Deciphering evolutionary drivers of biosynthetic pathways could enhance bioactive compound production. In Angelica, interspecific variation in furanocoumarins (FCs) accumulation reflects divergent pathway evolution. Here, we conduct comparative genomics between high-FC Angelica sensu stricto (s.s.) and low-FC Angelica sensu lato (s.l.) species. We reveal an FC biosynthetic gene cluster (BGC) comprising core enzymes (p-coumaroyl-CoA 2'-hydroxylases (C2'Hs), prenyltransferases (PTs)) and peripheral O-methyltransferases (OMTs). The ancestral Angelica s.l. clade retains an FC BGC configuration with OMTs on separate chromosomes and PTs performing only C-prenylation. In contrast, Angelica s.s. evolves an FC BGC, where core enzymes and OMTs co-localise on the same chromosome, with C2'H copy number expansion correlating with elevated expression and PTs enabling both C- and O-prenylation, collectively enhancing FC production and structural diversity. These findings elucidate how BGC architecture, gene copy number and functional innovation collectively drive phytochemical innovation, providing a blueprint for engineering medicinal FC biosynthesis.

Furocoumarins (FCs), important natural compounds with biodefense roles and pharmacological activities, are notably abundant in medicinal plant Angelica dahurica. However, its accumulation patterns over development stages in FC-enriched tissue, biosynthetic pathways, and regulatory mechanisms in A. dahurica remain elusive. Here, we quantified the concentration dynamics of 17 coumarins across six developmental stages of root and found a gradual decrease in FC concentration as the roots develop. Using a de-novo assembled chromosome-level genome for A. dahurica, we conducted integrative multi-omics analyses to screen out candidate genes to fill in the sole missing step in the biosynthesis of imperatorin and isoimperatorin. This revealed that CYP71AZ18 catalyzes hydroxylation at the C-5 position of psoralen to generate bergaptol, while CYP71AZ19 and CYP83F95 catalyze hydroxylation at the C-8 position to produce xanthotoxol, notably indicating that a single step is catalyzed by two genes from distinct CYP450 subfamilies in this species. CYP71AZ19 originated from a proximal duplication event of CYP71AZ18, specific to A. dahurica, and subsequently underwent neofunctionalization. Accessible chromatin regions (ACRs), especially proximal ACRs, correlated with high gene expression levels, and the three validated genes exhibited strong signals of ACRs, showing the importance of chromosomal accessibility in regulating metabolite biosynthesis.

Postharvest lipid degradation is a major factor contributing to quality loss, off-flavors, and reduced shelf life in stored rice grains. We investigated whether inducing secondary dormancy using abscisic acid (ABA) can stabilize lipid metabolism in rice. Non-dormant japonica (Nanjing 46) and indica (Fengliangyou) rice grains were induced into secondary dormancy with ABA (800 and 1500 mg/L, respectively), then subjected to accelerated aging (35 °C, 75 ± 2 % RH, up to 45 days). Lipid metabolism was assessed by enzyme assays, UHPLC-MS/MS lipidomics, and RNA-seq. Dormancy induction significantly suppressed the activities of lipase, phospholipase D, and lipoxygenase, resulting in lower rates of lipid hydrolysis and oxidative deterioration. Lipidomic profiling revealed delayed degradation of triacylglycerols and membrane phospholipids (phosphatidylcholine and phosphatidylethanolamine), while transcriptomic analysis showed coordinated downregulation of lipid degradation genes (OsLOX1, OsPLDalpha3). This multi-level suppression led to decreased accumulation of free fatty acids, lipid peroxides, and volatile aldehydes associated with rancid off-flavors. These findings provide new insights into dormancy-mediated lipid regulation and suggest a novel physiological strategy to enhance the storage stability and sensory quality of rice and other lipid-rich cereals.

