for patient management and can reduce the number of biopsies considerably.
Changes of malate dehydrogenase isozyme in oyster exposed to different temperature, pH and salinity were investigated by polyacrylamide gel electrophoresis. MDH isozyme in control group was separated into two bands on the positive side. In case of temperature and pH stress, MDH isozyme was separated into only one band after 12 hours exposure but two bands after 24, 48 hours exposure on the positive side. In case of salinity stress, after 12 hours exposure, MDH isozyme bands were separated into two bands in 5 ppt, 30 ppt and three bands in 10 ppt, 40 ppt concentration on the positive side. After 24 hours and 48 hours exposure case in salinity stress, MDH isozyme bands was separated into two bands on the positive side in all concentration. Activities of isozyme bands show their characteristics according to the condition of experiment. In conclusion, changes of MDH isozyme was a biochemical defense mechanism in oyster and result from effect of environmental stress to oyster.
The variation of isoperoxidase band patterns in the zymograms in the leaves of ${\times}$P. albaglandulosa clones showing excellent growth were observed by starch gel electrophoresis in this study. The results are summerized as follows; The numbers of total bands in the clones were six to eleven. Four to seven were active and one to four were of trace in these bands, and also active bands appeared plentifully in all clones. The appearing pattern of the bands was more monotonous to the cathode than to the anode. Besides, the uniqueness of the isoenzyme forms in each clone made possible to identify the clones, and g and 1 bands were fixed in ${\times}$P. albaglandulosa, ${\times}$P. albaglandulosa being $F_1$ hybrid, the genetic variation of isoenzyme forms was significant statistically.
The Korean Society of Urogenital Radiology (KSUR) aimed to present a consensus statement for patient preparation, standard technique, and pain management in relation to transrectal ultrasound-guided prostate biopsy (TRUS-Bx) to reduce the variability in TRUS-Bx methodologies and suggest a nationwide guideline. The KSUR guideline development subcommittee constructed questionnaires assessing prebiopsy anticoagulation, the cleansing enema, antimicrobial prophylaxis, local anesthesia methods such as periprostatic neurovascular bundle block (PNB) or intrarectal lidocaine gel application (IRLA), opioid usage, and the number of biopsy cores and length and diameter of the biopsy needle. The survey was conducted using an Internet-based platform, and responses were solicited from the 90 members registered on the KSUR mailing list as of 2018. A comprehensive search of relevant literature from Medline database was conducted. The strength of each recommendation was graded on the basis of the level of evidence. Among the 90 registered members, 29 doctors (32.2%) responded to this online survey. Most KSUR members stopped anticoagulants (100%) and antiplatelets (76%) one week before the procedure. All respondents performed a cleansing enema before TRUS-Bx. Approximately 86% of respondents administered prophylactic antibiotics before TRUS-Bx. The most frequently used antibiotics were third-generation cephalosporins. PNB was the most widely used pain control method, followed by a combination of PNB plus IRLA. Opioids were rarely used (6.8%), and they were used only as an adjunctive pain management approach during TRUS-Bx. The KSUR members mainly chose the 12-core biopsy method (89.7%) and 18G 16-mm or 22-mm (96.5%) needles. The KSUR recommends the 12-core biopsy scheme with PNB with or without IRLA as the standard protocol for TRUS-Bx. Anticoagulants and antiplatelet agents should be discontinued at least 5 days prior to the procedure, and antibiotic prophylaxis is highly recommended to prevent infectious complications. Glycerin cleansing enemas and administration of opioid analogues before the procedure could be helpful in some situations. The choice of biopsy needle is dependent on the practitioners' situation and preferences.
Journal of the Korea Organic Resources Recycling Association
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v.13
no.2
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pp.85-90
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2005
Changes of biochemical and genetic diversity of microbial communities in the soil damaged by a forest fire were analyzed. Soil samples were collected from Gangnung area where a forest fire was broken out in 2000. Two soil samples were from the burnt area, one from the naturally restoring soil (NS) and the other from the artificially restoring soil (AS). A normal, unaffected soil sample (US) was also included as a control. For the biochemical diversity, each sample was directly applied to the BIOLOG system, and the cluster analysis through a statistic process (SPSS) were performed. Genetic diversity was analyzed through DGGE using 16S-rDNA amplified from soil DNA. Among the samples tested, top soils of US and NS, and sub soil of NS revealed more than 70% of the similarity value in biochemical diversity. In case of genetic diversity, however, the similarity value was found to be in the range of 53% to 68% in all samples. This result indicates that the biochemical diversity is not always correlated with the genetic diversity in the analysis of microbial communities.
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