Evaluation of mRNA markers for estimating blood deposition time : towards alibi testing from human forensic stains with rhythmic biomarkers

This study was supported by grant 27.011.001 by the Netherlands Organization for Scientific Research (NWO) Forensic Science Program, Erasmus MC University Medical Center Rotterdam, by the EU 6th Framework project EUCLOCK (018741), UK Biotechnology and Biological Sciences Research Council (BBSRC) Grant BB/I019405/1, and by a previous grant from the Netherlands Genomics Initiative (NGI)/Netherlands Organization for Scientific Research (NWO) within the framework of the Forensic Genomics Consortium Netherlands (FGCN). D.J.S. is a Royal Society Wolfson Research Merit Award holder.Determining the time a biological trace was left at a scene of crime reflects a crucial aspect of forensic investigations as - if possible - it would permit testing the sample donor's alibi directly from the trace evidence, helping to link (or not) the DNA-identified sample donor with the crime event. However, reliable and robust methodology is lacking thus far. In this study, we assessed the suitability of mRNA for the purpose of estimating blood deposition time, and its added value relative to melatonin and cortisol, two circadian hormones we previously introduced for this purpose. By analysing 21 candidate mRNA markers in blood samples from 12 individuals collected around the clock at 2 h intervals for 36 h under real-life, controlled conditions, we identified 11 mRNAs with statistically significant expression rhythms. We then used these 11 significantly rhythmic mRNA markers, with and without melatonin and cortisol also analysed in these samples, to establish statistical models for predicting day/night time categories. We found that although in general mRNA-based estimation of time categories was less accurate than hormone-based estimation, the use of three mRNA markers HSPA1B, MKNK2 and PER3 together with melatonin and cortisol generally enhanced the time prediction accuracy relative to the use of the two hormones alone. Our data best support a model that by using these five molecular biomarkers estimates three time categories, i.e., night/early morning, morning/noon, and afternoon/evening with prediction accuracies expressed as AUC values of 0.88, 0.88, and 0.95, respectively. For the first time, we demonstrate the value of mRNA for blood deposition timing and introduce a statistical model for estimating day/night time categories based on molecular biomarkers, which shall be further validated with additional samples in the future. Moreover, our work provides new leads for molecular approaches on time of death estimation using the significantly rhythmic mRNA markers established here.PostprintPeer reviewe

Investigation of metabolites for estimating blood deposition time

This study was supported by a UK Biotechnology and Biological Sciences Research Council (BBSRC) Grant (BB/I019405/1) to DJS, grant 727.011.001 from the Netherlands Organization for Scientific Research (NWO) Forensic Science Program to MK and by Erasmus MC University Medical Centre Rotterdam. DJS is a Royal Society Wolfson Research Merit Award holder. RAH and IH were funded by the Dutch applied research foundation (STW Perspectief Program ‘OnTime’ project 12185).Trace deposition timing reflects a novel concept in forensic molecular biology involving the use of rhythmic biomarkers for estimating the time within a 24-h day/night cycle a human biological sample was left at the crime scene, which in principle allows verifying a sample donor’s alibi. Previously, we introduced two circadian hormones for trace deposition timing and recently demonstrated that messenger RNA (mRNA) biomarkers significantly improve time prediction accuracy. Here, we investigate the suitability of metabolites measured using a targeted metabolomics approach, for trace deposition timing. Analysis of 171 plasma metabolites collected around the clock at 2-h intervals for 36 h from 12 male participants under controlled laboratory conditions identified 56 metabolites showing statistically significant oscillations, with peak times falling into three day/night time categories: morning/noon, afternoon/evening and night/early morning. Time prediction modelling identified 10 independently contributing metabolite biomarkers, which together achieved prediction accuracies expressed as AUC of 0.81, 0.86 and 0.90 for these three time categories respectively. Combining metabolites with previously established hormone and mRNA biomarkers in time prediction modelling resulted in an improved prediction accuracy reaching AUCs of 0.85, 0.89 and 0.96 respectively. The additional impact of metabolite biomarkers, however, was rather minor as the previously established model with melatonin, cortisol and three mRNA biomarkers achieved AUC values of 0.88, 0.88 and 0.95 for the same three time categories respectively. Nevertheless, the selected metabolites could become practically useful in scenarios where RNA marker information is unavailable such as due to RNA degradation. This is the first metabolomics study investigating circulating metabolites for trace deposition timing, and more work is needed to fully establish their usefulness for this forensic purpose.Publisher PDFPeer reviewe

Effect of Single and Combined Monochromatic Light on the Human Pupillary Light Response

