A practical approach to supported liquid extraction and measurement of 18 steroids in plasma and serum by targeted liquid chromatography tandem mass spectrometry.

Chromatography combined with mass spectrometry is a gold standard technique for steroid measurement, however the type of sample preparation, the dynamic range and reliability of the calibration curve, the chromatographic separation and mass spectrometry settings ultimately determine the success of the method. The steroid biosynthetic pathway is conserved in higher mammals and literature demonstrates that the concentration ranges of different steroid groups are relatively comparable across species. We sought to develop a robust and reliable multi steroid targeted analysis method for blood that would have wide application across higher mammals. The method was developed following bioanalytical method validation guidelines to standards typically applied to human clinical studies, including isotopically labelled internal standards where at all possible. Here we describe the practical approach to a 96-well supported liquid extraction (SLE) method of extraction from plasma (200 µL) using an Extrahera liquid handling robot (Biotage, Sweden), including quality control samples, followed by a comprehensive separation and targeted LC-MS/MS analysis of 18 steroids in plasma (pregnenolone, progesterone, 17α-hydroxyprogesterone, 11-deoxycorticosterone, corticosterone, 11-dehydrocorticosterone, aldosterone, 11-deoxycortisol, 21-deoxycortisol, cortisol, cortisone, androstenedione, testosterone, 5α-dihydrotestosterone, dehydroepiandrosterone, estrone, 17ß-estradiol and estriol). •SLE in a 96-well format of up to 74 biological plasma samples, enriched with multiple isotopically labelled internal standards, a 12-point aqueous calibration curve, and 6 serum quality controls, designed to monitor long-term performance of the method•Chromatographic separation of multiple steroids along the gradient, with ammonium fluoride mobile phase additive to improve sensitivity, followed by electrospray ionisation and constant polarity switching•Aqueous calibration standards that cover physiologically relevant ranges - high nanomolar glucocorticoids, low nanomolar androgens and picomolar ranges for estrogens and steroid intermediates.

ATP-binding cassette family C member 1 constrains metabolic responses to high-fat diet in male mice.

Glucocorticoids modulate glucose homeostasis, acting on metabolically active tissues such as liver, skeletal muscle, and adipose tissue. Intracellular regulation of glucocorticoid action in adipose tissue impacts metabolic responses to obesity. ATP-binding cassette family C member 1 (ABCC1) is a transmembrane glucocorticoid transporter known to limit the accumulation of exogenously administered corticosterone in adipose tissue. However, the role of ABCC1 in the regulation of endogenous glucocorticoid action and its impact on fuel metabolism has not been studied. Here, we investigate the impact of Abcc1 deficiency on glucocorticoid action and high-fat-diet (HFD)-induced obesity. In lean male mice, deficiency of Abcc1 increased endogenous corticosterone levels in skeletal muscle and adipose tissue but did not impact insulin sensitivity. In contrast, Abcc1-deficient male mice on HFD displayed impaired glucose and insulin tolerance, and fasting hyperinsulinaemia, without alterations in tissue corticosterone levels. Proteomics and bulk RNA sequencing revealed that Abcc1 deficiency amplified the transcriptional response to an obesogenic diet in adipose tissue but not in skeletal muscle. Moreover, Abcc1 deficiency impairs key signalling pathways related to glucose metabolism in both skeletal muscle and adipose tissue, in particular those related to OXPHOS machinery and Glut4. Together, our results highlight a role for ABCC1 in regulating glucose homeostasis, demonstrating diet-dependent effects that are not associated with altered tissue glucocorticoid concentrations.

Unpacking workplace stress and forensic expert decision-making: From theory to practice.

Workplace stress can affect forensic experts' job satisfaction and performance, which holds financial and other implications for forensic service providers. Therefore, it is important to understand and manage workplace stress, but that is not simple or straightforward. This paper explores stress as a human factor that influences forensic expert decision-making. First, we identify and highlight three factors that mitigate decisions under stress conditions: nature of decision, individual differences, and context of decision. Second, we situate workplace stress in forensic science within the Challenge-Hindrance Stressor Framework. We argue that stressors in forensic science workplaces can have a positive or a negative impact, depending on the type, level, and context of stress. Developing an understanding of the stressors, their sources, and their possible impact can help forensic service providers and researchers to implement context-specific interventions to manage stress at work and optimize expert performance.

