• Korean Journal of Microbiology
• /
• v.44 no.1
• /
• pp.14-21
• /
• 2008

Bovine blood, cell, tissue, and organ are used as raw materials for manufacturing biopharmaceuticals, tissue engineered products, and cell therapy. Manufacturing processes for the biologicals using bovine materials have the risk of viral contamination. Therefore viral validation is, essential in ensuring the safety of the products. Bovine herpesvirus type 1 (BHV-1) is the most common bovine pathogen found in bovine blood, cell, tissue, and organ. In order to establish the validation system for the BHV-1 safety of the products, a real-time PCR method was developed for quantitative detection of BHV-1 in raw materials, manufacturing processes, and final products as well as BHV-1 clearance validation. Specific primers for amplification of BHV-1 DNA was selected, and BHV-1 DNA was quantified by use of SYBR Green I. The sensitivity of the assay was calculated to be $2\;TCID_{50}/ml$. The real-time PCR method was validated to be reproducible and very specific to BHV-1. The established real-time PCR assay was successfully applied to the validation of Chinese hamster ovary (CHO) cell artificially infected with BHV-1. BHV-1 DNA could be quantified in CHO cell as well as culture supernatant. Also the real-time PCR assay could detect $10\;TCID_{50}/ml$ of BHV-1 artificially contaminated in bovine collagen. The overall results indicated that this rapid, specific, sensitive, and robust assay can be reliably used for quantitative detection of BHV-1 contamination during the manufacture of biologics.

• Journal of Food Hygiene and Safety
• /
• v.15 no.3
• /
• pp.262-266
• /
• 2000

The polymerase chain reaction was used to selectively detect sequences within the fimbrial antigen of Salmonella enteritidis. Sterile milk was artificially inoculated with known amount of S. enteritidis and then DNA was extracted with guanidine thiocyanate/phenol/chloroform, followed by PCR. A detection limit of as few as 100 colony forming unit (cfu) per 0.5 ml milk was obtained with this method. For the whole procedure, it took only 5 h. A semi-quantitative polymerase chain reaction assay which allows an estimation of colony forming unit of S. enteritidis was developed. Known amount of standard plasmid pGem-4Z-Sef B(-) containing cloned S. enteritidis fimbrial antigen gene was co-amplified with Salmonella genomic DNA isolated from artificially inoculated milk. The same set of primers were used for the amplification and the products were cleaved with Bam HI. The concentration of the target DNA could be estimated by comparing the intensity of the two bands after electrophoresis. The PCR-based protocol described in this paper provides a rapid, simple, and sensitive method for detecting S. enteritidis in milk.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
• /
• v.30 no.6
• /
• pp.488-496
• /
• 2004

Oral & Maxillofacial surgery can lead to complications that result in abnormal sensation or movement. Inferior alveolar nerve(IAN) injury can result in dysesthesia, paresthsia of the lower lip and chin, so patients presenting with IAN damage suffer from sensory loss. But diagnosis of the nerve injury is largely limited to the subjective statements made by the patient. Distribution of sympathetic nerves parallels the distribution of the somatosensory nerves. Loss of sensory tone causes a concomitant loss of sympathetic activity, resulting in vasodilation of the cutaneous blood vessels that demonstrates greater heat loss. Digital infrared thermographic imaging(DITI) detects infra-red radiation given off by body. DITI can detect minute difference in temperature from different parts of the body and translates the amount of heat into quantitative data. The area of different temperature correlated with pain or disease can be visualized by corresponding color. The objective of this study was to determine the efficacy of DITI in objectively assessing IAN injury. The 19 normal subjects and the 14 patients underwent DITI scan. The normal subjects received unilateral IAN block anesthesia with 2 ml of 2% lidocaine (IAN bolck group) to evaluate temporary alteration in nerve function. Patient group were patients with unilateral IAN damage (dysesthesia or paresthesia) after surgical treatment(Mn. 3rd molar Extraction, etc.). The surgical procedure performed within 6 months of test. The results were as follows. 1. No significant differences in temperature were found between left and right sides of the lower lip and chin in the control group. 2. Significant temperature differences were found between the anesthetized and non-anesthetized sides of the lower lip and chin in the IAN block group. 3. Significant temperature differences were found between the involved and uninvolved sides of the lower lip and chin areas of the experimental group. The results of the study show that DITI can be an useful and effective means of objectively assessing and visualizing IAN damage.

