Cell cycle arrest combined with CDK1 inhibition suppresses genome-wide mutations by activating alternative DNA repair genes during genome editing.

Cells regularly repair numerous mutations. However, the effects of CRISPR/Cas9-induced double-strand DNA breaks on the repair processes of naturally occurring genome-wide mutations is unclear. In this study, we used TSCE5 cells with the heterozygous thymidine kinase genotype (TK+/-) to examine these effects. We strategically inserted the target sites for guide RNA (gRNA)/Cas9 and I-SceI into the functional allele and designed the experiment such that deletions of > 81 bp or base substitutions within exon 5 disrupted the TK gene, resulting in a TK-/- genotype. TSCE5 cells in the resting state exhibited 16 genome-wide mutations that affected cellular functions. After gRNA/Cas9 editing, these cells produced 859 mutations, including 67 high-impact variants that severely affected cellular functions under standard culture conditions. Mutation profile analysis indicated a significant accumulation of C to A substitutions, underscoring the widespread induction of characteristic mutations by gRNA/Cas9. In contrast, gRNA/Cas9-edited cells under conditions of S∼G2/M arrest and cyclin dependent kinase 1 inhibition showed only 5 mutations. Transcriptomic analysis revealed the downregulation of DNA replication genes and upregulation of alternative DNA repair genes, such as zinc finger protein 384 (ZNF384) and dual specificity phosphatase (DUSP), under S∼G2/M conditions. Additionally, activation of nucleotide and base excision repair gene, including O-6-methylguanine-DNA methyltransferase (MGMT) and xeroderma pigmentosum complementation group C (XPC), was observed. This study highlights the profound impact of CRISPR/Cas9 editing on genome-wide mutation processes and underscores the emergence of novel DNA repair pathways, Finally, our findings provide significant insights into the maintenance of genome integrity during genome editing.

Loop-mediated isothermal amplification (LAMP) for rapid and easy identification of Omphalotus japonicus.

Omphalotus japonicus is a major toxic mushroom in Japan. When food poisoning caused by O. japonicus occurs, quick and accurate identification using a method that does not rely on morphological discrimination is required. Because the loop-mediated isothermal amplification (LAMP) method meets these requirements, we developed a LAMP method for detecting O. japonicus. Amplification occurred within 60 min, and the presence or absence of O. japonicus was confirmed within 2 h, including the DNA extraction protocol. The LAMP method did not show cross-reactivity with 13 species of edible mushrooms, had high specificity toward O. japonicus, and had sufficient detection sensitivity even in a mixed mushroom sample containing 1% O. japonicus. Additionally, O. japonicus could be detected in simulated food poisoning samples of heated and digested mushrooms, and in actual food poisoning residual samples.

[Qualitative Real-Time PCR Method for Poisonous Entoloma rhodopolium-Related Species in Japan: Real-Time PCR Method for Entoloma Mushrooms].

Qualitative real-time PCR method for three poisonous Entoloma rhodopolium-related species in Japan was established using specific primers and FAM, VIC, Texas Red, Cy5-labeled probes. The use of multicolor probes can extend the method to simultaneous detection of different targets. Standard plasmids were constructed as reference materials. Designed primers and probes in the method detect only a target species, and the detection limit was 12.5 copies or below. This indicates it is highly specific and sensitive enough to detect the poisonous mushrooms in food residues. Next, we applied the method to four food residue samples obtained from food poisoning cases. The real-time PCR method did identify all of four samples as E. subrhodopolium and E. pseudorhodopolium, whereas PCR-RFLP did not. The method established here revealed Entoloma rhodopolium-related species in Hokkaido were different species such as E. eminens and unknown species.

Molecular phylogenetic analysis of new Entoloma rhodopolium-related species in Japan and its identification method using PCR-RFLP.

Poisonous Entoloma rhodopolium and other similar species including edible E. sarcopum are morphologically diverse. People mistake poisonous species for edible species. Classification and the detection method of these species need to be defined. The morphological and phylogenetic studies have been reported in northern Europe. In Japan, the genetic study remains unsolved. Thus, phylogenetic analysis of E. rhodopolium was conducted using ITS and RPB2 sequences, and the result was compared with that of European species. Japanese E. rhodopolium was classified into three clades, none of which belonged to the true European E. rhodopolium and other known species. Three species were defined as new species. Entoloma rhodopolium clade-I (named E. lacus) was genetically close to but morphologically separated from E. majaloides. Clade-II (E. subrhodopolium) was classified to the same group as E. sinuatum and E. subsinuatum, but distinct from these species. Clade-III was segregated from known Entoloma species including E. lupinum, and named E. pseudorhodopolium. Based on the classification, a simple identification method PCR-RFLP was developed to discriminate between poisonous species and edible E. sarcopum, which is very similar in morphology. The study can help to clarify the taxonomy of complex E. rhodopolium-related species, and to prevent food poisoning.