Comparison of DNA recovery methods and locations from regularly-worn hooded jumpers before and after use by a second wearer.

Items of worn clothing are routinely examined for DNA in forensic casework, commonly with the expectation that at least some of the DNA will come from a wearer of the item, so-called 'wearer DNA'. This study investigated DNA recovered from hooded jumpers that were regularly worn and laundered for four weeks and then subsequently worn by a different individual for four hours. This study also systematically investigated whether using different recovery methods or sampling locations on the jumpers might distinguish between DNA deposited by the regular and most recent wearers of clothing. Four volunteers each wore a new hooded jumper regularly (6 h/day, 2 days/week, washed at weekends) during two 4-week periods. At the end of each month, DNA was first recovered by cutting out and mini-taping the inside left cuff, half-collar, pocket and underarm fabric. The jumpers were then worn by a different individual for four hours, and DNA was again recovered by cutting out and mini-taping, but this time from the inside right cuff, half-collar, pocket and underarm fabric. All DNA samples (n = 128) were quantified and profiled. DNA quantities ranged from 0 to âˆ¼40 ng with an outlier of âˆ¼150 ng, and no significant differences were observed among recovery methods and sampling locations, nor whether one or two wearers had worn the jumpers. However, one volunteer consistently deposited significantly more DNA to their jumpers than two other volunteers, confirming the impact of 'shedder status' on DNA deposition during wearing of clothing. When jumpers were regularly worn by one wearer, the majority (72.7-83.3 %) of the samples for all wearers across both months comprised a major profile of the wearer with a minor profile of non-wearer alleles. When jumpers were then worn by a second wearer, the composition of the profiles obtained were generally reproducible across the recovery methods used, the sampling locations and the two replicates of the experiment for each pairing of wearers. However, profile compositions differed between wearer pairings. Overall, ∼60 % of profiles obtained gave a major profile of the regular wearer, whereas âˆ¼30 % gave a major profile of the second wearer. The remaining profiles comprised other much less frequent observations of single-source profiles of each wearer and equal proportions of DNA from both wearers. Non-wearer DNA was also observed in the majority of samples, both before and after jumpers were worn by a second wearer. For one volunteer's jumpers, a recurring non-wearer DNA profile was observed that could be attributed to their romantic partner, and this DNA persisted on the jumpers even after being worn by the second wearer. This study provides insight on the impact of shedder status, multiple wearers, different recovery methods and sampling locations on the quantities of DNA and compositions of DNA profiles recovered from authentically regularly-worn hooded jumpers. The findings also provide a preliminary dataset that can be used to infer activity level probabilities in casework.

Human identification: an investigation of 3D models of paranasal sinuses to establish a biological profile on a modern UK population.

Medical imaging is a valuable source for facilitating empirical research and provides an accessible gateway for developing novel forensic anthropological methods for analysis including 3D modelling. This is especially critical for the United Kingdom (UK), where methods developed from modern UK populations do not currently exist. This study introduces a new approach to assist in human identification using 3D models of the paranasal sinuses. The models were produced from a database of 500 modern CT scans provided by University College London Hospital. Linear measurements and elliptic Fourier coefficients taken from 1500 three-dimensional models across six ethnic groups assessed by one-way ANOVA and discriminant function analysis showed a range of classification rates with certain rates reaching 75-85.7% (p < 0.05) in correctly classifying age and sex according to size and shape. The findings offer insights into the potential for employing paranasal sinuses as an attribute for establishing the identification of unknown human remains in future crime reconstructions.

Plasma cortisol-linked gene networks in hepatic and adipose tissues implicate corticosteroid-binding globulin in modulating tissue glucocorticoid action and cardiovascular risk.