• BMB Reports
• /
• v.30 no.6
• /
• pp.390-396
• /
• 1997

A sandwich-type enzyme-linked immunosorbent assay (ELISA) for the quantification of human apolipoprotein A-I (apoA-I) was developed using monoclonal antibodies. For this assay, we used three monoclonal antibodies to trap and detect apo A-I. HDAI16 and HDA15 monoclonal antibodies were used for trapping apoA-I and HDAI8 monoclonal antibody was for detecting apoA-I. These three monoclonal antibodies were produced by immunizing mice with high density lipoprotein (HDL) isolated from human plasma. By immunoblot analysis, these three monoclonal antibodies were specific to apoA-I and showed no cross-reactivities with other plasma proteins. The results of competition assays for epitope cross-reactivity test also verified that these monoclonal antibodies identified separate and distinct epitopes on HDL and apoA-I. Affinity constants of monoclonal antibodies were measured by ELISA. Their association constants ranged from $10^7$ to $10^8$ $M^{-1}$. For this assay, pure apoA-I was isolated by affinity chromatography using monoclonal antibodies. In this sandwich assay, the amount of HRP-labeled HDAI8 bound to apoA-I trapped by HDAI16 and HDAI5 was proportional to apoA-I concentration in the range of 0 to 500ng/ml. ApoA-I concentration in plasma was calculated from the linear regression equation of standard curve. The precision and reliability of the assays are reflected in the low intra-and interassay coefficients of variation that averaged 3.25% and 4.30%, respectively. This assay is sensitive, simple, reproducible, convenient in incubation interval, and does not use radioisotope: thus it can be widely applied in clinical laboratories.

• Journal of Microbiology and Biotechnology
• /
• v.27 no.1
• /
• pp.19-25
• /
• 2017

Staphylococcus aureus (S. aureus) is a common gram-positive bacterium that causes serious infections in humans and animals. With the continuous emergence of methicillin-resistant S. aureus (MRSA) strains, antibiotics have limited efficacy in treating MRSA infections. Accordingly, novel agents that act on new targets are desperately needed to combat these infections. S. aureus alpha-hemolysin plays an indispensable role in its pathogenicity. In this study, we demonstrate that sclareol, a fragrant chemical compound found in clary sage, can prominently decrease alpha-hemolysin secretion in S. aureus strain USA300 at sub-inhibitory concentrations. Hemolysis assays, western-blotting, and RT-PCR were used to detect the production of alpha-hemolysin in the culture supernatant. When USA300 was co-cultured with A549 epithelial cells, sclareol could protect the A549 cells at a final concentration of $8{\mu}g/ml$. The protective capability of sclareol against the USA300-mediated injury of A549 cells was further shown by cytotoxicity assays and live/dead analysis. In conclusion, sclareol was shown to inhibit the production of S. aureus alpha-hemolysin. Sclareol has potential for development as a new agent to treat S. aureus infections.

• Proceedings of the KSAR Conference
• /
• 2003.06a
• /
• pp.43-43
• /
• 2003

This study was conducted to produce transgenic pig harboring human tissue plasminogene activator (tPA) gene. Two different tPA genes containing bovine $\beta$-casein promoter and mouse uroplakin promoter were prepared for microinjection and confirmed the expression level of tPA protein from the CHO (Chinese hamster ovary) cell lines by gene transfection. Concentration of tPA expression from the six cell lines (all of CHO cells) were average 212.4 ng/ml. Reconstructed DNA to used the CHO cell were microinjected into the pronuclei of in vivo embryos The total of 2,307 zygotes were collected from 95 donors and 1,851 embryos were in 1-cell stage which were visualized the pronuclei for DNA microinjection. The concentration of linear DNA was 2.0 ng per microliter and injected into zygotes with two pronuclei on an inverted Nikon microscope equipped with narishige micromanipulator and modulation contrast optics. The 541 embryos injected with bovine $\beta$-casein promoter-tPA were transferred to 22 recipients. The 1,154 embryos injected with mouse uroplakin promoter-tPA were transferred to 51 recipients. Sixty nine offspring from 9 delivered sows were produced. We analysed the transgenes with PCR methods from 69 offsprings, but could not detect the PCR product from piglet tails DNA.

• Journal of Korean Ophthalmic Optics Society
• /
• v.18 no.2
• /
• pp.93-99
• /
• 2013

Purpose: The aim of this study was to develop a dry eye test method using a paper based microfluidic device that improves inaccuracy caused by using one of current point-of-care dry eye tests such as Shirmer's. Methods: Wax printed hydrophilic chromatography papers were dyed with anthocyanin extracts to detect colorimetric display of liquid samples with varying pH. Fluid distribution rates were measured using artificial tears and human tears directly from 32 subjects. Results: With Shirmer's, fluid distribution rates with small amount of samples (less than $0.5{\mu}l$) were not displayed. However, with paper based microfluidic device, fluid imbibition distances over time were clearly showed. Also clinical results of dry eye from newly developed paper based microfluidic device showed correlation with the results from tear break up time tests. Conclusions: The newly developed paper based microfluidic devices were easy to use and exhibited more accurate clinical results than current dry eye point of care tests such as Shirmer's.

