Targeted next-generation sequencing for detection of PIK3CA mutations in archival tissues from patients with Klippel-Trenaunay syndrome in an Asian population : List the full names and institutional addresses for all authors.

BACKGROUND: Klippel-Trenaunay syndrome (KTS) is a rare slow-flow combined vascular malformation with limb hypertrophy. KTS is thought to lie on the PIK3CA-related overgrowth spectrum, but reports are limited. PIK3CA encodes p110α, a catalytic subunit of phosphatidylinositol 3-kinase (PI3K) that plays an essential role in the PI3K/AKT/mammalian target of rapamycin (mTOR) signaling pathway. We aimed to demonstrate the clinical utility of targeted next-generation sequencing (NGS) in identifying PIK3CA mosaicism in archival formalin-fixed paraffin-embedded (FFPE) tissues from patients with KTS. RESULTS: Participants were 9 female and 5 male patients with KTS diagnosed as capillaro-venous malformation (CVM) or capillaro-lymphatico-venous malformation (CLVM). Median age at resection was 14 years (range, 5-57 years). Median archival period before DNA extraction from FFPE tissues was 5.4 years (range, 3-7 years). NGS-based sequencing of PIK3CA achieved an amplicon mean coverage of 119,000x. PIK3CA missense mutations were found in 12 of 14 patients (85.7%; 6/8 CVM and 6/6 CLVM), with 8 patients showing the hotspot variants E542K, E545K, H1047R, and H1047L. The non-hotspot PIK3CA variants C420R, Q546K, and Q546R were identified in 4 patients. Overall, the mean variant allele frequency for identified PIK3CA variants was 6.9% (range, 1.6-17.4%). All patients with geographic capillary malformation, histopathological lymphatic malformation or macrodactyly of the foot had PIK3CA variants. No genotype-phenotype association between hotspot and non-hotspot PIK3CA variants was found. Histologically, the vessels and adipose tissues of the lesions showed phosphorylation of the proteins in the PI3K/AKT/mTOR signaling pathway, including p-AKT, p-mTOR, and p-4EBP1. CONCLUSIONS: The PI3K/AKT/mTOR pathway in mesenchymal tissues was activated in patients with KTS. Amplicon-based targeted NGS could identify low-level mosaicism from low-input DNA extracted from FFPE tissues, potentially providing a diagnostic option for personalized medicine with inhibitors of the PI3K/AKT/mTOR signaling pathway.

Molecular Interactions between an Enzyme and Its Inhibitor for Selective Detection of Limonene.

Researchers widely apply enzyme inhibition to chemicals such as pesticides, nerve gases, and anti-Alzheimer's drugs. However, application of enzyme inhibition to odorant sensors is less common because the corresponding reaction mechanisms have not yet been clarified in detail. In this study, we propose a new strategy for highly selective detection of odorant molecules by using an inhibitor-specific enzyme. As an example, we analyzed the selective interactions between acetylcholinesterase (AChE) and limonene─the major odorant of citrus and an AChE inhibitor─using molecular dynamics simulations. In these simulations, limonene was found to be captured at specific binding sites of AChE by modifying the binding site of acetylcholine (ACh), which induced inhibition of the catalytic activity of AChE toward ACh hydrolysis. We confirmed the simulation results by experiments using an ion-sensitive field-effect transistor, and the degree of inhibition of ACh hydrolysis depended on the limonene concentration. Accordingly, we quantitatively detected limonene at a detection limit of 5.7 µM. We furthermore distinguished the response signals to limonene from those to other odorants, such as pinene and perillic acid. Researchers will use our proposed odorant detection method for other odorant-enzyme combinations and applications of miniaturized odorant-sensing systems based on rapid testing.

Wafer-scalable chemical modification of amino groups on graphene biosensors.

Graphene's remarkable attributes make it suitable for application to biosensors for biomolecular recognition. Specific and precise target detection is realized by designing robust methods for immobilization of probe molecules, such as oligonucleotides, antibodies, receptors, and sugar chains, to a device surface. In this research, we developed a chemical modification method with a plasma treatment of amino groups on natural defects of graphene, which is compatible with a wafer-scalable semiconductor process, to prevent deterioration of the carrier mobility. The plasma treatment was optimized in terms of the efficiency of the amino radical generation, length of the mean free path, and reaction energy on graphene. The density of the modified amino groups on graphene was approximately 0.065 groups/nm2, and the change in the ΔId/ΔVg characteristic of the graphene field-effect transistor (FET) was negligible. DNA probes were then attached to the amino groups on the graphene FET. The target complementary DNA was detected at 1 nM after hybridization using the graphene FET devices. The plasma-assisted modification of the amino groups on the graphene surface was developed for immobilization of the DNA probes, and hybridization with the target DNA was demonstrated without deterioration of the carrier mobility.

Immunological Gene Signature Associated With the Tumor Microenvironment of Pancreatic Cancer After Neoadjuvant Chemotherapy.