Genome-wide association meta-analysis (GWAMA) by the Cortisol Network (CORNET) consortium identified genetic variants spanning the SERPINA6/SERPINA1 locus on chromosome 14 associated with morning plasma cortisol, cardiovascular disease (CVD), and SERPINA6 mRNA expression encoding corticosteroid-binding globulin (CBG) in the liver. These and other findings indicate that higher plasma cortisol levels are causally associated with CVD; however, the mechanisms by which variations in CBG lead to CVD are undetermined. Using genomic and transcriptomic data from The Stockholm Tartu Atherosclerosis Reverse Networks Engineering Task (STARNET) study, we identified plasma cortisol-linked single-nucleotide polymorphisms (SNPs) that are trans-associated with genes from seven different vascular and metabolic tissues, finding the highest representation of trans-genes in the liver, subcutaneous fat, and visceral abdominal fat, [false discovery rate (FDR) = 15%]. We identified a subset of cortisol-associated trans-genes that are putatively regulated by the glucocorticoid receptor (GR), the primary transcription factor activated by cortisol. Using causal inference, we identified GR-regulated trans-genes that are responsible for the regulation of tissue-specific gene networks. Cis-expression Quantitative Trait Loci (eQTLs) were used as genetic instruments for identification of pairwise causal relationships from which gene networks could be reconstructed. Gene networks were identified in the liver, subcutaneous fat, and visceral abdominal fat, including a high confidence gene network specific to subcutaneous adipose (FDR = 10%) under the regulation of the interferon regulatory transcription factor, IRF2. These data identify a plausible pathway through which variation in the liver CBG production perturbs cortisol-regulated gene networks in peripheral tissues and thereby promote CVD.

Sex estimation from virtual models: exploring the potential of stereolithic 3D crania models for morphoscopic trait scoring.

Modern computed tomography (CT) databases are becoming an accepted resource for the practice and development of identification methods in forensic anthropology. However, the utility of 3D models created using free and open-source visualization software such as 3D Slicer has not yet been thoroughly assessed for morphoscopic biological profiling methods where virtual methods of analysis are becoming more common. This paper presents a study that builds on the initial findings from Robles et al. (2020) to determine the feasibility of estimating sex on stereolithic (STL) 3D cranial models produced from CT scans from a modern, living UK population (n = 80) using equation 2 from the Walker's (2008) morphoscopic method. Kendall's coefficients of concordance (KCC) indicated substantial agreement using cranial features scores in an inter-observer test and a video-inter-observer test. Fleiss' Kappa scores showed moderate agreement (0.50) overall between inter-observer sex estimations, and for observer sex estimations in comparison to recorded sexes (0.56). It was found that novice users could virtually employ morphoscopic sex estimation methods effectively on STL 3D cranial models from modern individuals. This study also highlights the potential that digital databases of modern living populations can offer forensic anthropology. Key points: First example of Walker's (2008) method applied to a living UK population.Open-source software is a valuable resource for crime reconstruction approaches.Male scoring bias was observed in method application.Forensic anthropologists would benefit from virtual anthropology training to use and interpret 3D models.Digital databases offer more ethical, diverse, modern populations for future research.

Regaining creativity in science: insights from conversation.

The 'early modern' (Renaissance) workshop was predicated on the idea that informal, open-ended cooperation enables participants to experience difference and develop new insights, which can lead to new ways of thinking and doing. This paper presents the insights that emerged from a conversation event that brought wide-ranging voices together from different domains in science, and across the arts and industry, to consider science leadership as we look to the future in a time of interlocking crises. The core theme identified was a need to regain creativity in science; in the methods of scientific endeavours, in the way science is produced and communicated, and in how science is experienced in society. Three key challenges for re-establishing a culture of creativity in science emerged: (i) how scientists communicate what science is and what it is for, (ii) what scientists value, and (iii) how scientists create and co-create science with and for society. Furthermore, the value of open-ended and ongoing conversation between different perspectives as a means of achieving this culture was identified and demonstrated.

Drilling down into ethics: A thematic review of ethical considerations for the creation and use of 3D printed human remains in crime reconstruction.