Rapid Identification Method of Omphalotus japonicus by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP).

Omphalotus japonicus is a poisonous mushroom that grows in Japan. It can be mistaken for edible mushrooms (Shiitake, Hiratake and Mukitake), and if ingested, it causes food poisoning within 30 min to 1 hr. We established a rapid detection method using PCR-RFLP to identify O. japonicus by restriction digestion of the amplified ITS region. By using Sau96I, Bpu10I, SfcI or DrdI/HincII as a restriction enzyme, it was possible to rapidly identify and discriminate O. japonicus based on the fragment length. This study also provided a short PCR-RFLP system comprising amplification and digestion of a short 200-bp DNA fragment within the ITS region. The system could identify and discriminate O. japonicus after in vitro gastric digestion of native and heated mushroom samples as a model of food poisoning. In addition, a confirmatory assay using real-time PCR was developed to achieve more sensitive detection of O. japonicus.

Optimization method of MRI scan parameters of a double inversion recovery sequence using a T1 map and a developed analysis algorithm.

BACKGROUND: Optimizing scan parameters for double inversion recovery (DIR) sequences remains difficult. OBJECTIVE: To evaluate a new method for optimizing DIR sequence scan parameters using T1 mapping and a newly developed analysis algorithm. METHODS: Twelve healthy volunteers underwent T1 mapping and DIR magnetic resonance imaging. The following steps were used for image optimization including: 1) measurement of gray matter (GM), white matter (WM), and cerebrospinal fluid (CSF) T1 values to create a T1 map; 2) calculation of optimized scan parameters by using a developed analysis algorithm; 3) performance assessment of DIR magnetic resonance imaging by using the calculated optimized imaging parameters. Additionally, we used scan parameters from previous studies to obtain DIR images in order to evaluate our new method. The contrast between GM and suppressed tissues was compared between these images and those obtained using the optimized parameters. RESULTS: Using our optimization method, WM and CSF regions were suppressed uniformly for all scan conditions. The contrast was significantly higher in images obtained using this optimization method compared to those obtained using previously published parameters (p < 0.01). CONCLUSIONS: It is possible to obtain superior DIR images by using an optimization method that involves T1 mapping and a newly developed analysis algorithm.

MTF Measurement of MR Blurring in Liver Dynamic MRI with Gd-EOB-DTPA.

During the arterial phase acquisition of Gd-EOB-DTPA examinations, use of a small volume of the Gd-EOB-DTPA may make it difficult the encoding center of the k-space, and produce blurring. The previous studies revealed the encoding technique of the k-space was one of the most important reasons. However, there is no report to discuss the reasons with quantitative evaluations. The purpose of this study was to quantitatively evaluate the characteristics of the artifacts using different k-space encoding techniques (centric-view ordering (CVO) and sequential-view ordering (SVO)) for liver dynamic MRI in computer simulation study. This simulation study consists of the following steps. First of all, the creation of a time intensity curve, and original simulation images at certain points among the one phase dynamic scanning. Secondly, creation-simulated MR echo data from the created original images using FFT, and encoding simulated k-space using the simulated MR echo data. Finally, a reconstruction of simulated dynamic MR images from the simulated k-space, and to evaluate each simulated MR images, we measured modulation transfer functions (MTFs) from the bar patterns of the reconstructed images. The results of the CVO simulation indicated that the bar patterns were blurring compared to the images encoded by the SVO. The results of the SVO simulation indicated that the bar patterns were not enhanced at late scan timings. In addition, the results of MTFs indicated that there was no edge enhancement at all scan timings and both encoding techniques. In conclusion, it is possible to quantitatively evaluate the characteristics of artifacts using MTF, which was measured by the bar patterns, in liver dynamic MRI.

Interlaboratory validation data on real-time polymerase chain reaction detection for unauthorized genetically modified papaya line PRSV-YK.