The pupillary light reflex (PLR) is a neurological reflex driven by rods, cones, and melanopsin-containing retinal ganglion cells. Our aim was to achieve a more precise picture of the effects of 5-min duration monochromatic light stimuli, alone or in combination, on the human PLR, to determine its spectral sensitivity and to assess the importance of photon flux. Using pupillometry, the PLR was assessed in 13 participants (6 women) aged 27.2 ± 5.41 years (mean ± SD) during 5-min light stimuli of purple (437 nm), blue (479 nm), red (627 nm), and combinations of red+purple or red+blue light. In addition, nine 5-min, photon-matched light stimuli, ranging in 10 nm increments peaking between 420 and 500 nm were tested in 15 participants (8 women) aged 25.7 ± 8.90 years. Maximum pupil constriction, time to achieve this, constriction velocity, area under the curve (AUC) at short (0–60 s), and longer duration (240–300 s) light exposures, and 6-s post-illumination pupillary response (6-s PIPR) were assessed. Photoreceptor activation was estimated by mathematical modeling. The velocity of constriction was significantly faster with blue monochromatic light than with red or purple light. Within the blue light spectrum (between 420 and 500 nm), the velocity of constriction was significantly faster with the 480 nm light stimulus, while the slowest pupil constriction was observed with 430 nm light. Maximum pupil constriction was achieved with 470 nm light, and the greatest AUC0−60 and AUC240−300 was observed with 490 and 460 nm light, respectively. The 6-s PIPR was maximum after 490 nm light stimulus. Both the transient (AUC0−60) and sustained (AUC240−300) response was significantly correlated with melanopic activation. Higher photon fluxes for both purple and blue light produced greater amplitude sustained pupillary constriction. The findings confirm human PLR dependence on wavelength, monochromatic or bichromatic light and photon flux under 5-min duration light stimuli. Since the most rapid and high amplitude PLR occurred within the 460–490 nm light range (alone or combined), our results suggest that color discrimination should be studied under total or partial substitution of this blue light range (460–490 nm) by shorter wavelengths (~440 nm). Thus for nocturnal lighting, replacement of blue light with purple light might be a plausible solution to preserve color discrimination while minimizing melanopic activation

Pregnancy and neonatal outcomes of COVID-19: The PAN-COVID study

Objective To assess perinatal outcomes for pregnancies affected by suspected or confirmed SARS-CoV-2 infection. Methods Prospective, web-based registry. Pregnant women were invited to participate if they had suspected or confirmed SARS-CoV-2 infection between 1st January 2020 and 31st March 2021 to assess the impact of infection on maternal and perinatal outcomes including miscarriage, stillbirth, fetal growth restriction, pre-term birth and transmission to the infant. Results Between April 2020 and March 2021, the study recruited 8239 participants who had suspected or confirmed SARs-CoV-2 infection episodes in pregnancy between January 2020 and March 2021. Maternal death affected 14/8197 (0.2%) participants, 176/8187 (2.2%) of participants required ventilatory support. Pre-eclampsia affected 389/8189 (4.8%) participants, eclampsia was reported in 40/ 8024 (0.5%) of all participants. Stillbirth affected 35/8187 (0.4 %) participants. In participants delivering within 2 weeks of delivery 21/2686 (0.8 %) were affected by stillbirth compared with 8/4596 (0.2 %) delivering ≥ 2 weeks after infection (95 % CI 0.3–1.0). SGA affected 744/7696 (9.3 %) of livebirths, FGR affected 360/8175 (4.4 %) of all pregnancies. Pre-term birth occurred in 922/8066 (11.5%), the majority of these were indicated pre-term births, 220/7987 (2.8%) participants experienced spontaneous pre-term births. Early neonatal deaths affected 11/8050 livebirths. Of all neonates, 80/7993 (1.0%) tested positive for SARS-CoV-2. Conclusions Infection was associated with indicated pre-term birth, most commonly for fetal compromise. The overall proportions of women affected by SGA and FGR were not higher than expected, however there was the proportion affected by stillbirth in participants delivering within 2 weeks of infection was significantly higher than those delivering ≥ 2 weeks after infection. We suggest that clinicians’ threshold for delivery should be low if there are concerns with fetal movements or fetal heart rate monitoring in the time around infection