Cuticular hydrocarbons (CHCs) in blow flies vary with species, age, and environmental factors. Blow fly puparia are often found associated with decomposing remains, but their utility in post-mortem interval (PMI) estimation in forensic cases is limited by challenges in accurately ageing them using conventional methods. This study builds on prior research by successfully distinguishing the CHC profiles of Lucilia sericata full puparia aged 1- and 5- days post-pupation. The results indicate that CHC analysis could serve as a valuable tool, either as a complement to or as a potential alternative for traditional forensic entomology methods.

Cuticular hydrocarbons (CHCs) are long-chain lipids found on the exoskeletons of insects, serving primarily as a protective barrier against water loss and environmental factors. In the last few decades, the qualitative and quantitative analysis of CHCs, particularly in blow flies, has emerged as a valuable tool in forensic entomology, offering promising potential for species identification and age estimation of forensically important insects. This review examines the current application of CHC analysis in forensic investigations and highlights the significant advancements in the field over the past few years. Studies have demonstrated that CHC profiles vary with insect development, and while intra-species variability exists due to factors such as age, sex, geographical location, and environmental conditions, these variations can be harnessed to refine post-mortem interval (PMI) estimations and improve the accuracy of forensic entomological evidence. Notably, CHC analysis can also aid in distinguishing between multiple generations of insects on a body, providing insights into post-mortem body movement and aiding in the interpretation of PMI in complex cases. Furthermore, recent studies have investigated the variability and degradation of CHCs over time, revealing how environmental factors—such as temperature, humidity, UV light exposure, and toxicological substances—affect CHC composition, providing valuable insights for forensic investigations. Despite the promise of CHC profiling, several challenges remain, and this review also aims to highlight future research directions to enhance the reliability of this technique in forensic casework.

Little is known about the prevalence of incapacitating substances present in drug facilitated sexual assaults (DFSA). Presented here is a literature review conducted to provide background information, such as symptoms, exacerbations, and drug interactions, on drugs typically implicated in DFSA, namely gamma-hydroxybutyrate (GHB), gamma-butyrolactone (GBL), 1,4-butanediol (1,4-BD), ketamine, diazepam, oxycodone, methamphetamine, and alcohol.Literature found through Scopus and Pubmed was reviewed to determine the current prevalence of these substances in DFSA with a focus on Australian data.The global literature revealed that there is a wide variety of substances used in DFSA and the prevalence varied by country. For example, it was found that in Northern Ireland, opioids were most prevalent whereas in France, benzodiazepines were most prevalent. In Australia the review revealed a lack of contemporary data with the most recent report in Victoria using data collected during 2011–2013. The literature also revealed there can be an important difference between self-reported substance use and substances discovered via toxicological analysis. This can be due to the challenges of biological detection, reliability of self-reporting, and the possibility of a substance being introduced to a person's food or drink without their knowledge.This review highlights the need for the collection and analysis of current data pertaining to DFSA reports and the drugs detected, and due to the constantly evolving picture of both licit and illicit drug use an assessment of the role of prescription medications in DFSA due to drug-drug interactions as well as potential to incapacitate is warranted.

Deceased human remains are often buried as a forensic countermeasure or method of disposal by homicide perpetrators. Owing to this, the excavation of clandestine grave sites is a task that forensic crime scene teams may only encounter a few times a year. Not all crime scene units have specialised teams for this task, and even those that do, may not have specific protocols for the optimal recovery of forensic traces retained within grave fill as procedures such as sieving require optimisation for the specific soil conditions of the jurisdiction. This study aimed to define the optimal sieving conditions for a sandy environment when searching for minute traces of paint, glass, hair and fibres. Furthermore, this study justifies the practice of retaining grave fill and examining it under controlled laboratory conditions, rather than in-situ adjacent to the grave site. The results demonstrate that using sieve mesh sizes as fine as 0.1mm can recover up to 82% of the deposited traces and almost all paint, hair and glass traces. The processing of grave fill in the laboratory lead to increased yield of forensic evidence, which on a case-basis may warrant the increased time needed. These findings merit consideration for clandestine grave crime scenes where evidence is scarce or the case is likely to become cold.

