Investigation of metabolites for estimating blood deposition time.

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.

Unraveling the functions of type II-prohibitins in Arabidopsis mitochondria.

In yeast and mammals, prohibitins (PHBs) are considered as structural proteins that form a scaffold-like structure for interacting with a set of proteins involved in various processes occurring in the mitochondria. The role of PHB in plant mitochondria is poorly understood. In the study, the model organism Arabidopsis thaliana was used to identify the possible roles of type-II PHBs (homologs of yeast Phb2p) in plant mitochondria. The obtained results suggest that the plant PHB complex participates in the assembly of multisubunit complexes; namely, respiratory complex I and enzymatic complexes carrying lipoic acid as a cofactor (pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and glycine decarboxylase). PHBs physically interact with subunits of these complexes. Knockout of two Arabidopsis type-II prohibitins (AtPHB2 and AtPHB6) results in a decreased abundance of these complexes along with a reduction in mitochondrial acyl carrier proteins. Also, the absence of AtPHB2 and AtPHB6 influences the expression of the mitochondrial genome and leads to the activation of alternative respiratory pathways, namely alternative oxidase and external NADH-dependent alternative dehydrogenases.

Understanding the Effect of Drilling Parameters on Hole Quality of Fiber-Reinforced Polymer Structures.

Hole quality in composite materials is gaining interest in aerospace, automotive, and marine industries, especially for structural applications. This paper aims to investigate the quality of holes performed without a backup plate, in thin plates of glass fiber-reinforced polymer (GFRP). The samples were manufactured by two different technologies: vacuum bagging and an innovative method named vacuum mold pressing. Three experiments were designed choosing the control factors that affect the maximum cutting force, delamination factor, and surface roughness of drilled holes in composite materials based on twill fabric layers. Quality analysis of the hole features was performed by microscopy investigations. The effects of the main factors on the targets are investigated using the statistical design of experiments, considering control factors, such as support opening width, weight fraction (wf), feed per tooth, and hole area. The results showed that the feed per tooth and hole area had a more significant influence on the delamination factors and surface roughness (Sa). The best quality of the holes drilled in twill-based GFRP was achieved for a lower feed rate of 0.04 mm/tooth and used a support opening width of 55 mm.

Effect of Psyllium Husk Addition on the Structural and Physical Properties of Biodegradable Thermoplastic Starch Film.

The research subject was the analysis of the microstructure, barrier properties, and mechanical resistance of the psyllium husk (PH)-modified thermoplastic starch films. The tensile tests under various static loading conditions were not performed by researchers for this type of material before and are essential for a more precise assessment of the material's behavior under the conditions of its subsequent use. The film samples were manufactured by the casting method. PH addition improved starch gelatinization and caused a decrease in failure strain by 86% and an increase in failure stress by 48% compared to pure films. Fourier transform infrared spectroscopy results showed the formation of additional hydrogen bonds between polysaccharides in starch and PH. An increase in the number of hydrophilic groups in the modified films resulted in a faster contact angle decrease (27.4% compared to 12.8% for pure ones within the first 5 s); however, it increased the energy of water binding and surface complexity. The modified films showed the opacity at 600 nm, 43% higher than in the pure starch film, and lower transmittance, suggesting effectively improving barrier properties to UV light, a potent lipid-oxidizing agent in food systems.

Effect of Nesfatin-1 on Rat Humerus Mechanical Properties under Quasi-Static and Impact Loading Conditions.

The investigations on the response of bone tissue under different loading conditions are important from clinical and engineering points of view. In this paper, the influence of nesfatin-1 administration on rat humerus mechanical properties was analyzed. The classical three-point bending and impact tests were carried out for three rat bone groups: control (SHO), the humerus of animals under the conditions of established osteopenia (OVX), and bones of rats receiving nesfatin-1 after ovariectomy (NES). The experiments proved that the bone strength parameters measured under various mechanical loading conditions increased after the nesfatin-1 administration. The OVX bones were most susceptible to deformation and had the smallest fracture toughness. The SEM images of humerus fracture surface in this group showed that ovariectomized rats had a much looser bone structure compared to the SHO and NES females. Loosening of the bone structure was also confirmed by the densitometric and qualitative EDS analysis, showing a decrease in the OVX bones' mineral content. The samples of the NES group were characterized by the largest values of maximum force obtained under both quasi-static and impact conditions. The energies absorbed during the impact and the critical energy for fracture (from the three-point bending test) were similar for the SHO and NES groups. Statistically significant differences were observed between the mean Fi max values of all analyzed sample groups. The obtained results suggest that the impact test was more sensitive than the classical quasi-static three-point bending one. Hence, Fi max could be used as a parameter to predict bone fracture toughness.

Role of polyamines in determining the cellular response to chemotherapeutic agents: modulation of protein kinase CK2 expression and activity.

Numerous studies have shown that platinum compounds stimulate the expression of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase resulting in anti-proliferative activity and apoptosis. As many cancer cell types including pancreatic cancer cells express high levels of polyamines, the possibility to develop anti-tumor strategies to deplete polyamine pools has drawn considerable attention in recent years. This has been effectively accomplished by treating cells with platinum drugs in combination with polyamine analogs such as N(1),N(11)-diethylnorspermine (DENSPM). The present study, examined the cytotoxic effects of oxaliplatin in combination with stimulators of polyamine catabolism in human pancreatic cancer cells, that are notoriously resistant to chemotherapeutic treatment, and colorectal cancer cells. Additionally, as protein kinase CK2 has been shown to be an anti-apoptotic and pro-survival enzyme regulated by the intracellular polyamine pools, we aimed to investigate the effect of combined DENSPM and oxaliplatin treatment on CK2-mRNA and -protein levels. Results reported here show that treatment with oxaliplatin and DENSPM in combination impairs cell viability particularly in the case of colorectal cancer cells. The analysis of CK2 expression and activity indicates that the response to a specific treatment may depend on the impact that individual compounds exert on pro-survival and pro-death proteins at the transcription and translation levels that should be carefully evaluated in view of subsequent clinical studies.