A three pulse phase response curve to three milligrams of melatonin in humans

Exogenous melatonin is increasingly used for its phase shifting and soporific effects. We generated a three pulse phase response curve (PRC) to exogenous melatonin (3 mg) by administering it to free-running subjects. Young healthy subjects (n = 27) participated in two 5 day laboratory sessions, each preceded by at least a week of habitual, but fixed sleep. Each 5 day laboratory session started and ended with a phase assessment to measure the circadian rhythm of endogenous melatonin in dim light using 30 min saliva samples. In between were three days in an ultradian dim light (< 150 lux)–dark cycle (LD 2.5 : 1.5) during which each subject took one pill per day at the same clock time (3 mg melatonin or placebo, double blind, counterbalanced). Each individual's phase shift to exogenous melatonin was corrected by subtracting their phase shift to placebo (a free-run). The resulting PRC has a phase advance portion peaking about 5 h before the dim light melatonin onset, in the afternoon. The phase delay portion peaks about 11 h after the dim light melatonin onset, shortly after the usual time of morning awakening. A dead zone of minimal phase shifts occurred around the first half of habitual sleep. The fitted maximum advance and delay shifts were 1.8 h and 1.3 h, respectively. This new PRC will aid in determining the optimal time to administer exogenous melatonin to achieve desired phase shifts and demonstrates that using exogenous melatonin as a sleep aid at night has minimal phase shifting effects

Predicting human nocturnal nonvisual responses to monochromatic and polychromatic light with a melanopsin photosensitivity function

\u3cp\u3eThe short-wavelength (blue) light sensitivity of human circadian, neurobehavioral, neuroendocrine, and neurophysiological responses is attributed to melanopsin. Whether melanopsin is the sole factor in determining the efficacy of a polychromatic light source in driving nonvisual responses, however, remains to be established. Monochromatic (λ\u3csub\u3emax\u3c/sub\u3e 437, 479, and 532nm administered singly and in combination with 479nm light) and polychromatic (color temperature: 4000 K and 17000 K) light stimuli were photon matched for their predicted ability to stimulate melanopsin, and their capacity to affect nocturnal melatonin levels, auditory reaction time, and subjective alertness and mood was assessed. Young, healthy male participants aged 18-35 yrs (23.6±3.6 yrs [mean±SD]; n = 12) participated in 12 overnight sessions that included an individually timed 30-min nocturnal light stimulus on the rising limb of the melatonin profile. At regular intervals before, during, and after the light stimulus, subjective mood and alertness were verbally assessed, blood samples were taken for analysis of plasma melatonin levels, and an auditory reaction time task (psychomotor vigilance task; PVT) was performed. Proc GLM (general linear model) repeated-measures ANOVA (analysis of variance) revealed significantly lower melatonin suppression with the polychromatic light conditions (4000 and 17000 K) compared to the melanopsin photon-matched monochromatic light conditions (p&lt;.05). In contrast, subjective alertness was significantly lower under the 479nm monochromatic light condition compared to the 437 and 532nm monochromatic and both polychromatic light conditions. The alerting responses more reflected the total photon content of the light stimulus. The demonstration that the melatonin suppression response to polychromatic light was significantly lower than predicted by the melanopsin photosensitivity function suggests this function is not the sole consideration when trying to predict the efficacy of broadband lighting. The different spectral sensitivity of subjective alertness and melatonin suppression responses may imply a differential involvement of the cone photopigments. An analysis of the photon densities in specific wavelength bands for the polychromatic lights used in this and the authors' previous study suggests the spectral composition of a polychromatic light source, and particularly the very short-wavelength content, may be critical in determining response magnitude for the neuroendocrine and neurobehavioral effects of nocturnal light.\u3c/p\u3

Blue-light phase shifts PER3 gene expression in human leukocytes

The timing of clock gene expression in human leukocytes was investigated following a phase-advancing light stimulus to determine whether the response is wavelength- and/or age-dependent. PERIOD3 (PER3) clock gene expression in leukocytes and plasma melatonin were analyzed before and after monochromatic blue and green light exposure. Significant phase advances were observed in the peak timing of both PER3 expression and melatonin following blue but not green light. The amplitude of the PER3 rhythm at baseline was significantly reduced with age. However, age did not affect the response of the PER3 rhythm to light.</p

Effect of sleep deprivation on rhythms of clock gene expression and melatonin in humans

This study investigated the impact of sleep deprivation on the human circadian system. Plasma melatonin and cortisol levels and leukocyte expression levels of 12 genes were examined over 48 h (sleep vs. no-sleep nights) in 12 young males (mean ± SD: 23 ± 5 yrs). During one night of total sleep deprivation, BMAL1 expression was suppressed, the heat shock gene HSPA1B expression was induced, and the amplitude of the melatonin rhythm increased, whereas other high-amplitude clock gene rhythms (e.g., PER1-3, REV-ERBα) remained unaffected. These data suggest that the core clock mechanism in peripheral oscillators is compromised during acute sleep deprivation