The existing literature contains some exploration of the ethics concerning human remains in forensic and virtual anthropology. However, previous work has stopped short of interrogating the underlying ethical concepts. The question of how people understand and apply these concepts in practice, and what it means to act ethically, remain underexplored. This thematic review explores the ethical considerations that contribute to the creation and use of 3D printed human remains for forensic purposes. The three main branches of ethical theory are outlined to explore how they may apply to forensic practice. Key themes relating to 3D printing human remains in forensic contexts were explored to better understand the ethics landscape, ethical challenges, and the current guidelines in place. Through this thematic review, nine ethics principles were identified as key principles for guiding best practice: anonymity, autonomy, beneficence, consent, context, justice, non-maleficence, proportionality, and transparency. It is suggested that these principles could be incorporated into adaptable guidelines going forward to support ethical practice. The findings also suggest that holistic ethics cognition training may have value in supporting forensic scientists in ethical decision-making, together with procedural and structural design that may promote best practice and reduce cognitive load.

The effect of season, management and endocrinopathies on vitamin D status in horses.

BACKGROUND: Vitamin D deficiency is common in humans and is increasingly linked to the pathogenesis of a multitude of diseases including obesity and metabolic syndrome. The biology of vitamin D in horses is poorly described; the relative contribution of the diet and skin synthesis to circulating concentrations is unclear and associations with the endocrine disease have not been explored. OBJECTIVES: To determine the relationship between management, season and endocrine disease and vitamin D status in horses. STUDY DESIGN: Cross-sectional cohort study. METHODS: Plasma concentrations of 25-hydroxyvitamin D2 (25(OH)D2 ) and D3 (25(OH)D3 ) were measured by liquid chromatography-tandem mass spectrometry in 34 healthy unsupplemented grazing ponies and 22 stabled Thoroughbreds receiving supplementary vitamin D3 in feed. A nested group of 18 grazing ponies were sampled on long and short days (>12 and <12 h of light/day) to determine the effect of sunlight exposure. In addition, the relationships between age, sex, adiposity, serum insulin, adrenocorticotropic hormone and vitamin D status were assessed in a mixed group of 107 horses using a linear regression model. RESULTS: All animals had a measurable level of 25(OH)D2 (median 10.7 nmol/L) whilst 25(OH)D3 was only detected in Thoroughbreds receiving D3 supplementation. Thoroughbreds had lower concentrations of 25(OH)D2 than ponies (7.4 vs. 12.6 nmol/L, p < 0.01). In grazing ponies, 25(OH)D2 concentrations were significantly higher on long days compared to short days (14.4 vs. 8.7 nmol/L, p < 0.01), whilst 25(OH)D3 was undetectable. Measures of increased adiposity, but not basal insulin, were associated with higher 25(OH)D2 concentrations, conversely to humans. Increasing ACTH was associated with lower 25(OH)D2 (p < 0.01). MAIN LIMITATIONS: Vitamin D2 concentrations were not measured in grass or forage. CONCLUSIONS: In horses 25(OH)D2 is the predominant vitamin D metabolite, and there is an apparent lack of endogenous vitamin D3 production. The relationship between vitamin D and endocrine disorders in horses does not reflect that of other species and warrants further investigation.

A method to induce stress in human subjects in online research environments.

This paper presents a method to induce stress in human subjects during online participation in research studies without the presence of researchers. In this study, participants in the stress-inducing condition (N = 52, 44%) were asked to answer general knowledge and mathematical questions which people often get wrong, and did so under time pressure as well as receiving feedback. In contrast, participants in the control condition (N = 66, 56%) did not have time pressure or receive feedback. The stress manipulation was found to be effective, as the reported state anxiety and visual analog scale on stress scores were higher for the stress group than for the non-stress group (both findings, p < 0.001). Consistent findings were found when accounting for trait anxiety as a moderator, with the exception of the state anxiety levels in high trait anxiety group. This stressing method combines the established stress conditions of uncontrollability (such as time pressures) and social evaluative threats (such as negative feedback). In addition, the method contains specific measures (such as a commitment statement and attention check questions) to enhance the internal validity by preventing and detecting cheating or random responses. This method can be deployed through any commonly available online software. It offers a simple and cost-effective way to collect data online - which fits the increasing need to carry out research in virtual and online environments.

Comparison of mechanisms of angiostasis caused by the anti-inflammatory steroid 5α-tetrahydrocorticosterone versus conventional glucocorticoids.

