Synthesis and herbicidal activity of optically active cinmethylin, its enantiomer, and C3-substituted cinmethylin analogs.

We investigated the synthesis and herbicidal activity of optically active cinmethylin, its enantiomer, and C3-substituted cinmethylin analogs. Optically active cinmethylin could be obtained in seven steps with the Sharpless asymmetric dihydroxylation of α-terpinene. The synthesized cinmethylin and its enantiomer showed similar herbicidal activity, which was independent of the stereochemistry. Next, we synthesized cinmethylin analogs with various substituents at the C3 position. We found that analogs with methylene, oxime, ketone, or methyl groups at the C3 position show excellent herbicidal activity.

Efficient and accurate KRAS genotyping using digital PCR combined with melting curve analysis for ctDNA from pancreatic cancer patients.

A highly sensitive and highly multiplexed quantification technique for nucleic acids is necessary to predict and evaluate cancer treatment by liquid biopsy. Digital PCR (dPCR) is a highly sensitive quantification technique, but conventional dPCR discriminates multiple targets by the color of the fluorescent dye of the probe, which limits multiplexing beyond the number of colors of fluorescent dyes. We previously developed a highly multiplexed dPCR technique combined with melting curve analysis. Herein, we improved the detection efficiency and accuracy of multiplexed dPCR with melting curve analysis to detect KRAS mutations in circulating tumor DNA (ctDNA) prepared from clinical samples. The mutation detection efficiency was increased from 25.9% of the input DNA to 45.2% by shortening the amplicon size. The limit of detection of mutation was improved from 0.41 to 0.06% by changing the mutation type determination algorithm for G12A, resulting in a limit of detection of less than 0.2% for all the target mutations. Then, ctDNA in plasma from pancreatic cancer patients was measured and genotyped. The measured mutation frequencies correlated well with those measured by conventional dPCR, which can measure only the total frequency of KRAS mutants. KRAS mutations were detected in 82.3% of patients with liver or lung metastasis, which was consistent with other reports. Accordingly, this study demonstrated the clinical utility of multiplex dPCR with melting curve analysis to detect and genotype ctDNA from plasma with sufficient sensitivity.

10-Plex Digital Polymerase Chain Reaction with Four-Color Melting Curve Analysis for Simultaneous KRAS and BRAF Genotyping.

Digital PCR (dPCR) is a promising method for performing liquid biopsies that quantifies nucleic acids more sensitively than real-time PCR. However, dPCR shows large fluctuations in the fluorescence intensity of droplets or wells due to insufficient PCR amplification in the small partitions, limiting the multiplexing capability of using the fluorescence intensity. In this study, we propose a measurement method that combines dPCR with melting curve analysis for highly multiplexed genotyping. A sample was digitized into a silicon chip with up to 2 × 104 wells in which asymmetric PCR was performed to obtain more single-stranded amplicons that were complementary to molecular beacon probes. Fluorescence images were captured while controlling the temperature of the chip, and the melting curve was measured for each well. Then, genotyping was performed by using the fluorescence intensity, the dye color of the probe, and the melting temperature (Tm). Because the Tm of the PCR products is not highly dependent on the amplification efficiency of PCR, genotyping accuracy is improved by using Tm values, enabling highly multiplexed genotyping. The concept was confirmed by simultaneously identifying wild-type KRAS, BRAF, and eight mutants of these genes (G12D, G12R, G12V, G13D, G12A, G12C, G12S, and V600E) through four-color melting curve analysis. To the best of our knowledge, this is the first demonstration of the genotyping of 10 DNA groups including single mutations of cancer-related genes by combining dPCR with four-color melting curve analysis.

KRAS genotyping by digital PCR combined with melting curve analysis.

Digital PCR (dPCR) has been developed as a method that can quantify nucleic acids more sensitively than real-time PCR. However, dPCR exhibits large fluctuations in the fluorescence intensity of the compartment, resulting in low accuracy. The main cause is most likely due to insufficient PCR. In this study, we proposed a new method that combines dPCR with melting curve analysis and applied that method to KRAS genotyping. Since the melting temperature (Tm) of the PCR product hardly depends on the amplification efficiency, genotyping accuracy is improved by using the Tm value. The results showed that the peaks of the distribution of the Tm values of DNA in the wells were 68.7, 66.3, and 62.6 °C for wild-type KRAS, the G12R mutant, and the G12D mutant, respectively, and the standard deviation of the Tm values was 0.2 °C for each genotype. This result indicates that the proposed method is capable of discriminating between the wild-type sequence and the two mutants. To the best of our knowledge, this is the first demonstration of the genotyping of single mutations by combining melting curve analysis and dPCR. The application of this approach could be useful for the quantification and genotyping of cancer-related genes in low-abundance samples.