Whilst wound repair in severe burns has received substantial research attention, non-severe burns (<20% total body surface area) remain relatively understudied, despite causing considerable physiological impact and constituting most of the hospital admissions for burns. Early prediction of healing outcomes would decrease financial and patient burden, and aid in preventing long-term complications from poor wound healing. Lipids have been implicated in inflammation and tissue repair and may play essential roles in burn wound healing. In this study, plasma samples were collected from 20 non-severe burn patients over 6 weeks from admission, including surgery, and analysed by liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance spectroscopy to detect 850 lipids and 112 lipoproteins. Orthogonal projections to latent structures-discriminant analysis was performed to identify changes associated with re-epithelialisation and delayed re-epithelisation. We demonstrated that the lipid and lipoprotein profiles at admission could predict re-epithelisation outcomes at 2 weeks post-surgery, and that these discriminatory profiles were maintained up to 6 weeks post-burn. Inflammatory markers GlycB and C-reactive protein indicated divergent systemic responses to the burn injury at admission. Triacylglycerols, diacylglycerols and low-density lipoprotein subfractions were associated with delayed wound closure (p-value <0.02, Cliff’s delta >0.7), whilst high-density lipoprotein subfractions, phosphatidylinositols, phosphatidylcholines, and phosphatidylserines were associated with re-epithelisation at 2 weeks post-surgery (p-value <0.01, Cliff’s delta <-0.7). Further model validation will potentially lead to personalised intervention strategies to reduce the risk of chronic complications post-burn injury.

Cold case reviews within police and law enforcement agencies are challenging, not the least owing to the amount of time required to carefully review documentation, forensic exhibit holdings and various other casefile information. Most federal and state agencies are time poor, meaning there are very few dedicated cold case teams fortunate enough to have an abundance of police and expert staff resources. Universities and education organisations, however, have large troves of various expertise, alongside expansive human resources, by way of their academic and student body. In certain circumstances, the academic expertise and course offerings of a university may be well suited to assisting law enforcement in reviewing cold cases.There is growing desire for university courses to generate job ready graduates. In the field of law enforcement and policing this is difficult, as safety and the security of sensitive material and evidence is paramount. Educators strive to create workplace simulations, and with the correct mix of academic expertise, course offerings and industry linkages, the emerging opportunity for real cold case collaboration is possible. One such example is the Cold Case Review @ Murdoch (CCR) initiative. Since 2020, CCR has worked with the Lower Saxony Police Academy in Germany to develop the novel International Cold Case Analysis Project (ICCAP), now incorporating over 25 member institutions, to assist in solving real cases from both Niedersachsen (Lower Saxony) and federal police jurisdictions. One case, known as “The North Sea Man” has shown great success and demonstrates the power of joining forces between law enforcement and external agencies to help advance cold cases.

Drink spiking in social settings is one of the most pervasive forms of drug-facilitated sexual assault (DFSA). There are no current data in Australia on the rates of drink spiking or their associated assaults. There is also little known about the prevalence of different substances involved and how the current substance use trends compare to sexual assault trends. To explore this, a recalculation of sexual assault trends to estimate substance related sexual assault was performed. Data about recent trends of mental health prescriptions and sexual assault were obtained from the Australian Institute of Health and Welfare (AIHW). The analysis of these datasets highlighted that females are the highest consumers of antidepressants and benzodiazepines. Results also indicated a statistically significant positive correlation between females and a higher incidence of sexual assault (r = 0.996, p < .001). This paper demonstrates that females are at most risk of drug-drug interactions (e.g., diazepam and ketamine) with their medications due to the higher rate of prescriptions amongst this population, and therefore more vulnerable to both opportunistic and proactive DFSA. While these findings are preliminary and not causal, they highlight trends in need of further study.

