Generation of “Virtual” Control Groups for Single Arm Prostate Cancer Adjuvant Trials

It is difficult to construct a control group for trials of adjuvant therapy (Rx) of prostate cancer after radical prostatectomy (RP) due to ethical issues and patient acceptance. We utilized 8 curve-fitting models to estimate the time to 60%, 65%, … 95% chance of progression free survival (PFS) based on the data derived from Kattan post-RP nomogram. The 8 models were systematically applied to a training set of 153 post-RP cases without adjuvant Rx to develop 8 subsets of cases (reference case sets) whose observed PFS times were most accurately predicted by each model. To prepare a virtual control group for a single-arm adjuvant Rx trial, we first select the optimal model for the trial cases based on the minimum weighted Euclidean distance between the trial case set and the reference case set in terms of clinical features, and then compare the virtual PFS times calculated by the optimum model with the observed PFSs of the trial cases by the logrank test. The method was validated using an independent dataset of 155 post-RP patients without adjuvant Rx. We then applied the method to patients on a Phase II trial of adjuvant chemo-hormonal Rx post RP, which indicated that the adjuvant Rx is highly effective in prolonging PFS after RP in patients at high risk for prostate cancer recurrence. The method can accurately generate control groups for single-arm, post-RP adjuvant Rx trials for prostate cancer, facilitating development of new therapeutic strategies.     

Comprehensive Analysis to Improve the Validation Rate for Single Nucleotide Variants Detected by Next-Generation Sequencing

Next-generation sequencing (NGS) has enabled the high-throughput discovery of germline and somatic mutations. However, NGS-based variant detection is still prone to errors, resulting in inaccurate variant calls. Here, we categorized the variants detected by NGS according to total read depth (TD) and SNP quality (SNPQ), and performed Sanger sequencing with 348 selected non-synonymous single nucleotide variants (SNVs) for validation. Using the SAMtools and GATK algorithms, the validation rate was positively correlated with SNPQ but showed no correlation with TD. In addition, common variants called by both programs had a higher validation rate than caller-specific variants. We further examined several parameters to improve the validation rate, and found that strand bias (SB) was a key parameter. SB in NGS data showed a strong difference between the variants passing validation and those that failed validation, showing a validation rate of more than 92% (filtering cutoff value: alternate allele forward [AF]≥20 and AF<80 in SAMtools, SB<–10 in GATK). Moreover, the validation rate increased significantly (up to 97–99%) when the variant was filtered together with the suggested values of mapping quality (MQ), SNPQ and SB. This detailed and systematic study provides comprehensive recommendations for improving validation rates, saving time and lowering cost in NGS analyses.     

The species range‐size patterns for vascular plants of Seorak Mountain (Korea): Relationship between group of life forms and phytogeography affinity along the elevational gradient

Research on species richness patterns and the advanced elevational Rapoport rule (ERR) has been widespread in recent years; however, there is a lack of such research for the temperate mountainous regions in northeast Asia. Here, we collected plant species from the Seorak Mountain in northeast Asia through field surveys. The species were divided into 11 groups according to the life‐form types and phytogeography affinities of each species. The ERR was evaluated using Steven''s method and by examining the species richness patterns of each group. The species richness patterns revealed a positive multimodal pattern along the elevation gradient, but phytogeography affinities (increasing trend) and life‐form analysis (unimodal) exhibited different patterns. The elevation gradients (1,350 m for the mean elevation–range relationships), which are affected by the boundary effect and different life forms, did not consistently support the ERR. However, herbs as well as rare, endemic, and red list species showed consistent support for the ERR, which could be attributed to the influence by phytogeography affinities. Therefore, the results from Seorak Mountain showed that the ERR was not consistent for different plant life forms in the same area; however, phytogeography affinities could support and explain ERR.     

