Discussion regarding the regulatory status of genome-edited crops has focused on precision of editing and on doubts regarding the feasibility of analytical monitoring compliant with existing GMO regulations. Effective detection methods are important, both for regulatory enforcement and traceability in case of biosafety, environmental or socio-economic impacts. Here, we approach the analysis question for the first time in the laboratory and report the successful development of a quantitative PCR detection method for the first commercialized genome-edited crop, a canola with a single base pair edit conferring herbicide tolerance. The method is highly sensitive and specific (quantification limit, 0.05%), compatible with the standards of practice, equipment and expertise typical in GMO laboratories, and readily integrable into their analytical workflows, including use of the matrix approach. The method, validated by an independent laboratory, meets all legal requirements for GMO analytical methods in jurisdictions such as the EU, is consistent with ISO17025 accreditation standards and has been placed in the public domain. Having developed a qPCR method for the most challenging class of genome edits, single-nucleotide variants, this research suggests that qPCR-based method development may be applicable to virtually any genome-edited organism. This advance resolves doubts regarding the feasibility of extending the regulatory approach currently employed for recombinant DNA-based GMOs to genome-edited organisms.

Cleistanthin B, one of the toxic constituents of Cleistanthus collinus, was found to be cytotoxic to normal and tumour cells. In comparison with normal cells, tumour cells were sensitive to lower doses of toxin. The 50‰ growth inhibition GI₅₀ values for normal cell lines were from 2 × 10⁻⁵ to 4.7×10⁻⁴ M and for tumour cells the values ranged from 1.6×10⁻⁶ to 4×10⁻⁵ M. Short exposure 30 min of Chinese hamster ovary CHO cells to cleistanthin B at 1–6 μg/ml resulted in extensive chromatid and isochromatid breaks and gaps. However there was no significant increase in cell death and DNA strand breaks in cells treated under the above conditions. Cleistanthin B induced micronucleus formation in cultured lymphocytes in a dose-dependent manner. CHO cells treated with high doses of cleistanthin B showed a decrease in cell viability and a concomitant increase in DNA strand-breaks. The cell death appears to be due to apoptosis since nucleosome-like ladders were observed in the treated cells when the DNA was electrophorized in agarose gels.

The MLL gene at chromosome band 11q23 is specifically cleaved at a unique site within its breakpoint cluster region (bcr) during the higher order chromatin fragmentation associated with apoptosis. We now show that the same specific DNA cleavage event can be detected in an exogenous MLL bcr fragment that is integrated into the genome outside of its normal chromosomal context, as well as in an extrachromosomal episome containing an MLL bcr fragment. We also show that episomal or randomly integrated copies of the MLL bcr behave similar to the endogenous MLL bcr when tested in a scaffold-associated region (SAR) assay. Furthermore, an episomal murine MLL bcr introduced into human cells is cleaved at the same site as the endogenous murine MLL bcr; this episomal murine MLL bcr also functions as a SAR in human cells. We conclude that both nuclear DNA scaffold attachment as well as site-specific DNA cleavage can be directed by sequences contained within the MLL bcr, and that it is feasible to study these events using episomal shuttle vectors.

Aim: Dental diseases can be prevented by reducing early bacterial colonization in biofilm, a precursor to mature dental plaque. Most studies on dental disease pathogenesis focus on mature plaque and fail to address the impact of oral cleaning on biofilm formation. Here we used next-generation metagenomics to assess the effects of a new method of regular, simple biofilm disruption on the oral metagenome. Materials and Methods: This was a randomized, controlled study of 45 healthy children divided into three groups. Participants avoided oral cleaning for 3 days and then performed 10 days of oral cleaning either by: (1) brushing and tongue cleaning twice a day (BT) with toothpaste; (2) Gum and tooth rubbing with Index Finger Tongue cleaning and water Swishing (GIFTS) after each meal, snack, and drink; or (3) GIFTS twice a day with nano-charcoal and tongue cleaning (CT) (n = 15 per group). Saliva, plaque, and tongue scraping samples were collected on day 0 and 10 for quantitative polymerase chain reaction (qPCR) and next-generation metagenomics sequencing to analyze microbiome taxa differences between groups. Results: GIFTS more significantly reduced (P < 0.004) total bacteria in saliva than BT (P < 0.02). Metagenomics revealed a significant reduction in Firmicutes in GIFTS and CT tongue samples compared to BT samples. BT and CT saliva samples showed significantly more Streptococcus species than GIFTS saliva samples. In the plaque samples, GIFTS cleaning significantly reduced early colonizers, including Streptococcus, compared to the BT and CT methods. Conclusion: Here, we introduce the “frequent disruption of biofilm” concept for enhanced oral hygiene. GIFTS can be used to prevent early bacterial colonization of biofilm and plaque formation in both small children and adults. Frequent biofilm disturbance more effectively disrupts early bacterial colonization than twice oral cleaning, is nonabrasive, and is, therefore, a practical and straightforward complement to regular toothbrushing for improved oral hygiene and disease prevention.