The immortalised human hepatocellular HepG2 cell line is commonly used for toxicology studies as an alternative to animal testing due to its characteristic liver-distinctive functions. However, little is known about the baseline metabolic changes within these cells upon toxin exposure. We have applied 1H Nuclear Magnetic Resonance (NMR) spectroscopy to characterise the biochemical composition of HepG2 cells at baseline and post-exposure to hydrogen peroxide (H2O2). Metabolic profiles of live cells, cell extracts, and their spent media supernatants were obtained using 1H high-resolution magic angle spinning (HR-MAS) NMR and 1H NMR spectroscopic techniques. Orthogonal partial least squares discriminant analysis (O-PLS-DA) was used to characterise the metabolites that differed between the baseline and H2O2 treated groups. The results showed that H2O2 caused alterations to 10 metabolites, including acetate, glutamate, lipids, phosphocholine, and creatine in the live cells; 25 metabolites, including acetate, alanine, adenosine diphosphate (ADP), aspartate, citrate, creatine, glucose, glutamine, glutathione, and lactate in the cell extracts, and 22 metabolites, including acetate, alanine, formate, glucose, pyruvate, phenylalanine, threonine, tryptophan, tyrosine, and valine in the cell supernatants. At least 10 biochemical pathways associated with these metabolites were disrupted upon toxin exposure, including those involved in energy, lipid, and amino acid metabolism. Our findings illustrate the ability of NMR-based metabolic profiling of immortalised human cells to detect metabolic effects on central metabolism due to toxin exposure. The established data sets will enable more subtle biochemical changes in the HepG2 model cell system to be identified in future toxicity testing.

Furocoumarins (FCs) are crucial natural products playing a dual role as plant defense molecules and pharmacologically active substances. Angelica dahurica is a renowned herb with diverse and abundant FCs. However, the accumulation pattern over developmental stages, biosynthesis pathway and regulatory mechanisms of FCs in A. dahurica remain elusive, hindering the production of FCs via synthetic biology approaches. Here, we constructed a chromosome-level reference genome for A. dahurica and quantified the content dynamics of 17 coumarins across six developmental stages of its medicinal organ, root. It showed a gradual decrease in FC concentration with root enlargement. The combined analyses of transcriptomic and metabolomic data, together with in vivo enzymatic assay, confirmed that CYP71AZ18 was involved in the biosynthesis of bergaptol, whereas CYP71AZ19 and CYP83F95 contributed to the biosynthesis of xanthotoxol. Notably, CYP71AZ19 originated from a proximal duplication event of CYP71AZ18, specific to A. dahurica , subsequently undergoing neofunctionalization. Accessible chromatin regions (ACRs), especially proximal ACRs, are correlated with higher gene expression levels, including the three validated genes involved in FC biosynthesis, showing potential to regulate metabolite biosynthesis. Our findings provide new insights into the biosynthetic pathway of FCs and the epigenetic regulation of metabolite biosynthesis. ### Competing Interest Statement The authors have declared no competing interest.

Angelica sinensis (Oliv.) Diels belongs to the Apiaceae family. The root of A. sinensis, is used in traditional Chinese medicine for its antioxidant and immune regulation properties. The main active compounds in A. sinensis include organic acids, phthalides and coumarins, and their biosynthetic pathways are the focus of international attention. A. sinensis is prone to early flowering and bolting, which negatively impacts production for several reasons, including germplasm degradation and quality instability in artificial cultivation. The identification of top-geoherbalism of A. sinensis has also become the focus of recent research, as it would allow selection for breeds with excellent medicinal quality and remarkable curative effects. Advances in sequencing technology and bioinformatic methodologies have enabled extensive molecular and genetic studies in A. sinensis. In this review, we summarize the latest molecular research advances related to A. sinensis, including biosynthetic pathways and regulation of active compounds, and molecular underpinnings of early bolting and flowering and top-geoherbalism. We discuss limitations of the current research and propose prospective topics in need of further exploration.

