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Viral load monitoring in human immunodeficiency virus type 1 (HIV-1) infection is often performed using reverse transcription quantitative PCR (RT-qPCR) to observe response to treatment and identify the development of resistance. Traceability is achieved using a calibration hierarchy traceable to the International Unit (IU). IU values are determined using consensus agreement derived from estimations by different laboratories. Such a consensus approach is necessary due to the fact that there are currently no reference measurement procedures available that can independently assign a reference value to viral reference materials for molecular in vitro diagnostic tests. Digital PCR (dPCR) is a technique that has the potential to be used for this purpose. In this paper, we investigate the ability of reverse transcriptase dPCR (RT-dPCR) to quantify HIV-1 genomic RNA without calibration. Criteria investigated included the performance of HIV-1 RNA extraction steps, choice of reverse transcription approach and selection of target gene with assays performed in both single and duplex format. We developed a protocol which was subsequently applied by two independent laboratories as part of an external quality assurance (EQA) scheme for HIV-1 genome detection. Our findings suggest that RT-dPCR could be used as reference measurement procedure to aid the value assignment of HIV-1 reference materials to support routine calibration of HIV-1 viral load testing by RT-qPCR.

A candidate digital PCR (dPCR)-based reference measurement procedure for quantification of human cytomegalovirus (hCMV) was evaluated in 10 viral load comparison schemes (seven external quality assessment (EQA) and three additional training schemes) organized by INSTAND e.V. over four years (between September 2014 and March 2018). Four metrology institutes participated in these schemes using the same extraction method and dPCR measurement procedure for the hCMV specific target sequence of UL54 gene. The calibration independent reference measurement procedure results from the metrology institutes were compared to the results of the clinical diagnostic laboratories applying hCMV qPCR measurement procedures calibrated to reference materials. While the criteria for the acceptable deviation from the target value interval for INSTAND's EQA schemes is from-0.8 log10 to +0.8 log(10), the majority of dPCR results were between-0.2 log10 to +0.2 log(10). Only 4 out of 45 results exceeded this interval with the maximum deviation of-0.542 log(10). In the training schemes containing samples with lower hCMV concentrations, more than half of the results deviated less than +/- 0.2 log(10) from the target value, while more than 95% deviated less than +/- 0.4 log(10) from the target value. Evaluation of intra-and inter-laboratory variation of dPCR results confirmed high reproducibility and trueness of the method. This work demonstrates that dPCR has the potential to act as a calibration independent reference measurement procedure for the value assignment of hCMV calibration and reference materials to support qPCR calibration as well as ultimately for routine hCMV load testing. This study was financially supported by the INFECTMET project (the EMRP project; jointly funded by the EMRP participating countries within EURAMET and the European Union, project no. HLT08); AntiMicroResist project (which has received funding from the EMPIR program co-financed by the Participating States and from the European Union’s Horizon 2020 research and innovation program, project no. 15HLT07); the Slovenian Research Agency (contract no. P4-0165 and 1000-13-0105); Ministry of Education, Science and Sport of republic of Slovenia (contract no. 3330-14-500133) and supported by the UK National Measurement System. The dPCR equipment used at NIB in this study was financed by the Metrology Institute of the Republic of Slovenia (MIRS), with financial support from the European Regional Development Fund. The funding source(s) had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. This publication reflects only the authors’ s views and EURAMET is not responsible for any use that may be made of the information it contains.

Resveratrol has gained tremendous interest owing to multiple reported health-beneficial effects. However, the underlying key mechanism of action of resveratrol remained largely controversial. Here, we demonstrate that under physiologically relevant conditions major biological effects of resveratrol can be attributed to the generation of oxidation products such as reactive oxygen species (ROS). At low hormetic concentrations (< 50 μM), treatment with resveratrol increased cell viability in a set of representative cell models, whereas application of quenchers of ROS completely truncated these beneficial effects. Notably, application of resveratrol led to mild, Nrf2-specific cellular gene expression reprogramming. For example, in primary human epidermal keratinocytes this resulted in a 1.3-fold increase of endogenous metabolites such as gluthathione (GSH) and subsequently in a quantitative reduction of the cellular redox environment by 2.61 mV mmol GSH (g protein)-1. In particular in resveratrol pre-treated cells, after external application of oxidative stress by using 0.8 % ethanol, endogenous ROS generation was consequently reduced by 24 %. In contrast to the common perception that resveratrol acts mainly as a chemical antioxidant or as a target protein-specific ligand, we propose that effects from resveratrol treatment are essentially based on oxidative triggering of cells. In relevant physiological microenvironments this effect can lead to hormetic shifting of cellular defence towards a more reductive state to improve resilience to oxidative stress in a manner that can be exactly defined by the redox-environment of the cell.

The modern term nutraceutical sounds extremely promising to the health-conscious consumers and to the broad audience. But what are the scientific foundations of this hybrid between nutrient and pharmaceutical? Still, most compelling evidence for potential health-beneficial effects of nutraceuticals seem to derive from descriptive and correlative epidemiological data. Here, we will take an inventory of the general concepts of research to assess the current plethora of health claims that were made for nutraceutical products. Thereby, we will discuss the limitations of current experimental approaches to advance in establishing mechanistic or causal links to the possible benefits of bioactive dietary molecules. Finally, we aim to provide perspectives to generate potential scientific evidence for the beneficial effects of nutraceuticals on human physiology.

Amorfrutins are isoprenoid-substituted benzoic acid derivatives, which were found in Amorpha fruticosa L. (bastard indigo) and in Glycyrrhiza foetida Desf. (licorice). Recently, amorfrutins were shown to be selective activators of the nuclear receptor PPARgamma. Here, we investigated the effects and PPARgamma-based mechanisms of reducing inflammation in colon cells by treatment with amorfrutins. In TNF-alpha-stimulated colon cells amorfrutin A (1) reduced significantly the expression and secretion of several inflammation mediators, in part due to interaction with PPARgamma. These results support the hypothesis that amorfrutins may have the potential to treat inflammation disorders such as chronic inflammatory bowel diseases.