Application of a Combination of a Knowledge-Based Algorithm and 2-Stage Screening to Hypothesis-Free Genomic Data on Irinotecan-Treated Patients for Identification of a Candidate Single Nucleotide Polymorphism Related to an Adverse Effect

Interindividual variation in a drug response among patients is known to cause serious problems in medicine. Genomic information has been proposed as the basis for “personalized” health care. The genome-wide association study (GWAS) is a powerful technique for examining single nucleotide polymorphisms (SNPs) and their relationship with drug response variation; however, when using only GWAS, it often happens that no useful SNPs are identified due to multiple testing problems. Therefore, in a previous study, we proposed a combined method consisting of a knowledge-based algorithm, 2 stages of screening, and a permutation test for identifying SNPs. In the present study, we applied this method to a pharmacogenomics study where 109,365 SNPs were genotyped using Illumina Human-1 BeadChip in 168 cancer patients treated with irinotecan chemotherapy. We identified the SNP rs9351963 in potassium voltage-gated channel subfamily KQT member 5 (KCNQ5) as a candidate factor related to incidence of irinotecan-induced diarrhea. The p value for rs9351963 was 3.31×10⁻⁵ in Fisher''s exact test and 0.0289 in the permutation test (when multiple testing problems were corrected). Additionally, rs9351963 was clearly superior to the clinical parameters and the model involving rs9351963 showed sensitivity of 77.8% and specificity of 57.6% in the evaluation by means of logistic regression. Recent studies showed that KCNQ4 and KCNQ5 genes encode members of the M channel expressed in gastrointestinal smooth muscle and suggested that these genes are associated with irritable bowel syndrome and similar peristalsis diseases. These results suggest that rs9351963 in KCNQ5 is a possible predictive factor of incidence of diarrhea in cancer patients treated with irinotecan chemotherapy and for selecting chemotherapy regimens, such as irinotecan alone or a combination of irinotecan with a KCNQ5 opener. Nonetheless, clinical importance of rs9351963 should be further elucidated.     

Facile preparation of DNA-tagged carbohydrates

We report here that unprotected carbohydrates (maltose, lactose, cellobiose, and maltoheptaose) can be attached to the aminoalkylated oligonucleotides under mild reductive-amination conditions (aqueous borate buffer, pH 8.0, NaBH(3)CN, 60 degrees C) without notable side reactions. Quadruplex-forming G-rich oligonucleotide, 5'-aminoalkyl d(TGGGGT), is glycosylated with maltoheptaose to afford a novel DNA-assisted tetrasaccharide cluster motif.     

An efficient and practical method for solid-phase synthesis of tripeptide-bearing glycopeptide antibiotics: combinatorial parallel synthesis of carboxamide derivatives of chloroorienticin B

An efficient and practical method was established for solid-phase parallel synthesis of the peptide-bearing carboxamide derivatives of chloroorienticin B, and over 80 compounds were synthesized simultaneously. Among the derivatives prepared, compounds having both tryptophan and tyrosine residues (1-3) were found to possess potent antibacterial activity against VRE.     

Involvement of cytochrome c and caspases in apoptotic cell death of human submandibular gland ductal cells induced by concanamycin A

In the present study, we found that a specific inhibitor of vacuolar type H+-ATPase (V-ATPase), concanamycin A, induced apoptosis in a human submandibular gland ductal cancer cell line, HSG. Immunoblot analysis revealed that cytochrome c was released from mitochondria into the cytoplasm when HSG cells were cultured with concanamycin A for 6 h. The maximum activities of caspase-3 and -9 were reached in HSG cells after 18 and 12 h culture of concanamycin A, respectively. Both caspase-3 and -9 were cleaved to an active form in HSG cells cultured with concanamycin A. Interestingly, concanamycin A decreased the level of heat shock protein 27 (HSP27) in HSG cells. Taken together, these findings suggest that apoptosis in HSG cells induced by concanamycin A is regulated by cytochrome c released from mitochondria into cytoplasm and the subsequent activation of caspases, and that HSP27 may interfere with caspase-dependent apoptotic cell death induced by concanamycin A.     