Dysregulated lipid metabolism underpins many chronic diseases including cardiometabolic diseases. Mass spectrometry-based lipidomics is an important tool for understanding mechanisms of lipid dysfunction and is widely applied in epidemiology and clinical studies. With ever-increasing sample numbers, single batch acquisition is often unfeasible, requiring advanced methods that are accurate and robust to batch-to-batch and interday analytical variation. Herein, an optimized comprehensive targeted workflow for plasma and serum lipid quantification is presented, combining stable isotope internal standard dilution, automated sample preparation, and ultrahigh performance liquid chromatography-tandem mass spectrometry with rapid polarity switching to target 1163 lipid species spanning 20 subclasses. The resultant method is robust to common sources of analytical variation including blood collection tubes, hemolysis, freeze–thaw cycles, storage stability, analyte extraction technique, interinstrument variation, and batch-to-batch variation with 820 lipids reporting a relative standard deviation of <30% in 1048 replicate quality control plasma samples acquired across 16 independent batches (total injection count = 6142). However, sample hemolysis of ≥0.4% impacted lipid concentrations, specifically for phosphatidylethanolamines (PEs). Low interinstrument variability across two identical LC-MS systems indicated feasibility for intra/inter-lab parallelization of the assay. In summary, we have optimized a comprehensive lipidomic protocol to support rigorous analysis for large-scale, multibatch applications in precision medicine. The mass spectrometry lipidomics data have been deposited to massIVE: data set identifiers MSV000090952 and 10.25345/C5NP1WQ4S.

Forensic samples for DNA analysis are limited by physical size and volume as DNA extraction methods require small portions placed into 1.5 or 2.0 mL tubes for lysis. Apart from tape lifting techniques, this precludes large sample areas from being harvested of cellular material by any means other than washing or wet vacuuming. In addition, forensically savvy perpetrators may employ clean-up methods to remove DNA evidence by way of wash basins and sinks, although evidence may be retained within the p-trap of plumbing. Both outcomes still leave forensic biologists with samples incompatible with most commercial DNA extraction methods. Fields of microbiology and ecology have long used environmental DNA (eDNA) collection methods to overcome the similar challenges of sampling biota within large volumes of water or liquid samples. This study is the first application of eDNA techniques to capture such dilute traces of human cellular material from large water volumes using a microfiltration method. The technique was able to isolate human DNA from as little as 100 cells in 1 L of water. While future optimization is required to determine ideal filter specifications, this research demonstrates a proof of concept for forensic application in cases with challenging substrates that can be washed or contain water. This research was conducted under the Murdoch University Human Research Ethics Committee approval 2019/025.

Globally, burns are a significant cause of injury that can cause substantial acute trauma as well as lead to increased incidence of chronic comorbidity and disease. To date, research has primarily focused on the systemic response to severe injury, with little in the literature reported on the impact of nonsevere injuries (<15% total burn surface area; TBSA). To elucidate the metabolic consequences of a nonsevere burn injury, longitudinal plasma was collected from adults (n = 35) who presented at hospital with a nonsevere burn injury at admission, and at 6 week follow up. A cross-sectional baseline sample was also collected from nonburn control participants (n = 14). Samples underwent multiplatform metabolic phenotyping using 1H nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry to quantify 112 lipoprotein and glycoprotein signatures and 852 lipid species from across 20 subclasses. Multivariate data modeling (orthogonal projections to latent structures-discriminate analysis; OPLS-DA) revealed alterations in lipoprotein and lipid metabolism when comparing the baseline control to hospital admission samples, with the phenotypic signature found to be sustained at follow up. Univariate (Mann–Whitney U) testing and OPLS-DA indicated specific increases in GlycB (p-value < 1.0e–4), low density lipoprotein-2 subfractions (variable importance in projection score; VIP > 6.83e–1) and monoacyglyceride (20:4) (p-value < 1.0e–4) and decreases in circulating anti-inflammatory high-density lipoprotein-4 subfractions (VIP > 7.75e–1), phosphatidylcholines, phosphatidylglycerols, phosphatidylinositols, and phosphatidylserines. The results indicate a persistent systemic metabolic phenotype that occurs even in cases of a nonsevere burn injury. The phenotype is indicative of an acute inflammatory profile that continues to be sustained postinjury, suggesting an impact on systems health beyond the site of injury. The phenotypes contained metabolic signatures consistent with chronic inflammatory states reported to have an elevated incidence postburn injury. Such phenotypic signatures may provide patient stratification opportunities, to identify individual responses to injury, personalize intervention strategies, and improve acute care, reducing the risk of chronic comorbidity.

