• Genomics & Informatics
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• v.13 no.2
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• pp.31-39
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• 2015

Sequencing depth, which is directly related to the cost and time required for the generation, processing, and maintenance of next-generation sequencing data, is an important factor in the practical utilization of such data in clinical fields. Unfortunately, identifying an exome sequencing depth adequate for clinical use is a challenge that has not been addressed extensively. Here, we investigate the effect of exome sequencing depth on the discovery of sequence variants for clinical use. Toward this, we sequenced ten germ-line blood samples from breast cancer patients on the Illumina platform GAII(x) at a high depth of ${\sim}200{\times}$. We observed that most function-related diverse variants in the human exonic regions could be detected at a sequencing depth of $120{\times}$. Furthermore, investigation using a diagnostic gene set showed that the number of clinical variants identified using exome sequencing reached a plateau at an average sequencing depth of about $120{\times}$. Moreover, the phenomena were consistent across the breast cancer samples.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.8
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• pp.3713-3716
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• 2012

Objective: Transmembrane protein 166 (TMEM166) expression in esophageal squamous cell carcinoma (ESCC) and remote normal esophageal tissues was examined to assess any role in tumour biology. Methods: TMEM166 mRNA expression in 36 cases with ESCC (36 tumour samples, 36 remote normal esophageal tissue samples) was detected by RT-PCR. TMEM166 protein expression was analysed in paraffin-embedded tissue samples from the same cases by immunohistochemistry. Results: Semi-quantitative analysis showed TMEM166 mRNA expression in ESCCs to be significantly lower than in remote normal esophageal tissues ($0.759{\pm}0.713$ vs. $2.622{\pm}1.690$, P=0.014). TMEM166 protein expression was also significantly reduced (69.4% vs. 94.4%, P<0.01). Conclusion: TMEM166 mRNA and protein expression demonstrated significant reduction in ESCCs compared with remote esophageal tissues, albeit with no correlation with tumour size, differentiation, stage, and lymph node metastasis, suggesting a role in regulating autophagic and apoptotic processes in the ESCC.

• Journal of Microbiology
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• v.43 no.3
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• pp.257-259
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• 2005

Although automated continuous-monitoring blood culture systems are both rapid and sensitive, false-positive and false-negative results still occur. The objective of this study, then, was to evaluate negative results occurring with BacT/Alert 3D blood culture systems. A total of 1032 samples were cultured with the BacT/Alert 3D automated blood culture system, using both aerobic (BPA) and anaerobic (BPN) media, and 128 of these samples yielded positive results. A total of 904 negative blood samples were then subcultured in $5\%$ sheep blood agar, eosin methylene blue, chocolate agar, and sabouraud-dextrose agar. Organisms growing on these subcultures were subsequently identified using both Vitek32 (bioMerieux, Durham, NC) and conventional methods. Twenty four $(2.6\%)$ of the 904 subcultures grew on the subculture media. The majority $(83.3\%)$ of these were determined to be gram-positive microorganisms. Fourteen $(58.3\%)$ were coagulase-negative staphylococci, two $(8.3\%)$ were Bacillus spp., one $(4.2\%)$ was Staphylococcus aureus, and one $(4.2\%)$ was identified as Enterococcus faecium. Streptococcus pneumoniae and Neisseria spp. were isolated together in two $(8.3\%)$ vials. Gram-negative microorganisms comprised $12.5\%$ of the subcultures, of which two $(8.3\%)$ were found to be Pseudomonas aeruginosa, and one $(4.2\%)$ was Pseudomonas fluorescens. The other isolate $(4.2\%)$ was identified as Candida albicans. We conclude that the subculture of negative results is valuable in the BacT/Alert 3D system, especially in situations in which only one set of blood cultures is taken.

• Korean Journal of Clinical Laboratory Science
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• v.36 no.2
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• pp.127-130
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• 2004

Analytical sensitivity on TDM test is the lowest concentration that can be distinguished from background noise. The aim of study was to evaluate analytical sensitivity that is also referred to as the lower limit of detection(LLD) about difference between zero calibrator and isotonic saline sample. We tested for 10 days with zero calibrators and 0.85% saline samples while running trilevel control samples under control. Raw data divided by two groups calculated mean and standard deviation from two sample populations and analytical sensitivity by ${\bar{X}}+2SD$. In comparison with isotonic saline samples and zero calibrators, there were significant differences in phenytoin, phenobarbital and vancomycin, etc. Especially analytical sensitivity on phenytoin is at the same level as the upper limit of analytical measurement range with $40{\mu}g/mL$. We think the cause of this is matrix interference. In conclusion, we were sure that standard protocol for analytical sensitivity as lower limit of analytical measurement range on TDM test must be measured with zero standard rather than an isotonic saline sample and type 1 reagent DW for reducing matrix effects within interactions between different materials in a mixture.