This article is referred to research article entitled "Whole genome sequence analysis of unidentified genetically modified papaya for development of a specific detection method" (Nakamura et al., 2016) [1]. Real-time polymerase chain reaction (PCR) detection method for unauthorized genetically modified (GM) papaya (Carica papaya L.) line PRSV-YK (PRSV-YK detection method) was developed using whole genome sequence data (DDBJ Sequenced Read Archive under accession No. PRJDB3976). Interlaboratory validation datasets for PRSV-YK detection method were provided. Data indicating homogeneity of samples prepared for interlaboratory validation were included. Specificity and sensitivity test data for PRSV-YK detection method were also provided.

Whole genome sequence analysis of unidentified genetically modified papaya for development of a specific detection method.

Identification of transgenic sequences in an unknown genetically modified (GM) papaya (Carica papaya L.) by whole genome sequence analysis was demonstrated. Whole genome sequence data were generated for a GM-positive fresh papaya fruit commodity detected in monitoring using real-time polymerase chain reaction (PCR). The sequences obtained were mapped against an open database for papaya genome sequence. Transgenic construct- and event-specific sequences were identified as a GM papaya developed to resist infection from a Papaya ringspot virus. Based on the transgenic sequences, a specific real-time PCR detection method for GM papaya applicable to various food commodities was developed. Whole genome sequence analysis enabled identifying unknown transgenic construct- and event-specific sequences in GM papaya and development of a reliable method for detecting them in papaya food commodities.

Development and Interlaboratory Validation of a Simple Screening Method for Genetically Modified Maize Using a ΔΔC(q)-Based Multiplex Real-Time PCR Assay.

A number of genetically modified (GM) maize events have been developed and approved worldwide for commercial cultivation. A screening method is needed to monitor GM maize approved for commercialization in countries that mandate the labeling of foods containing a specified threshold level of GM crops. In Japan, a screening method has been implemented to monitor approved GM maize since 2001. However, the screening method currently used in Japan is time-consuming and requires generation of a calibration curve and experimental conversion factor (C(f)) value. We developed a simple screening method that avoids the need for a calibration curve and C(f) value. In this method, ΔC(q) values between the target sequences and the endogenous gene are calculated using multiplex real-time PCR, and the ΔΔC(q) value between the analytical and control samples is used as the criterion for determining analytical samples in which the GM organism content is below the threshold level for labeling of GM crops. An interlaboratory study indicated that the method is applicable independently with at least two models of PCR instruments used in this study.

[Development of an Optimizing Program of Scanning Parameters for Double Inversion Recovery MRI].

The purpose of this study was to develop an optimizing program of scanning parameters for double inversion recovery (DIR) MRI. The optimization algorithm consists of the following steps: (1) obtaining the initial parameters (TR, TE, and T1 values of the two attenuated tissues); (2) iterative calculation for minimization of errors; and (3) determination of the optimized TI(1st) and TI(2nd). To evaluate the developed algorithm, we performed the phantom and simulation studies using the phantoms which were imitated T1 values of white and gray matters and cerebrospinal fluid. In addition, white matter attenuated inversion recovery (WAIR) and gray matter attenuated inversion recovery (GAIR) images were obtained by optimized scan parameters in one volunteer. The developed algorithm could calculate the optimized TI(1st) and TI(2nd) values at once. Results of summation of signal intensity (SI) of two attenuated tissues shows that the SI of the two tissues were well-attenuated using the theoretical values which were calculated using the developed algorithm. The correlation coefficient of the SI of the phantom of the gray matter between actual and simulation measurements was r=0.997. The SI obtained by actual measurements well correlated with the SI obtained by the simulation measurements. The WAIR and GAIR images in the volunteer were well enhanced gray or white matters. We thus conclude that it is possible to calculate the optimized parameters for the DIR-MRI using the developed algorithm.

Interlaboratory validation study of an event-specific real-time polymerase chain reaction detection method for genetically modified 55-1 papaya.

Genetically modified (GM) papaya line 55-1 (55-1) is resistant to papaya ringspot virus infection, and is commercially available in several countries. A specific detection method for 55-1 is required for mandatory labeling regulations. An event-specific real-time PCR method was developed by our laboratory. To validate the method, interlaboratory validation of event-specific qualitative real-time PCR analysis for 55-1 was performed in collaboration with 12 laboratories. DNA extraction and real-time PCR reaction methods were evaluated using 12 blind samples: six non-GM papayas and six GM papayas in each laboratory. Genomic DNA was highly purified from all papayas using an ion-exchange column, and the resulting DNA sample was analyzed using real-time PCR. Papaya endogenous reference gene chymopapain (CHY) and the event-specific 55-1 targeted sequence were detected in GM papayas whereas CHYalone was detected in non-GM papayas in all laboratories. The cycle threshold values of CHYand the 55-1 targeted sequence showed high repeatability (RSD, 0.6-0.8%) and reproducibility (RSDR 2.2-3.6%). This study demonstrates that the 55-1 real-time PCR detection method is a useful and reliable method to monitor 55-1 papaya in foods.