Ion mobility (IM) separations, especially when combined with mass spectrometry, offer the opportunity for the rapid analysis and characterization of mixtures. However, the limited resolution afforded by many IM systems means that in practice applications may be limited. Here we have employed an IM separation on a highresolution cyclic IM device with MS/MS to separate and characterize mixtures of sulfated isomers of tyrosine and associated metabolites containing multiple sulfated isoforms present in reaction mixtures. The cIMS device allowed ions, not resolved using a single pass, to be subjected to multiple passes, enabling the resolution of those with similar collision cross sections (CCS). Predicted single pass CCS values calculated for the isomers likely to be present in these mixtures showed only small differences between them, ranging between of between 0.1 - 0.7 % depending on structure. These small differences highlight the high degree of mobility resolution required for separating the isomers. Experimentally different isoforms of tyrosine sulfate and sulfated tyrosine metabolites could be sufficiently resolved via multipass separations (3-35 passes). This degree of separation provided resolving powers of up to 384 CCS/Delta CCS for sulfated dopamine which enabled good MS/MS spectra to be generated. In human urine the presence of a single sulfated form of tyrosine was detected and identified as the Osulfate after 3 passes based on the synthetic standard. Of the other tyrosine-related sulfates for which synthetic standards had been prepared only dopamine sulfate was detected in this sample.

Ozone is widely used to control pests in grain and impacts seed germination, a crucial stage in crop establishment which involves metabolic alterations. In this study, dormancy was overcome through after-ripening (AR) in dry barley seed storage of more than 4 weeks; alternatively, a 15-min ozone treatment could break the dormancy of barley immediately after harvest, with accelerated germination efficiency remaining around 96% until 4 weeks. Headspace solid-phase microextraction (HS-SPME) and liquid absorption coupled with gas chromatography mass spectrometry (GC-MS) were utilized for metabolite profiling of 2-, 4- and 7-day germinating seeds. Metabolic changes during barley germination are reflected by time-dependent characteristics. Alcohols, fatty acids, and ketones were major contributors to time-driven changes during germination. In addition, greater fatty acids were released at the early germination stage when subjected to ozone treatment.

Ozone (O3) is a potential fumigant to control pests in stored grain since it can safely and rapidly auto-decompose without leaving residues. In this study, the efficacy of O3 on all life stages of Rhyzopertha dominica (Fabricius) and Tribolium castaneum (Herbst) in barley and the physiological effects on barley and its quality were investigated. Complete control of all life stages of pests was obtained at 700 ppm for 1440 min of ozone exposure without negatively impacting the contents of soluble protein, moisture content, seed colour, hardness, and the weight of thousand barley seeds. The eggs and pupae of these two insects were the more tolerant stages than their larvae and adults. Prolonged exposure times (40 to 1440 min) and mortality assessment intervals (1, 2, and 7 days) increased O3 efficacy due to the reaction characteristics and delayed toxicity. Aging barley seeds appeared to be more sensitive to prolonged ozone duration than new seeds. A total of 20 and 40 min could promote germination rate, and longer O3 exposure (1440 min) was unfavourable for germination and seedling growth. Thus, it is imperative to select an optimal O3 exposure time to transfer ozone into quality contributors of final products and achieve the desired functional outcomes.