• YAKHAK HOEJI
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• v.42 no.5
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• pp.473-479
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• 1998

Lovastatin (LOVA), a fungal metabolite isolated from cultures of Aspergillus terreus, is a competitive HMG-CoA reductase inhibitor used for the treatment of primary hyper cholesterolemia, and has also been shown to suppress growth in a variety of non-glioma tumor cell lines. A sensitive reversed-phase high-perfonnance liquid chromatographic method with ultraviolet (UV) absorbance detection has been developed to quantitate LOVA in human plasma and urine samples using liquid-liquid extraction procedure. Baseline separation of LOVA and internal standard, simvastatin was achieved on a Novapak $C_{18}$ analytical column with a mobile phase containing 0.025M $NaH_2PO_4$: CAN (35:65, v/v%), adjusted pH to 4.5. The flow rate was set at 1.5ml/min, and the column effluent was monitored by a UV detection at 238nm. The limit of quantification was determined to be 0.5${\mu}$g/ml while extraction efficiency of LOVA ranged from 73.4-82.9% at LOVA concentrations of 0.5 to 10${\mu}$g/ml. Good linearity with correlation coefficients greater than 0.999 was obtained in the range of LOVA concentrations from 0.5 to 10${\mu}$g/ml. The accuracy and the precision were proven excellent with relative standard deviation (RSD, %) and relative error (RE, %) of less than 4.2 and 4.0, respectively. Intraday precision, evaluated at five LOVA concentrations (0.5, 1, 2, 5, 10${\mu}$g/ml) and expressed as RSD ranged from 0-1.82% while the interday precision at the same concentrations ranged from 0.7-10.5%. The analytical method described was then successfully employed for the determination of LOVA concentrations in plasma samples obtained during a phase II clinical trial using high doses of LOVA (30-40mg/kg/day). This method could be further utilized for the ongoing pharmacolkinetic studies and therapeutic drug monitoring of the high-dose LOVA therapy in adenocarcinoma patients.

• Biomolecules & Therapeutics
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• v.32 no.3
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• pp.379-389
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• 2024

This study was aimed to evaluate endogenous metabolic changes before and after cisplatin and radiation therapy in patients with cervical cancer via untargeted metabolomic analysis using plasma samples. A total of 13 cervical cancer patients were enrolled in this study. Plasma samples were collected from each patient on two occasions: approximately one week before therapy (P1) and after completion of cisplatin and radiation therapy (P2). Of the 13 patients, 12 patients received both cisplatin and radiation therapy, whereas one patient received radiation therapy alone. The samples were analyzed using the Ultimate 3000 coupled with Q Exactive^TM Focus Hybrid Quadrupole-Orbitrap^TM mass spectrometry (Thermo Fisher Scientific, Waltham, MA, USA). Chromatographic separation utilized a Kinetex C18 column 2.1×100 mm (2.6 ㎛) (Phenomenex, Torrance, CA, USA), and the temperature was maintained at 40℃. Following P2, there were statistically significant increases in the concentrations of indoxyl sulfate, phenylacetylglutamine, Lysophosphatidyethanolamine (LysoPE) (18:1), and indole-3-acetic acid compared with the concentrations observed at P1. Specifically, in the human papillomavirus (HPV) noninfection group, indoxyl sulfate, LysoPE (18:1), and phenylacetylglutamine showed statistically significant increases at P2 compared with P1. No significant changes in metabolite concentrations were observed in the HPV infection group. Indoxyl sulfate, LysoPE (18:1), phenylacetylglutamine, and indole-3-acetic acid were significantly increased following cisplatin and radiation therapy.