OBJECTIVES: Neoadjuvant chemotherapy (NAC) has improved overall survival in patients with pancreatic ductal adenocarcinoma (PDAC), but its effects on immune gene signatures are unknown. Here, we examined the immune transcriptome after NAC for PDAC. METHODS: Resected tumor specimens were obtained from 140 patients with PDAC who received surgery first (n = 93) or NAC (n = 47). Six patients were randomly selected from each group, and RNA was extracted from tumor tissues. We compared 770 immune-related genes among the 2 groups using nCounterPanCancer Immune Profiling (NanoString Technologies, Seattle, Wash). Gene clusters were classified into 14 immune function groups based on gene ontology argolism by nSolver 4.0 software (NanoString Technologies), and corresponding immune cell function scores were compared. RESULTS: Eleven genes (LY86, SH2D1A, CD247, TIGIT, CR2, CD83, LAMP3, CXCR4, DUSP4, SELL, and IL2RA) were significantly downregulated in the NAC group. Gene expression analysis showed that the functions of regulatory T cells, B cells, and natural killer CD56 dim cells were significantly decreased in the NAC group. CONCLUSIONS: Neoadjuvant chemotherapy may suppress regulatory T cells and B-cell function in the PDAC microenvironment. The 11 identified genes could be useful for predicting the efficacy of NAC and could be therapeutic targets for PDAC.

Development, validation, and comparison of gene analysis methods for detecting EGFR mutation from non-small cell lung cancer patients-derived circulating free DNA.

The feasibility and required sensitivity of circulating free DNA (cfDNA)-based detection methods in second-line epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) treatment are not well elucidated. We examined T790M and other activating mutations of EGFR by cfDNA to assess the clinical usability. In 45 non-small cell lung cancer (NSCLC) patients harboring activating EGFR mutations, cfDNAs were prepared from the plasma samples. EGFR mutations in cfDNA were detected using highly sensitive methods and originally developed assays and these results were compared to tissue-based definitive diagnoses. The specificity of each cfDNA-based method ranged 96-100% whereas the sensitivity ranged 56-67%, indicating its low pseudo-positive rate. In EGFR-TKI failure cohort, 41-46% samples were positive for T790M by each cfDNA-based method, which was comparable to re-biopsy tissue-based T790M positive rates in literature. The concordance of the results for each EGFR mutation ranged from 83-95%. In eight patients, the results of the cfDNA-based assays and re-biopsy-derived tissue-based test were compared. The observed overall agreement ranged in 50-63% in T790M, and in 63-100% in activating EGFR mutations. In this study, we have newly developed three types of assay which have enough sensitivity to detect cfDNA. We also detected T790M in 44% of patients who failed prior EGFR-TKI treatment, indicating that cfDNA-based assay has clinical relevance for detecting acquired mutations of EGFR.

Ionic Liquids with Wafer-Scalable Graphene Sensors for Biological Detection.

Ionic liquids, known as non-volatile solvents, have potential for realizing microanalysis with a minute quantity of sample. Here we report the measurement of the Id-Vg characteristics during the enzymatic catalytic reactions and streptavidin-biotin binding in ionic liquids by using the graphene FET sensors we fabricated, and successfully monitored the biological reactions in much smaller amount of solvents. These findings suggest the possibility of ionic liquids for application in bio-microanalysis with high sensitivity.

Interrelation among the handling, mechanical, and wear properties of the newly developed flowable resin composites.

OBJECTIVES: This study investigates the handling, mechanical, and wear properties of the newly developed flowable resin composites and elucidate the interrelations among the tested parameters. METHODS: Six flowable and two conventional resin composites are used. Five measurements are performed per resin composite to obtain the average inorganic filler content. Ten specimens per material are used to obtain the flexural strength, flexural modulus, and resilience. For sliding impact wear testing, twelve specimens are prepared. Noncontact profilometer and confocal laser scanning microscopy are used to determine the maximum facet depth and volume loss. Extrusion force and thread formation are used to measure the handling properties of the flowable resin composites. Six measurements are performed per flowable resin composite. Data evaluation is performed using analysis of variance and Tukey's honestly significant difference test at an α-level of 0.05. The correlation between the tested parameters is verified using the Pearson product-moment correlation coefficient. RESULTS: A subset of flowable resin composites exhibits higher flexural properties and wear resistance as compared to the conventional resin composites. The handling properties of the flowable resin composites are material dependent. CONCLUSION: While the resilience parameters exhibit an extremely strong and statistically significant correlation with the wear parameters, the handling properties exhibit no interrelation with the remaining parameters. SIGNIFICANCE: While the handling properties of the newly developed flowable resin composites did not correlate with the mechanical and wear properties, some new flowable resin composites have the potential for use in high-stress bearing areas, such as posterior lesions, because of the enhanced mechanical properties and wear resistance.

Regulation of replication at the R/G chromosomal band boundary and pericentromeric heterochromatin of mammalian cells.

Mammalian chromosomes consist of multiple replicons; however, in contrast to yeast, the details of this replication process (origin firing, fork progression and termination) relative to specific chromosomal domains remain unclear. Using direct visualization of DNA fibers, here we show that the rate of replication fork movement typically decreases in the early-mid S phase when the replication fork proceeds through the R/G chromosomal band boundary and pericentromeric heterochromatin. To support this, fluorescence in situ hybridization (FISH)-based replication profiles at the human 1q31.1 (R-band)-32.1 (G-band) regions revealed that replication timing switched around at the putative R/G chromosomal band boundary predicted by marked changes in GC content at the sequence level. Thus, the slowdown of replication fork movement is thought to be the general property of the band boundaries separating the functionally different chromosomal domains. By simultaneous visualization of replication fork movement and pericentromeric heterochromatin sequences on DNA fibers, we observed that this region is duplicated by many replication forks, some of which proceed unidirectionally, that originate from clustered replication origins. We showed that histone hyperacetylation is tightly associated with changes in the replication timing of pericentromeric heterochromatin induced by 5-aza-2'-deoxycytidine treatment. These results suggest that, similar to the yeast system, histone modification is involved in controlling the timing of origin firing in mammals.