5α-Tetrahydrocorticosterone (5αTHB) is an effective topical anti-inflammatory agent in mouse, with less propensity to cause skin thinning and impede new blood vessel growth compared with corticosterone. Its anti-inflammatory effects were not prevented by RU38486, a glucocorticoid receptor antagonist, suggesting alternative mechanisms. The hypothesis that 5αTHB directly inhibits angiogenesis to a lesser extent than hydrocortisone was tested, focussing on glucocorticoid receptor mediated actions. New vessel growth from aortae from C57BL/6 male mice was monitored in culture, in the presence of 5αTHB, hydrocortisone (mixed glucocorticoid/mineralocorticoid receptor agonist) or the selective glucocorticoid receptor agonist dexamethasone. Transcript profiles were studied, as was the role of the glucocorticoid receptor, using the antagonist, RU38486. Ex vivo, 5αTHB suppressed vessel growth from aortic rings, but was less potent than hydrocortisone (EC50 2512 nM 5αTHB, versus 762 nM hydrocortisone). In contrast to conventional glucocorticoids, 5αTHB did not alter expression of genes related to extracellular matrix integrity or inflammatory signalling, but caused a small increase in Per1 transcript, and decreased transcript abundance of Pecam1 gene. RU38486 did not antagonise the residual effects of 5αTHB to suppress vessel growth or regulate gene expression, but modified effects of dexamethasone. 5αTHB did not alter expression of glucocorticoid-regulated genes Fkbp51 and Hsd11b1, unlike hydrocortisone and dexamethasone. In conclusion, compared with hydrocortisone, 5αTHB exhibits limited suppression of angiogenesis, at least directly in blood vessels and probably independent of the glucocorticoid receptor. Discriminating the mechanisms employed by 5αTHB may provide the basis for the development of novel safer anti-inflammatory drugs for topical use.

Investigating the uses of mobile phone evidence in China criminal proceedings.

Data from mobile phones are regularly used in the investigation of crime and court proceedings. Previously published research has primarily addressed technical issues or provided operational manuals for using forensic science evidence, rather than analysing human factors and the implementation of forensic tools in investigation settings. Moreover, previous research has focused almost entirely on western countries, and there is a dearth of research into the uses of forensic evidence in China. In this study, a review was carried out of court sentencing documents referring to mobile phone evidence in China over the period 2013-2018. Automated content analysis was used to identify the specific evidence types utilised and the sentencing outcome for each case. Results show that mobile phone evidence was used in 3.3% of criminal proceedings. Among various data types mentioned in criminal proceedings, call records sustained as the most frequently used type of data. After which, instant messaging tools (e.g. WeChat) are an increasing proportion of all mobile phone evidence, from 1% in 2015 to 25% in 2018. For cases that utilised mobile phone data, the analysis of instant messaging and online transaction tools is routine, with little variation in the use of each application (WeChat, Alipay, QQ) for investigations of different types of crime. However, in the majority of criminal cases, mobile phone data function as subsidiary evidence and posed limited impacts on verdict reached. The current findings indicate that a large amount of mobile phone evidence was transformed into other evidence formats or filtered out directly before court proceedings.

An investigation into the accuracy of follow-on GPRS/mobile data CDRs.

This study investigated the accuracy of 3G and 4G follow-on GPRS (General Packet Radio Service)/mobile data CDRs (Call Detail Records) from three UK mobile network operators (EE, Vodafone and Three). Follow-on GPRS/mobile data CDRs are currently considered to be more open to misinterpretation than voice/SMS CDRs as uncertainties exist regarding the correspondence between the timestamp and the Cell ID presented within the CDRs. Consequently, follow-on GPRS/mobile CDRs may be disregarded during criminal investigations, potentially losing valuable intelligence and evidence. To assess the accuracy of follow-on GPRS/mobile data CDRs, connected mode RF (Radio Frequency) surveys were conducted while simultaneously producing follow-on GPRS/mobile data CDRs in a travelling vehicle. This allowed a comparison of the start Cell ID presented in the CDR and the Cell ID that provided coverage to the device at the start time of the CDR to assess the correspondence between the timestamp and the Cell ID presented within the CDRs, and to consider the validity of the terminology used by experts. It was found that individual follow-on GPRS/mobile data CDRs cannot consistently place a device within the coverage area of the start Cell ID at the start time of the CDR. Instead, the results indicate that a terminology which places the device within the coverage area of the start Cell ID 'at or before' the start time of the CDR is appropriate. It is crucial that follow-on GPRS/mobile data CDRs are analysed with this consideration in mind so to interpret the evidence correctly.