Differentiation of neural cells in the fetal cerebral cortex of cynomolgus monkeys (Macaca fascicularis)

Proliferation and programmed cell death are important in the formation of morphologic structures and functional activity during CNS development. We used immunohistochemical and TUNEL methods to examine the proliferation and differentiation of neural cells in, distribution of apoptotic cells in, and microglial cell involvement in the removal of apoptotic cells from the fetal cerebral cortex of cynomolgus monkeys. At embryonic day (E) 50 and E80, the neuroepithelium contained many mitotic cells. Cells staining for PCNA (a nuclear marker of proliferating cells) were prominent in the proliferative zone, whereas cells positive for NeuN (a neuron-specific marker) were absent. GFAP staining for glial cells was positive in the neuroepithelium and radial glial fibers. Iba1-positive cells (that is, macrophages and microglia) were distributed throughout all regions at all time points but accumulated especially in the ventricular zone at E80. Apoptotic morphology (at E80) and TUNEL-positive cells (that is, containing DNA fragmentation; at E50 and E80) were observed also. At E120 and E150, most PCNA-positive cells were in the ventricular zone, and NeuN-positive cells were prominent in all layers except layer I-II at E120. GFAP immunoreactivity was detected mainly in cells with fine processes in the white matter. Neither apoptosis nor TUNEL-positive cells were detected at either E120 or E150. These results suggest that proliferation, migration, and neural cell death occur during midgestation (that is, E50 to E80) in fetal brain of cynomolgus macaques, whereas differentiation and maturation of neural cells occur after midgestation (E80).     

Phylogeographical structure in Zelkova serrata in Japan and phylogeny in the genus Zelkova using the polymorphisms of chloroplast DNA

The genetic differentiation inherent in Zelkova serrata in Japan and the southern portion of the Korean Peninsula was examined by comparing a chloroplast DNA (cpDNA) sequence over a 16?k baselength in 40 individual samples collected from an area covering the natural distribution range of Z. serrata in Japan and the southern portion of the Korean Peninsula. We detected over 50 single-nucleotide polymorphisms in the protein-coding and intergenic regions, and over 30 insertions/deletions in the intergenic region. From the polymorphisms detected in the cpDNA, 14 haplotypes were identified. These 14 haplotypes had cluster-like structures and genetic differentiation between the clusters was large. Closely related haplotypes existed in adjacent regions. One haplotype existed in both Japan and the Korean Peninsula. By comparison with other Zelkova species, Z. serrata is apparently distinct from European and East Asian Zelkova species and Z. serrata is closest to the Ulmus species in the genus Zelkova. The effects of the analyzed length of the cpDNA sequence on the detection of polymorphisms were analyzed by re-sampling simulation.     

Color standardization method and system for whole slide imaging based on spectral sensing

In the field of whole slide imaging, the imaging device or staining process cause color variations for each slide that affect the result of image analysis made by pathologist. In order to stabilize the analysis, we developed a color standardization method and system as described below. (1) Color standardization method based on RGB imaging and multi spectral sensing, which utilize less band (16 bands) than conventional method (60 bands). (2) High speed spectral sensing module. As a result, we confirmed the following effect. (1) We confirmed the performance improvement of nucleus detection by the color standardization. And we can conduct without training data set which is needed in conventional method. (2) We can get detection performance of H&E component equivalent to conventional method (60 bands). And measurement process is more than 255 times faster.     

Immunohistochemical profile for unknown primary adenocarcinoma

Background

Development of tailored treatment based on immunohistochemical profiles (IPs) of tumors for cancers of unknown primary is needed.

Methodology/Principal Findings

We developed an algorithm based on primary known adenocarcinoma for testing sensitivity and specificity. Formalin-fixed paraffin-embedded tissue samples from 71 patients of unfavorable subsets of unknown primary adenocarcinoma were obtained. We examined 15 molecular markers using the algorithm incorporating these IPs and classified the tumours into 9 subsets based on the primary tumour site. The sensitivity and specificity of this algorithm were 80.3% and 97.6%, respectively. Apparent primary sites were lung in 17 patients, digestive organs in 13, gynecological organs in 9, prostate in 7, liver or kidney in 6, breast in 4, urothelial organ in 2, biliary tract and pancreatic profile in none, and unclassified in 13. The response rate to chemotherapy was highest for the gynecological IPs. Patients with gynecological or lung cancer IPs had longer median progression-free survival than those with others: 11.2 months for gynecological IPs (p<0.001) and 6.8 months for lung IPs (p = 0.05). Lung, digestive, prostate, and gynecological profiles were associated with significantly longer median survival time than the other profiles. Multivariate analysis confirmed that the IPs were independent prognostic factors for survival.

Conclusions/Significance

The IPs identified in this study can be used to further stratify patient prognosis for unfavorable subsets of unknown primary adenocarcinoma.