Extracellular Zn2+ Is Essential for Amyloid ß1-42-Induced Cognitive Decline in the Normal Brain and Its Rescue.

Brain Aß1-42 accumulation is considered an upstream event in pathogenesis of Alzheimer's disease. However, accumulating evidence indicates that other neurochemical changes potentiate the toxicity of this constitutively generated peptide. Here we report that the interaction of Aß1-42 with extracellular Zn2+ is essential for in vivo rapid uptake of Aß1-42 and Zn2+ into dentate granule cells in the normal rat hippocampus. The uptake of both Aß1-42 and Zn2+ was blocked by CaEDTA, an extracellular Zn2+ chelator, and by Cd2+, a metal that displaces Zn2+ for Aß1-42 binding. In vivo perforant pathway LTP was unaffected by perfusion with 1000 nm Aß1-42 in ACSF without Zn2+ However, LTP was attenuated under preperfusion with 5 nm Aß1-42 in ACSF containing 10 nm Zn2+, recapitulating the concentration of extracellular Zn2+, but not with 5 nm Aß1-40 in ACSF containing 10 nm Zn2+ Aß1-40 and Zn2+ were not taken up into dentate granule cells under these conditions, consistent with lower affinity of Aß1-40 for Zn2+ than Aß1-42 Aß1-42-induced attenuation of LTP was rescued by both CaEDTA and CdCl2, and was observed even with 500 pm Aß1-42 Aß1-42 injected into the dentate granule cell layer of rats induced a rapid memory disturbance that was also rescued by coinjection of CdCl2 The present study supports blocking the formation of Zn-Aß1-42 in the extracellular compartment as an effective preventive strategy for Alzheimer's disease.SIGNIFICANCE STATEMENT Short-term memory loss occurs in normal elderly and increases in the predementia stage of Alzheimer's disease (AD). Amyloid-ß1-42 (Aß1-42), a possible causing peptide in AD, is bound to Zn2+ in the extracellular compartment in the hippocampus induced short-term memory loss in the normal rat brain, suggesting that extracellular Zn2+ is essential for Aß1-42-induced short-term memory loss. The evidence is important to find an effective preventive strategy for AD, which is blocking the formation of Zn-Aß1-42 in the extracellular compartment.

Preventive Effect of 3,5-dihydroxy-4-methoxybenzyl Alcohol (DHMBA) and Zinc, Components of the Pacific Oyster Crassostrea gigas, on Glutamatergic Neuron Activity in the Hippocampus.

The effects of 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), and zinc--both components of the Pacific oyster Crassostrea gigas--were examined by glutamatergic neuron activity in rats in an in vivo microdialysis experiment and an in vitro brain slice experiment. The basal concentration of extracellular glutamate in the hippocampus was decreased under hippocampal perfusion with DHMBA (1 mmol l(-1)) or ZnCl2 (µmol l(-1)), indicating that DHMBA and Zn(2+) suppress glutamatergic neuron activity under basal (static) conditions. To assess the preventive effect of DHMBA and Zn(2+) on glutamate release from neuron terminals, brain slices were pretreated with DHMBA (1 mmol l(-1)) or ZnCl2 (100 nmol l(-1)) for 1 h, then stimulated with high K(+). A high, K(+)-induced increase in extracellular Zn(2+) level, an index of glutamate release, was suppressed with pretreatment with DHMBA or zinc. A high, K(+)-induced increase in intracellular Ca(2+) level was also suppressed with pretreatment with DHMBA or Zn(2+). These results suggest that DHMBA and Zn(2+), previously taken up in the hippocampal cells, suppress high, K(+)-induced glutamate release in the hippocampus, probably via presynaptic suppression of intracellular Ca(2+) signaling. It is likely that Zn(2+) and DHMBA play a preventive role in suppressing excess glutamatergic neuron activity in rats and mice.

Amyloid ß-mediated Zn2+ influx into dentate granule cells transiently induces a short-term cognitive deficit.