Quantification and identification of genetically modified maize events in non-identity preserved maize samples in 2009 using an individual kernel detection system.

We investigated the GM maize grain content of non-identity preserved (non-IP) maize samples produced in 2009 in the USA using our individual kernel detection system, involving two multiplex qualitative PCR methods coupled to microchip electrophoresis and partially real-time PCR array analysis, to clarify how many GM event maize grains were present in the samples and which GM events frequently appeared in 2009. The average percentage and standard deviation of GM maize grains on a kernel basis in five non-IP sample lots were 81.9%±2.8%, the average percentage of single GM event grains was 46.9%, and the average percentage of stacked GM event grains was 35.0%. MON88017 grains and NK603 grains were the most frequently observed as single GM event grains. The most frequent stacked GM event grains were MON88017×MON810 grains. This study shows that our method can provide information about GM maize events present in imported maize samples on a kernel basis.

Immunological effects of Oenothein B, an ellagitannin dimer, on dendritic cells.

Oenothein B is a unique macrocyclic ellagitannin dimer that has been found in various medicinal plants belonging to Onagraceae, Lythraceae, and Myrtaceae, with diverse biological activities. The immunological effects of tannins in terms of cytokine-release from macrophages and monocytes have been discussed, while the effects on other immunocompetent cells have been the subject of minimal investigation. We evaluated the immunomodulatory effects induced by tannin treatment in human dendritic cells (DCs), which play a critical role in the initial immune response, by measuring the changes in cytokine production, cell differentiation, and cell viability. Oenothein B showed significant down-regulation of the expression of cell surface molecules, CD1a and CD83, suggesting the inhibition of DC differentiation and/or maturation. The suppressive effect on DCs was associated with the induction of apoptosis without the activation of caspase-3/7, 8, and 9, and this was supported by the morphological features indicating significant nuclear condensation. Oenothein B also markedly suppressed the production of inflammatory cytokines, such as IL-1ß and IL-6, in a dose-dependent manner. These data may, in part, be able to explain the traditional use of tannin-containing medicinal plants for the treatment of a variety of inflammatory diseases, including inflammatory bowel disease, celiac disease, and rheumatoid arthritis.

Interlaboratory study of DNA extraction from multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for individual kernel detection system of genetically modified maize.

In many countries, the labeling of grains, feed, and foodstuff is mandatory if the genetically modified (GM) organism content exceeds a certain level of approved GM varieties. We previously developed an individual kernel detection system consisting of grinding individual kernels, DNA extraction from the individually ground kernels, GM detection using multiplex real-time PCR, and GM event detection using multiplex qualitative PCR to analyze the precise commingling level and varieties of GM maize in real sample grains. We performed the interlaboratory study of the DNA extraction with multiple ground samples, multiplex real-time PCR detection, and multiplex qualitative PCR detection to evaluate its applicability, practicality, and ruggedness for the individual kernel detection system of GM maize. DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR were evaluated by five laboratories in Japan, and all results from these laboratories were consistent with the expected results in terms of the commingling level and event analysis. Thus, the DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for the individual kernel detection system is applicable and practicable in a laboratory to regulate the commingling level of GM maize grain for GM samples, including stacked GM maize.

Development and evaluation of event-specific quantitative PCR method for genetically modified soybean A2704-12.

A novel real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) soybean event; A2704-12. During the plant transformation, DNA fragments derived from pUC19 plasmid were integrated in A2704-12, and the region was found to be A2704-12 specific. The pUC19-derived DNA sequences were used as primers for the specific detection of A2704-12. We first tried to construct a standard plasmid for A2704-12 quantification using pUC19. However, non-specific signals appeared with both qualitative and quantitative PCR analyses using the specific primers with pUC19 as a template, and we then constructed a plasmid using pBR322. The conversion factor (C(f)), which is required to calculate the amount of the genetically modified organism (GMO), was experimentally determined with two real-time PCR instruments, the Applied Biosystems 7900HT and the Applied Biosystems 7500. The determined C(f) values were both 0.98. The quantitative method was evaluated by means of blind tests in multi-laboratory trials using the two real-time PCR instruments. The limit of quantitation for the method was estimated to be 0.1%. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSD(R)), and the determined bias and RSD(R) values for the method were each less than 20%. These results suggest that the developed method would be suitable for practical analyses for the detection and quantification of A2704-12.