A multi-method assessment of 3D printed micromorphological osteological features.

The evaluation of 3D printed osteological materials has highlighted the difficulties associated with accurately representing fine surface details on printed bones. Moreover, there is an increasing need for reconstructions to be demonstrably accurate and reliable for use in the criminal justice system. The aim of this study was to assess the surface quality of 3D prints (n = 9) that presented with micromorphological alterations from trauma, taphonomy and pathology processes. The archaeological bones were imaged using micro-CT scanning and 3D printed with selective laser sintering (SLS) printing. A multi-method experimental approach subsequently identified: (1) the 3D printed bones to be metrically accurate to within 1.0 mm; (2) good representation of micromorphological surface features overall, albeit with some loss of intricate details, depths, and fine textures that can be important for visual processing; (3) five of the nine 3D printed bones were quantitatively scored as accurate using the visual comparison method; and, (4) low mesh comparison distances (± 0.2 mm) between the original models and the digitised 3D print models. The findings offer empirical data that can be used to underpin 3D printed reconstructions of exhibits for use in courts of law. In addition, an adaptable pathway was presented that can be used to assess 3D print accuracy in future reconstructions.

ABCC1 modulates negative feedback control of the hypothalamic-pituitary-adrenal axis in vivo in humans.

BACKGROUND: Cortisol and corticosterone both circulate in human plasma and, due to differing export by ATP-binding cassette (ABC) transporters, may exert differential cellular effects. ABCB1 (expressed in brain) exports cortisol not corticosterone while ABCC1 (expressed in adipose and skeletal muscle) exports corticosterone not cortisol. We hypothesised that ABCC1 inhibition increases corticosteroid receptor occupancy by corticosterone but not cortisol in humans. METHODS: A randomised double-blind crossover study was conducted in 14 healthy men comparing placebo and ABCC1 inhibitor probenecid. Blood sampling, including from veins draining adipose and muscle, was undertaken before and after administration of mineralocorticoid receptor antagonist potassium canrenoate and glucocorticoid receptor antagonist mifepristone (RU486). RESULTS: During placebo, systemic plasma cortisol and corticosterone concentrations increased promptly after canrenoate. Cortisol uptake was detected from adipose but not muscle following canrenoate + RU486. Probenecid significantly increased systemic cortisol concentrations, and tended to increase corticosterone and ACTH concentrations, after combined receptor antagonism but had no effects on net glucocorticoid balance in either adipose or muscle. Using quantitative PCR in brain bank tissue, ABCC1 expression was 5-fold higher in human pituitary than hypothalamus and hippocampus. ABCB1 was more highly expressed in hypothalamus compared to pituitary. CONCLUSIONS: Although displacement of corticosterone and/or cortisol from receptors in adipose and skeletal muscle could not be measured with sufficient precision to detect effects of probenecid, ABCC1 inhibition induced a greater incremental activation of the hypothalamic-pituitary-adrenal axis after combined receptor blockade, consistent with ABCC1 exporting corticosterone from the pituitary and adding to the evidence that ABC transporters modulate tissue glucocorticoid sensitivity.

3D forensic science: A new field integrating 3D imaging and 3D printing in crime reconstruction.

3D techniques are increasingly being used by forensic scientists in crime reconstruction. The proliferation of 3D techniques, such as 3D imaging and printing being employed across the various stages of the forensic science process, means that the use of 3D should be considered as a distinct field within forensic science. '3D Forensic Science' ('3DFS') is therefore presented in this paper as a field that brings together a range of 3D techniques and approaches that have been developed within different areas of forensic science for achieving crime reconstructions and interpreting and presenting evidence. It is argued that by establishing this distinct field, defining its boundaries, and developing expertise, best practice and standards, the contribution of 3DFS to the criminal justice system can be maximised and the accuracy and robustness of crime reconstruction endeavours can be enhanced.