We examined an idea that short-term cognition is transiently affected by a state of confusion in Zn2+ transport system due to a local increase in amyloid-ß (Aß) concentration. A single injection of Aß (25 pmol) into the dentate gyrus affected dentate gyrus long-term potentiation (LTP) 1 h after the injection, but not 4 h after the injection. Simultaneously, 1-h memory of object recognition was affected when the training was performed 1 h after the injection, but not 4 h after the injection. Aß-mediated impairments of LTP and memory were rescued in the presence of zinc chelators, suggesting that Zn2+ is involved in Aß action. When Aß was injected into the dentate gyrus, intracellular Zn2+ levels were increased only in the injected area in the dentate gyrus, suggesting that Aß induces the influx of Zn2+ into cells in the injected area. When Aß was added to hippocampal slices, Aß did not increase intracellular Zn2+ levels in the dentate granule cell layer in ACSF without Zn2+, but in ACSF containing Zn2+. The increase in intracellular Zn2+ levels was inhibited in the presence of CaEDTA, an extracellular zinc chelator, but not in the presence of CNQX, an AMPA receptor antagonist. The present study indicates that Aß-mediated Zn2+ influx into dentate granule cells, which may occur without AMPA receptor activation, transiently induces a short-term cognitive deficit. Extracellular Zn2+ may play a key role for transiently Aß-induced cognition deficits.

Absolute quantification of the budding yeast transcriptome by means of competitive PCR between genomic and complementary DNAs.

BACKGROUND: An ideal format to describe transcriptome would be its composition measured on the scale of absolute numbers of individual mRNAs per cell. It would help not only to precisely grasp the structure of the transcriptome but also to accelerate data exchange and integration. RESULTS: We conceived an idea of competitive PCR between genomic DNA and cDNA. Since the former contains every gene exactly at the same copy number, it can serve as an ideal normalization standard for the latter to obtain stoichiometric composition data of the transcriptome. This data can then be easily converted to absolute quantification data provided with an appropriate calibration. To implement this idea, we improved adaptor-tagged competitive PCR, originally developed for relative quantification of the 3'-end restriction fragment of each cDNA, such that it can be applied to any restriction fragment. We demonstrated that this "generalized" adaptor-tagged competitive PCR (GATC-PCR) can be performed between genomic DNA and cDNA to accurately measure absolute expression level of each mRNA in the budding yeast Saccharomyces cerevisiae. Furthermore, we constructed a large-scale GATC-PCR system to measure absolute expression levels of 5,038 genes to show that the yeast contains more than 30,000 copies of mRNA molecules per cell. CONCLUSION: We developed a GATC-PCR method to accurately measure absolute expression levels of mRNAs by means of competitive amplification of genomic and cDNA copies of each gene. A large-scale application of GATC-PCR to the budding yeast transcriptome revealed that it is twice or more as large as previously estimated. This method is flexibly applicable to both targeted and genome-wide analyses of absolute expression levels of mRNAs.

A novel strategy to design highly specific PCR primers based on the stability and uniqueness of 3'-end subsequences.

MOTIVATION: In contrast with conventional PCR using a pair of specific primers, some applications utilize a single unique primer in combination with a common primer, thereby relying solely on the former for specificity. These applications include rapid amplification of cDNA ends (RACE), adaptor-tagged competitive PCR (ATAC-PCR), PCR-mediated genome walking and so forth. Since the primers designed by conventional methods often fail to work in these applications, an improved strategy is required, particularly, for a large-scale analysis. RESULTS: Based on the structure of 'off-target' products in the ATAC-PCR, we reasoned that the practical determinant of the specificity of primers may not be the uniqueness of entire sequence but that of the shortest 3'-end subsequence that exceeds a threshold of duplex stability. We termed such a subsequence as a 'specificity-determining subsequence' (SDSS) and developed a simple algorithm to predict the performance of the primer: the algorithm identifies the SDSS of each primer and examines its uniqueness in the target genome. The primers designed using this algorithm worked much better than those designed using a conventional method in both ATAC-PCR and 5'-RACE experiments. Thus, the algorithm will be generally useful for improving various PCR-based applications.

An automated liquid handling system for polymerase chain reaction sample preparation.

An automated liquid handling system for dispensing liquid samples of sub-microliter volume has been developed. The system has eight nozzles composed of glass capillaries connected to syringe-style pumps. The distance between the nozzles can be changed from 4.5 to 9 mm, which corresponds to the distance between the wells in 96- and 384-well microplates, respectively. The eight nozzles of the system can aspirate and dispense liquid samples in both 96- and 384-well microplates. Sub-microliter volumes of reagents and samples were transferred between 96- and 384-well microplates using the system. This system was successfully used for PCR sample preparation.

Real-time detection of PCR products for comparative analysis of expressed genes using module-shuffling TaqMan probes (MTPs).

A method for comparative analysis of gene expression was developed. It is based on competitive PCR amplification and real-time detection of PCR products and it uses module-shuffling sequences as "universal TaqMan probes". Namely, cDNA-tagged module-shuffling sequences, which derived from different sources, were amplified in one reaction tube by the same primer set. Two kinds of fluorescent TaqMan probes with different module-shuffling sequences (MTPs) detect their own targets. The method can detect different amounts of expressed genes derived from different sources; accordingly, it was successfully used for comparative analysis of expressed mouse genes.