Development of a monitoring vector for Leuconostoc mesenteroides using the green fluorescent protein gene

The vector pCW5 with plasmid pC7, originally isolated in Lactobacillus paraplantarum C7 derived from kimchi, was constructed using a p32 strong promoter, the pC7 replicon, and green fluorescent protein (GFP) as the reporter. The constructed vector was transformed into E. coli and Leuconostoc mesenteroides, and GFP expression detected using a Western blot analysis. GFP fluorescence was recognized in E. coli and Leuconostoc mesenteroides using a confocal microscope. In addition, GFP fluorescence was also clearly detected in several industrially important lactic acid bacteria (LAB), including Lactobacillus bulgaricus, Lactobacillus paraplantarum, and Lactobacillus plantarum. Thus, pCW5 was shown to be effective for Leuconostoc mesenteroides when using GFP as the reporter, and it can also be used as a broad-host-range vector for other lactic acid bacteria.     

Role of NKG2D in Obesity-Induced Adipose Tissue Inflammation and Insulin Resistance

The early events that initiate inflammation in the adipose tissue during obesity are not well defined. It is unclear whether the recruitment of CD8 T cells to the adipose tissue during onset of obesity occurs through antigen-dependent or -independent processes. We have previously shown that interaction between NKG2D (natural-killer group 2, member D) and its ligand Rae-1ε is sufficient to recruit cytotoxic T lymphocytes to the pancreas and induce insulitis. Here, we tested whether NKG2D–NKG2D ligand interaction is also involved in obesity-induced adipose tissue inflammation and insulin resistance. We observed a significant induction of NKG2D ligand expression in the adipose tissue of obese mice, especially during the early stages of obesity. However, mice lacking NKG2D developed similar levels of insulin resistance and adipose tissue inflammation compared to control mice when placed on a high-fat diet. Moreover, overexpression of Rae-1ε in the adipose tissue did not increase immune cell infiltration to the adipose tissue either in the setting of a normal or high-fat diet. These results indicate that, unlike in the pancreas, NKG2D–NKG2D ligand interaction does not play a critical role in obesity-induced inflammation in the adipose tissue.     

The Postnatal Development of the Cerebellum— A fMRI and Silver Study

Summary The aim of this study was to evaluate the postnatal development of the cerebella of the pig and to compare this with the activation of the fMRI. The cells in the cerebella were studied by silver technique and the activation of the fMRI in the cerebella was initiated by flexion and extension of the hind paw. Our results showed an increase of the branching of the cells of the cerebellar cortex postnatally, coordinated with registration of fMRI active sites in the cerebella at 6-month postnatal. We concluded that the full maturation of the cerebella was around 6-month postnatal in the pig.     

A rapid and simple respirometric biosensor with immobilized cells of Nitrosomonas europaea for detecting inhibitors of ammonia oxidation

As obligate chemolithotrophs, ammonia-oxidizing bacteria (AOB) grow very slowly and are known to be extremely sensitive to a wide variety of inhibitors. Since it is generally accepted that inhibition of ammonia oxidation by AOB results in a total failure of nitrogen removal, it is necessary to develop a method to detect inhibitors of ammonia oxidation in wastewater. Since ammonia oxidation accompanies oxygen consumption, ammonia oxidation can be easily evaluated by measuring oxygen consumption rate using a dissolved oxygen (DO) probe. In this study, a rapid and simple respirometric biosensor using the pure culture of Nitrosomonas europaea was developed. N. europaea was cultivated in a continuous fermentor operating at the dilution rate of 0.008 h(-1) to obtain physiologically constant cells and was immobilized onto the dialysis membrane through filtration. DO, determined by the biosensor, started to increase 30 s later after ammonia oxidation inhibitor was fed, and a new steady-state DO was obtained in 10-30 min. For this DO profile, steady-state kinetics was applied to evaluate ammonia oxidation efficiency. The concentration of a toxic compound causing 50% decrease of oxygen-consumption activity (EC50) was determined for different chemicals. The EC50 values obtained with the biosensor (0.018 mg l(-1) for allylthiourea, 0.027 mg l(-1) for thioacetamide, 1.10 mg l(-1) for phenol and 0.0 1mg l(-1) for thiourea) indicated that the developed biosensor was highly sensitive to a variety of the inhibitors. It was also shown that the biosensor is applicable for on-line real time monitoring.