Many molecular epidemiology studies focusing on high prevalent diseases, such as metabolic disorders and cancer, investigate metabolic and hormonal markers. In general, sampling for these markers can occur at any time-point during the day or after an overnight fast. However, environmental factors, such as light exposure and food intake might affect the levels of these markers, since they provide input for the internal time-keeping system. When diurnal variation is larger than the inter-individual variation, time of day should be taken into account. Importantly, heterogeneity in diurnal variation and disturbance of circadian rhythms among a study population might increasingly occur as a result of our increasing 24/7 economy and related variation in exposure to environmental factors (such as light and food). The aim of the present study was to determine whether a set of often used biomarkers shows diurnal variation in a setting resembling large molecular epidemiology studies, i.e., non-fasted and limited control possibilities for other environmental influences. We show that markers for which diurnal variation is not an issue are adrenocorticotropic hormone, follicle stimulating hormone, estradiol and high-density lipoprotein. For all other tested markers diurnal variation was observed in at least one gender (cholesterol, cortisol, dehydroepiandrosterone sulfate, free fatty acids, low-density lipoprotein, luteinizing hormone, prolactin, progesterone, testosterone, triglycerides, total triiodothyronine and thyroid-stimulating hormone) or could not reliably be detected (human growth hormone). Thus, studies investigating these markers should take diurnal variation into account, for which we provide some options. Furthermore, our study indicates the need for investigating diurnal variation (in literature or experimentally) before setting up studies measuring markers in routine and controlled settings, especially since time-of-day likely matters for many more markers than the ones investigated in the present study.

Background: Dietary non-digestible carbohydrates stimulate the gut microflora and are therefore presumed to improve host resistance to intestinal infections. However, several strictly controlled rat infection studies showed that non-digestible fructo-oligosaccharides (FOS) increase, rather than decrease, translocation ofSalmonellatowards extra-intestinal sites. In addition, it was shown that FOS increases intestinal permeability already before infection. The mechanism responsible for this adverse effect of FOS is unclear. Possible explanations are altered mucosal integrity due to changes in tight junctions or changes in expression of defense molecules such as antimicrobials and mucins. To examine the mechanisms underlying weakening of the intestinal barrier by FOS, a controlled dietary intervention study was performed. Two groups of 12 rats were adapted to a diet with or without FOS. mRNA was collected from colonic mucosa and changes in gene expression were assessed for each individual rat using Agilent rat whole genome microarrays.Results: Among the 997 FOS induced genes we observed less mucosal integrity related genes than expected with the clear permeability changes. FOS did not induce changes in tight junction genes and only 8 genes related to mucosal defense were induced by FOS. These small effects are unlikely the cause for the clear increase in intestinal permeability that is observed. FOS significantly increased expression of 177 mitochondria-related genes. More specifically, induced expression of genes involved in all five OXPHOS complexes and the TCA cycle was observed. These results indicate that dietary FOS influences intestinal mucosal energy metabolism. Furthermore, increased expression of 113 genes related to protein turnover, including proteasome genes, ribosomal genes and protein maturation related genes, was seen. FOS upregulated expression of the peptide hormone proglucagon gene, in agreement with previous studies, as well as three other peptide hormone genes; peptide YY, pancreatic polypeptide and cholecystokinin.Conclusion: We conclude that altered energy metabolism may underly colonic barrier function disruption due to FOS feeding in rats.

This study investigates whether a set of ten potential breast cancer serum biomarkers and cancer antigens (osteopontin (OPN), haptoglobin, cancer antigen 15-3 (CA15-3), carcinoembryonic antigen (CEA), cancer antigen 125 (CA-125), prolactin, cancer antigen 19-9 (CA19-9), α-fetoprotein (AFP), leptin and migration inhibitory factor (MIF)) can predict early stage breast cancer in samples collected before clinical diagnosis (phase III samples). We performed a nested case-control study within the Prospect-EPIC (European Prospective Investigation into Cancer and nutrition) cohort. We examined to what extent the biomarker panel could discriminate between 68 women diagnosed with breast cancer up to three years after enrollment and 68 matched healthy controls (all 56-64 years at baseline). Using a quantitative bead-based multiplexed assay, we determined protein concentrations in serum samples collected at enrollment. Principal Component Analysis (PCA) and Random Forest (RF) analysis revealed that on the basis of all ten proteins, early cases could not be separated from controls. When we combined serum protein concentrations and subject characteristics related to breast cancer risk in the RF analysis, this did not result in classification accuracy scores that could correctly classify the samples (sensitivity: 50%, specificity: 50%). Our findings indicate that this panel of selected tumor markers cannot be used for diagnosis of early breast cancer.

Isolation of RNA from skin biopsies presents a challenge, due to the tough nature of skin tissue and a high presence of RNases. As we lacked the dedicated equipment, i.e. homogenizer or bead-beater, needed for the available RNA from skin isolation methods, we adapted and tested our zebrafish single-embryo RNA-isolation protocol for RNA isolation from skin punch biopsies.

Frequent shift work causes disruption of the circadian rhythm and might on the long-term result in increased health risk. Current biomarkers evaluating the presence of circadian rhythm disturbance (CRD), including melatonin, cortisol and body temperature, require 24-hr (“around the clock”) measurements, which is tedious. Therefore, these markers are not eligible to be used in large-scale (human) studies. The aim of the present study was to identify universal biomarkers for CRD independent of time of day using a transcriptomics approach. Female FVB mice were exposed to six shifts in a clockwise (CW) and counterclockwise (CCW) CRD protocol and sacrificed at baseline and after 1 shift, 6 shifts, 5 days recovery and 14 days recovery, respectively. At six time-points during the day, livers were collected for mRNA microarray analysis. Using a classification approach, we identified a set of biomarkers able to classify samples into either CRD or non-disrupted based on the hepatic gene expression. Furthermore, we identified differentially expressed genes 14 days after the last shift compared to baseline for both CRD protocols. Non-circadian genes differentially expressed upon both CW and CCW protocol were considered useful, universal markers for CRD. One candidate marker i.e. CD36 was evaluated in serum samples of the CRD animals versus controls. These biomarkers might be useful to measure CRD and can be used later on for monitoring the effectiveness of intervention strategies aiming to prevent or minimize chronic adverse health effects.

Pharmacogenomic testing is a method to prevent adverse drug reactions. Pharmacogenomics could be relevant to optimize statin treatment, by identifying patients at high risk for adverse drug reactions. We aim to investigate the clinical validity and utility of pre-emptive pharmacogenomics screening in primary care, with SLCO1B1 c.521T>C as a risk factor for statin-induced adverse drug reactions. The focus was on changes in therapy as a proxy for adverse drug reactions observed in statin-users in a population-based Dutch cohort. In total, 1136 statin users were retrospectively genotyped for the SLCO1B1 c.521T>C polymorphism (rs4149056) and information on their statin dispensing was evaluated as cross-sectional research. Approximately half of the included participants discontinued or switched their statin treatment within three years. In our analyses, we could not confirm an association between the SLCO1B1 c.521T>C genotype and any change in statin therapy or arriving at a stable dose sooner in primary care. To be able to evaluate the predictive values of SLCO1B1 c.521T>C genotype on adverse drug reactions from statins, prospective data collection of actual adverse drug reactions and reasons to change statin treatment should be facilitated.