Differential detection of shrimp and crab for food labeling using polymerase chain reaction.

Shrimp and crab are well-known as allergenic ingredients. According to Japanese food allergy labeling regulations, shrimp species (including prawns, crayfishes, and lobsters) and crab species must be differentially declared when ≥10 ppm (total protein) of an allergenic ingredient is present. However, the commercial ELISA tests for the detection of crustacean proteins cannot differentiate between shrimp and crab. Therefore, two methods were developed to discriminate shrimp and crab: a shrimp-PCR method with postamplification digestion and a crab-PCR method that specifically amplifies a fragment of the 16S rRNA gene. The sensitivity and specificity of both PCR methods were verified by experiments using DNA extracted from 15 shrimp species, 13 crab species, krill, mysid, mantis shrimp, other food samples (cephalopod, shellfish, and fish), incurred foods, and commercial food products. Both PCR methods could detect 5 pg of DNA extracted from target species and 50 ng of genomic DNA extracted from incurred foods containing 10 ppm (µg/g) total protein of shrimp or crab. The two PCR methods were considered to be specific enough to separately detect species belonging to shrimp and crab. Although false-positive and false-negative results were obtained from some nontarget crustacean species, the proposed PCR methods, when used in conjunction with ELISA tests, would be a useful tool for confirmation of the validity of food allergy labeling and management of processed food safety for allergic patients.

Interlaboratory validation of an event-specific real time polymerase chain reaction detection method for genetically modified DAS59132 maize.

A real-time polymerase chain reaction (PCR) method specific for genetically modified (GM) maize event DAS59132 (E32) was adapted for qualitative detection of low level presence of E32. The method was validated by a collaborative trial with eight participating Japanese laboratories. Sensitivity was assessed with three different samples of corn flour fortified to 0%, 0.05% and 0.1% (w/w) E32 respectively. In addition, a 0.01% E32 DNA solution was used. The detection limit with DNA solution was estimated to be approximately 0.01%. In conclusion, the results of the study confirmed this real-time PCR method as a reliable tool for qualitative detection of E32 maize.

Dietary carotenoids inhibit oral sensitization and the development of food allergy.

Type-I allergic disorders and particularly food hypersensitivities are becoming increasingly common worldwide. This study investigated whether dietary enrichment with carotenoids inhibited oral sensitization to an antigen and the development of food allergies. The effects of a diet high in carotenoids were investigated in B10A mice that were orally sensitized to ovalbumin (OVA). The serum titers of OVA-specific immunoglobulin E (IgE), IgG1, and IgG2a were inhibited in mice fed ad libitum on a diet high in alpha- or beta-carotene compared to the control mice when orally sensitized to OVA. High alpha- and beta-carotene diets inhibited the immediate reduction in body temperature and rise in serum histamine associated with active systemic anaphylaxis in OVA-sensitized B10A mice. After re-stimulation with OVA in vitro, the production of T-helper 2-type cytokines by splenocytes from mice fed a diet high in carotenoids was lower than in control mice. Furthermore, the proportion of CD4(+) CD103(+) T cells in Peyer's patches of mice fed a carotenoid-rich diet was significantly lower than in control mice. These results suggest that an increased oral intake of carotenoids inhibits OVA-specific IgE and IgG1 production and antigen-induced anaphylactic responses by inhibiting specific T-cell activation in the mucosal immune system. A diet high in carotenoids might therefore prevent the development of food allergies.

Novel method to detect a construct-specific sequence of the acetolactate synthase gene in genetically-modified flax CDC Triffid (FP967).

During the fall of 2009, a trace of unauthorized genetically modified (GM) flax (Linum usitatissimum L.) line, CDC Triffid, which is resistant to sulfonylurea herbicides, was detected in many countries including Japan. A method to reliably identify the CDC Triffid line was urgently required. We developed a novel construct-specific real-time polymerase chain reaction (PCR) method to identify the mutant acetolactate synthase gene in the CDC Triffid line. We confirmed that the method can detect 0.001% GM flax in DNA mixing solution. The study shows that the developed method is specific, sensitive and reliable way to monitor a trace of CDC Triffid.