• Journal of Microbiology and Biotechnology
• /
• v.27 no.5
• /
• pp.995-1004
• /
• 2017

Culture-dependent methods, such as heterotrophic plate counting (HPC), are usually applied to evaluate the bacteriological quality of hemodialysis water. However, these methods cannot detect the uncultured or viable but non-culturable (VBNC) bacteria, both of which may be quantitatively predominant throughout the hemodialysis water treatment system. Therefore, propidium monoazide (PMA)-qPCR associated with HPC was used together to profile the distribution of the total viable bacteria in such a system. Moreover, high-throughput sequencing of 16S rRNA gene amplicons was utilized to analyze the microbial community structure and diversity. The HPC results indicated that the total bacterial counts conformed to the standards, yet the bacteria amounts were abruptly enhanced after carbon filter treatment. Nevertheless, the bacterial counts detected by PMA-qPCR, with the highest levels of $2.14{\times}10^7copies/100ml$ in softener water, were much higher than the corresponding HPC results, which demonstrated the occurrence of numerous uncultured or VBNC bacteria among the entire system before reverse osmosis (RO). In addition, the microbial community structure was very different and the diversity was enhanced after the carbon filter. Although the diversity was minimized after RO treatment, pathogens such as Escherichia could still be detected in the RO effluent. In general, both the amounts of bacteria and the complexity of microbial community in the hemodialysis water treatment system revealed by molecular approaches were much higher than by traditional method. These results suggested the higher health risk potential for hemodialysis patients from the up-to-standard water. The treatment process could also be optimized, based on the results of this study.

• The Journal of the Institute of Internet, Broadcasting and Communication
• /
• v.18 no.1
• /
• pp.211-216
• /
• 2018

In this paper, we studied the way that classify whether unknown PE file is malware or not. In the classification problem of malware detection domain, feature extraction and classifier are important. For that purpose, we studied what the feature is good for classifier and the which classifier is good for the selected feature. So, we try to find the good combination of feature and classifier for detecting malware. For it, we did experiments at two step. In step one, we compared the accuracy of features using Opcode only, Win. API only, the one with both. We founded that the feature, Opcode and Win. API, is better than others. In step two, we compared AUC value of classifiers, Bernoulli Naïve Bayes, K-nearest neighbor, Support Vector Machine and Decision Tree. We founded that Decision Tree is better than others.

• Asian Pacific Journal of Cancer Prevention
• /
• v.16 no.15
• /
• pp.6407-6412
• /
• 2015

Purpose: To predict prostatic carcinoma using a logistic regression model on prebiopsy peripheral blood samples. Materials and Methods: Data of a total of 873 patients who consulted Urology Outpatient Clinics of Fatih Sultan Mehmet Training and Research Hospital between February 2008 and April 2014 scheduled for prostate biopsy were screened retrospectively. PSA levels, prostate volumes, prebiopsy whole blood cell counts, neutrophil and platelet counts, neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), biopsy results and Gleason scores in patients who had established diagnosis of prostate cancer (PCa) were evaluated. Results: This study was performed on a total of 873 cases, with an age range 48-76 years, divided into three groups as for biopsy results. with diagnoses of benign prostatic hyperplasia (BPH) (n=304, 34.8 %), PCa (n=265, 30.4 %) and histological prostatitis (n=304; 34.8 %). Intra- and intergroup comparative evaluations were performed. White blood cell and neutrophil counts in the histological prostatitis group were significantly higher than those of the BPH and PCa groups (p=0.001; p=0.004; p<0.01). A statistically significant intergroup difference was found for PLR (p=0.041; p<0.05) but not lymphocyte count (p>0.05). According to pairwise comparisons, PLR were significantly higher in the PCa group relative to BPH group (p=0.018, p<0.05, respectively). Though not statistically significant, higher PLR in cases with PCa in comparison with the prostatitis group was remarkable (p=0.067, and p>0.05, respectively). Conclusions: Meta-analyses showed that in patients with PSA levels over 4 ng/ml, positive predictive value of PSA is only 25 percent. Therefore, novel markers which can both detect clinically significant prostate cancer, and also prevent unnecessary biopsies are needed. Relevant to this issue in addition to PSA density, velocity, and PCA3, various markers have been analyzed. In the present study, PLR were found to be the additional predictor of prostatic carcinoma.