• Clinical Endoscopy
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• v.57 no.3
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• pp.384-392
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• 2024

Background/Aims: Endoscopic ultrasound-guided tissue acquisition (EUS-TA) is a standard diagnostic method for biliary tract cancer (BTC), and samples obtained in this manner may be used for comprehensive genomic profiling (CGP). This study evaluated the utility of EUS-TA for CGP in a clinical setting and determined the factors associated with the adequacy of CGP in patients with BTC. Methods: CGP was attempted for 105 samples from 94 patients with BTC at the Aichi Cancer Center, Japan, from October 2019 to April 2022. Results: Overall, 77.1% (81/105) of the samples were adequate for CGP. For 22-G or 19-G fine-needle biopsy (FNB), the sample adequacy was 85.7% (36/42), which was similar to that of surgical specimens (94%, p=0.45). Univariate analysis revealed that 22-G or larger FNB needle usage (86%, p=0.003), the target primary lesions (88%, p=0.015), a target size ≥30 mm (100%, p=0.0013), and number of punctures (90%, p=0.016) were significantly positively associated with CGP sample adequacy. Conclusions: EUS-TA is useful for CGP tissue sampling in patients with BTC. In particular, the use of 22-G or larger FNB needles may allow for specimen adequacy comparable to that of surgical specimens.

• Journal of Periodontal and Implant Science
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• v.50 no.5
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• pp.313-326
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• 2020

Purpose: This study was conducted to analyze specific RNA expression profiles in gingival tissue and saliva samples in periodontitis patients and healthy individuals, and to determine their correlations in light of the potential use of microarray-based analyses of saliva samples as a periodontal monitoring tool. Methods: Gingival tissue biopsies and saliva samples from 22 patients (12 with severe periodontitis and 10 with a healthy periodontium) were analyzed using transcriptomic microarray analysis. Differential gene expression was assessed, and pathway and clustering analyses were conducted for the samples. The correlations between the results for the gingival tissue and saliva samples were analyzed at both the gene and pathway levels. Results: There were 621 differentially expressed genes (DEGs; 320 upregulated and 301 downregulated) in the gingival tissue samples of the periodontitis group, and 154 DEGs (44 upregulated and 110 downregulated) in the saliva samples. Nine of these genes overlapped between the sample types. The periodontitis patients formed a distinct cluster group based on gene expression profiles for both the tissue and saliva samples. Database for Annotation, Visualization and Integrated Discovery analysis revealed 159 enriched pathways from the tissue samples of the periodontitis patients, as well as 110 enriched pathways In the saliva samples. Thirty-four pathways overlapped between the sample types. Conclusions: The present results indicate the possibility of using the salivary transcriptome to distinguish periodontitis patients from healthy individuals. Further work is required to enhance the extraction of available RNA from saliva samples.

• Korean Journal of Clinical Laboratory Science
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• v.45 no.1
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• pp.16-20
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• 2013

Outbreaks of vancomycin-resistant enterococci (VRE) are being reported more frequently in many countries. While seven glycopeptide resistance genotypes have been described in Enterococci, vanA and vanB are the most common resistance genotypes. The aim of this study was to detect antibiotic susceptibilities of 23 Enterococcus faecium strains, which caused an outbreak in a University hospital by a disk diffusion test to investigate the presence of the species specific gene, and the resistant genotypes, vanA and vanB by duplex PCR. PCR for vanA and vanB was performed on 23 enterococci. Twenty three were identified as E. faecium and were tested positive for the vanA genotype. This study will report on the validation of a simple and accurate VRE detection method that can be easily incorporated into the daily routine of a clinical laboratory. Early detection of VRE strains, including those with susceptibility to vancomycin, is of paramount clinical importance as it allows rapid initiation of strict infection control practices, as well as the therapeutic guidance for confirmed infections. The PCR method developed in the present study is simple and reliable for the rapid characterization of VRE.

• Parasites, Hosts and Diseases
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• v.58 no.2
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• pp.129-134
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• 2020

In most developing countries, Dientamoeba fragilis infection is an obscure protozoan infection. We aimed to determine a frequency and clinical importance of D. fragilis infection in Taif, Saudi Arabia. A 1-year case control study included patients with gastrointestinal (cases, n=114) or non-gastrointestinal symptoms (controls, n=90). The fecal samples were examined with the classical parasitological methods for intestinal protozoa, and by real time PCR for D. fragilis. The infection by D. fragilis was detected in 5.8% by PCR and in 4.4% patients by microscopy. The infection was identified more in control group (n=9) than in cases (n=3); a sole infection in 11 patients and mixed with Giardia in 1 patient. The other enteric parasites detected were Blastocystis sp. (8.3%), Giardia sp. (5.3%), Cryptosporidium sp. (2.9%), Entamoeba histolytica (1.4%), Entamoeba coli (0.9%) and Hymenolepis nana (0.4%). Our results tend to reinforce the need to increase awareness of D. fragilis infection in Saudi Arabia.