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

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 2h intervals for 36h 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.

Dissecting Daily and Circadian Expression Rhythms of Clock-Controlled Genes in Human Blood.

The identification and investigation of novel clock-controlled genes (CCGs) has been conducted thus far mainly in model organisms such as nocturnal rodents, with limited information in humans. Here, we aimed to characterize daily and circadian expression rhythms of CCGs in human peripheral blood during a sleep/sleep deprivation (S/SD) study and a constant routine (CR) study. Blood expression levels of 9 candidate CCGs (SREBF1, TRIB1, USF1, THRA1, SIRT1, STAT3, CAPRIN1, MKNK2, and ROCK2), were measured across 48 h in 12 participants in the S/SD study and across 33 h in 12 participants in the CR study. Statistically significant rhythms in expression were observed for STAT3, SREBF1, TRIB1, and THRA1 in samples from both the S/SD and the CR studies, indicating that their rhythmicity is driven by the endogenous clock. The MKNK2 gene was significantly rhythmic in the S/SD but not the CR study, which implies its exogenously driven rhythmic expression. In addition, we confirmed the circadian expression of PER1, PER3, and REV-ERBα in the CR study samples, while BMAL1 and HSPA1B were not significantly rhythmic in the CR samples; all 5 genes previously showed significant expression in the S/SD study samples. Overall, our results demonstrate that rhythmic expression patterns of clock and selected clock-controlled genes in human blood cells are in part determined by exogenous factors (sleep and fasting state) and in part by the endogenous circadian timing system. Knowledge of the exogenous and endogenous regulation of gene expression rhythms is needed prior to the selection of potential candidate marker genes for future applications in medical and forensic settings.

An observational study on disturbed peripheral circadian rhythms in hemodialysis patients.

The quality of life of hemodialysis (HD) patients is hampered by reduced nocturnal sleep quality and excessive daytime sleepiness. In addition to the sleep/wake cycle, levels of circadian biomarkers (e.g. melatonin) are disturbed in end-stage renal disease (ESRD). This suggests impaired circadian clock performance in HD patients, but the underlying mechanism is unknown. In this observational study, diurnal rhythms of sleep, serum melatonin and cortisol concentrations and clock gene mRNA expression are compared between HD patients (n = 9) and healthy control subjects (n = 9). In addition, the presence of circulating factors that might affect circadian rhythmicity is tested in vitro with cell culture experiments. Reduced sleep quality (median sleep onset latency [interquartile range] of 23.9 [17.3] min for patients versus 5.0 [10] minutes for controls, p < 0.01; mean (± SD) sleep efficiency 70.2 ± 8.1% versus 82.9 ± 10.9%, p = 0.02 and mean awake minutes after sleep onset 104.8 ± 27.9 versus 54.6 ± 41.6 minutes, p = 0.01) and increased daytime sleepiness (mean Epworth Sleepiness Score of 10.0 ± 4.8 versus 3.9 ± 2.0, p < 0.01) were confirmed in HD patients. Reduced nocturnal melatonin concentrations (1 AM: 98.1 [122.9] pmol/L versus 12.5 [44.2] pmol/L, p = 0.019; 5 AM: 114.0 [131.6] pmol/L versus 11.8 [86.8] pmol/L, p = 0.031) and affected circadian control of cortisol rhythm and circadian expression of the clock gene REV-ERBα were found. HD patient serum had a higher capacity to synchronize cells in vitro, suggesting an accumulated level of clock resetting compounds in HD patients. These compounds were not cleared by hemodialysis treatment or related to frequently used medications. In conclusion, the abovementioned results strongly suggest a disturbance in circadian timekeeping in peripheral tissues of HD patients. Accumulation of clock resetting compounds possibly contributes to this. Future studies are needed for a better mechanistic understanding of the interaction between renal failure and perturbation of the circadian clock.

Assessing the suitability of miRNA-142-5p and miRNA-541 for bloodstain deposition timing.

A recent proof-of-concept pilot study proposed using microRNA (miRNA) markers for time of death determination. The markers - miRNA-142-5p and miRNA-541, were reported to show considerable expression differences in vitreous humor between individuals who died during the day or night. Here, we investigated whether these miRNA markers show the same diurnal expression pattern in blood, which would make them useful for estimating bloodstain deposition time to allow molecular alibi testing for forensic casework. We analyzed venous blood samples collected from 12 healthy individuals every 4h during the 24hday/night period under controlled sleep-laboratory conditions. MiRNA-142-5p normalized against miRNA-222 showed no statistically significant expression differences between blood samples collected during daytime and nighttime (one-way ANOVA p=0.81), and also no statistically significant rhythmicity during the 24hday/night period (cosine fit for all individuals p>0.05, averaged data p=0.932). MicroRNA-541 amplification in blood was above the 34-cycle threshold applied in the study, indicating too low quantities for obtaining reliable data. Overall, we conclude that the two miRNA markers previously suggested for time of death determination in vitreous humor are not suitable for estimating the deposition time of forensic bloodstains. Future studies may find out if miRNA markers with significant diurnal expression patterns can be identified and how useful they would be for forensic trace deposition timing.