Digital PCR (dPCR) has developed considerably since the publication of the Minimum Information for Publication of Digital PCR Experiments (dMIQE) guidelines in 2013, with advances in instrumentation, software, applications, and our understanding of its technological potential. Yet these developments also have associated challenges; data analysis steps, including threshold setting, can be difficult and preanalytical steps required to purify, concentrate, and modify nucleic acids can lead to measurement error. To assist independent corroboration of conclusions, comprehensive disclosure of all relevant experimental details is required. To support the community and reflect the growing use of dPCR, we present an update to dMIQE, dMIQE2020, including a simplified dMIQE table format to assist researchers in providing key experimental information and understanding of the associated experimental process. Adoption of dMIQE2020 by the scientific community will assist in standardizing experimental protocols, maximize efficient utilization of resources, and further enhance the impact of this powerful technology.

The MIQE (minimum information for the publication of quantitative real-time PCR) guidelines were published in 2009 with the twin aims of providing a blueprint for good real-time quantitative polymerase chain reaction (qPCR) assay design and encouraging the comprehensive reporting of qPCR protocols. It had become increasingly clear that variable pre-assay conditions, poor assay design, and incorrect data analysis were leading to the routine publication of data that were often inconsistent, inaccurate, and wrong. The problem was exacerbated by a lack of transparency of reporting, with the details of technical information inadequate for the purpose of assessing the validity of published qPCR data. This had, and continues to have serious implications for basic research, reducing the potential for translating fi ndings into valuable applications and potentially devastating consequences for clinical practice. Today, the rationale underlying the MIQE guidelines has become widely accepted, with more than 2,200 citations by March 2014 and editorials in Nature and related publications acknowledging the enormity of the problem. However, the problem we now face is rather serious: thousands of publications that report suspect data are populating and corrupting the peer-reviewed scientifi c literature. It will be some time before the many contradictions apparent in every area of the life sciences are corrected.

Aims:  Aeromonas hydrophila is recognized as a human pathogen following wound exposure or ingestion of contaminated water and food. For rapid identification of this bacterium, a TaqMan‐based real‐time PCR assay has been developed. Methods and Results:  Primers and probes that target specific sequences of the 16S rRNA gene and cytolytic enterotoxin gene (aerA) were combined in a duplex assay. Presence and size of PCR products were confirmed with microchannel fluidics electrophoresis analysis. After validation, using type strain CIP7614T DNA, the PCR assay was tested on 12 positive and negative controls. Twenty‐one Aeromonas strains were isolated from environmental samples and were identified with biochemical tests as Aer. sobria, Aer. caviae and Aer. hydrophila. Only Aer. hydrophila strains tested positive by PCR assay. Conclusions:  The PCR developed here was successfully applied for the identification of Aer. hydrophila from reference, clinical and environmental samples and showed a high discrimination between Aer. hydrophila and other Aeromonas species. Significance and Impact of the Study:  This molecular method is convenient, rapid (2·5 h vs 24 h), specific to identify Aer. hydrophila and usable for diagnosis in medical and veterinary laboratories.

Objective: To develop a method for the simultaneous measurement of 5-methylcytosine (5-metC) and 2’-deoxyuridine monophosphate (dU). Materials and Methods: Genomic DNA was extracted from the HepG2 cell line grown in experimental complete medium or in folate-depleted medium. Samples were treated with RNAse A and RNAse T1 to avoid any RNA contamination. High-performance liquid chromatography (HPLC)/electrospray ionization mass spectrometric (ESI-MS) method was used to separate nucleotides after enzymatic hydrolysis of DNA with nuclease P1, phosphodiesterase I and alkaline phosphatase. Results: Using this sensitive new methodology, we were able to quantify simultaneously the concentration of DNA-5-metC and DNA-uracil in DNA. The linear correlation coefficient (R²) between the MS signal and the concentration of 5-metC in a range of 0.5–5 μM or dU in a range of 10–100 μM was 0.9954 and 0.9999, respectively. The coefficient of variation was 16.94 and 14.77%, respectively. The applicability of this assay is demonstrated by detection of a decrease in 5-metC% and elevation of dU/thymidylate (dT) into genomic DNA extracted from the HepG2 cell line grown in a folate-depleted medium. Conclusion: Our results confirm that the HPLC/ESI-MS method reported earlier for measuring 5-metC allows measurement of uracil misincorporation into DNA.

Fumonisin B1 (FB1) induces apoptosis and decreases the cellular uptake of 5-methyltetrahydrofolate. Two folate transporters (folate receptor, FR, and Reduced Folate Carrier, hRFC1) are involved in the cell uptake of folate. We aimed to study whether FB1 modifies the expression of the FR and the hRFC1 and whether its apoptotic effect is influenced by folate. Incubation of HepG2 cells with FB1 induced apoptosis in concentration and time-dependent manner in complete medium (experimental control medium, ECM), as well as in folate-depleted medium (FDM). FDM increased the toxicity of FB1 as the cells developed apoptosis within 24 h at 1 microM of FB1 instead of 100 microM in ECM. Whereas FR protein expression in cells grown in ECM was significantly inhibited after apoptosis event, protein expression of the hRFC1 was rather increased. The hrfc1 transcription was decreased in the treated cells. Under folate-deficient conditions, dramatic changes were observed on both transcriptional and post-transcriptional expression of the two transporters. FDM alone reduced FR protein expression by 12 +/- 2% and 43 +/- 1% at 48 and 72 h, respectively. The 5-methytetrahydrofolate attenuates apoptosis in a greater extent than the folic acid. However, its effects in preventing decrease of both folate transporters have not been observed. In conclusion, this study shows that the changes in the expression of FR after FB1 addition are probably a consequence of the FB1 toxicity. The response to FB1 by HepG2 cell lines is influenced by folate status and by folate form. 5-methyltetrahydrofolate appears to be more effective in preventing apoptosis than folic acid.