Ambient ionisation mass spectrometry for drug and toxin analysis: A review of the recent literature.

Ambient ionisation mass spectrometry (AIMS) is a form of mass spectrometry whereby analyte ionisation occurs outside of a vacuum source under ambient conditions. This enables the direct analysis of samples in their native state, with little or no sample preparation and without chromatographic separation. The removal of these steps facilitates a much faster analytical process, enabling the direct analysis of samples within minutes if not seconds. Consequently, AIMS has gained rapid popularity across a diverse range of applications, in particular the analysis of drugs and toxins. Numerous fields rely upon mass spectrometry for the detection and identification of drugs, including clinical diagnostics, forensic chemistry, and food safety. However, all of these fields are hindered by the time-consuming and laboratory-confined nature of traditional techniques. As such, the potential for AIMS to resolve these challenges has resulted in a growing interest in ambient ionisation for drug and toxin analysis. Since the early 2000s, forensic science, diagnostic testing, anti-doping, pharmaceuticals, environmental analysis and food safety have all seen a marked increase in AIMS applications, foreshadowing a new future for drug testing. In this review, some of the most promising AIMS techniques for drug analysis will be discussed, alongside different applications of AIMS published over a 5-year period, to provide a summary of the recent research activity for ambient ionisation for drug and toxin analysis.

Biomarkers in Acute Myocarditis and Chronic Inflammatory Cardiomyopathy: An Updated Review of the Literature.

Myocarditis is a disease caused by cardiac inflammation that can progress to dilated cardiomyopathy, heart failure, and eventually death. Several etiologies, including autoimmune, drug-induced, and infectious, lead to inflammation, which causes damage to the myocardium, followed by remodeling and fibrosis. Although there has been an increasing understanding of pathophysiology, early and accurate diagnosis, and effective treatment remain challenging due to the high heterogeneity. As a result, many patients have poor prognosis, with those surviving at risk of long-term sequelae. Current diagnostic methods, including imaging and endomyocardial biopsy, are, at times, expensive, invasive, and not always performed early enough to affect disease progression. Therefore, the identification of accurate, cost-effective, and prognostically informative biomarkers is critical for screening and treatment. The review then focuses on the biomarkers currently associated with these conditions, which have been extensively studied via blood tests and imaging techniques. The information within this review was retrieved through extensive literature research conducted on major publicly accessible databases and has been collated and revised by an international panel of experts. The biomarkers discussed in the article have shown great promise in clinical research studies and provide clinicians with essential tools for early diagnosis and improved outcomes.

Obesity dysregulates the pulmonary antiviral immune response.

Obesity is a well-recognized risk factor for severe influenza infections but the mechanisms underlying susceptibility are poorly understood. Here, we identify that obese individuals have deficient pulmonary antiviral immune responses in bronchoalveolar lavage cells but not in bronchial epithelial cells or peripheral blood dendritic cells. We show that the obese human airway metabolome is perturbed with associated increases in the airway concentrations of the adipokine leptin which correlated negatively with the magnitude of ex vivo antiviral responses. Exogenous pulmonary leptin administration in mice directly impaired antiviral type I interferon responses in vivo and ex vivo in cultured airway macrophages. Obese individuals hospitalised with influenza showed dysregulated upper airway immune responses. These studies provide insight into mechanisms driving propensity to severe influenza infections in obesity and raise the potential for development of leptin manipulation or interferon administration as novel strategies for conferring protection from severe infections in obese higher risk individuals.

Cannabidiol Oil Ingested as Sublingual Drops or Within Gelatin Capsules Shows Similar Pharmacokinetic Profiles in Healthy Males.

Introduction: Cannabidiol (CBD) is a nonintoxicating phytocannabinoid used in clinical treatments and sold widely in consumer products. CBD products may be designed for sublingual or oral delivery, but it is unclear whether either is advantageous for CBD absorption. This study compared CBD pharmacokinetics after providing CBD oil as sublingual drops and within orally ingested gelatin capsules, at a dose relevant to consumer products. Materials and Methods: Eight males completed three conditions in a participant-blinded, randomized crossover design. Participants received the following combinations of placebo and CBD-containing (69 mg/mL) hemp oil in capsules and as sublingual drops: placebo capsules/placebo drops (Placebo), CBD capsules/placebo drops (CBD-Caps), and placebo capsules/CBD drops (CBD-Drops). Blood samples, blood pressure, and subjective scales were obtained/completed hourly for 6 h and at 24 h. Discussion: Plasma CBD concentrations were not different between CBD-Caps and CBD-Drops (interaction effect p=0.76). Peak CBD concentration (28.0±15.6 vs. 24.0±22.2 ng/mL), time of peak CBD concentration (4±1 vs. 4±2 h), and area under the concentration curve (45.3±20.3 vs. 41.8±23.3 ng/mL·6 h) were not different between conditions (p≥0.25). Cardiometabolic outcomes (plasma glucose/triacylglycerol, heart rate, blood pressure), liver function (plasma alanine aminotransferase/aspartate aminotransferase), kidney function (plasma creatinine), and subjective feelings/symptoms were not different between conditions (p≥0.07). Conclusions: Plasma CBD profiles were comparable between CBD-Caps and CBD-Drops, suggesting that there were not meaningful differences in routes of CBD absorption between conditions. This implies that CBD oil delivered sublingually is swallowed before oral mucosal CBD absorption occurs, which may have implications for research design, CBD product design, and consumer product choice.

Trimethylamine N-Oxide Concentration and Blood Pressure in Young Healthy Men and Women: A Replicated Crossover Study.

Trimethylamine N-oxide (TMAO), a gut-derived metabolite and marker of gut dysbiosis, has been linked to hypertension. Blood pressure is proposed to be elevated in hormonal contraceptive users and males compared to age-matched eumenorrheic females, but the extent to which TMAO differs between these populations has yet to be investigated. Peripheral and central blood pressure were measured, with the latter determined via applanation tonometry, and plasma TMAO concentration was assessed using liquid chromatography-tandem mass spectrometry. The following variables were assessed on two occasions in each of the following conditions: the early follicular phase (EFP) and mid-luteal phase (MLP) in eumenorrheic women (n = 13), and the pill-free interval (INACTIVE) and pill consumption days (ACTIVE) in women using oral contraceptive pills (n = 12), and in men (n = 22). Briefly, 17-ß-estradiol and progesterone concentrations were quantified via ELISA in all females. There were no differences in TMAO concentration between EFP (2.9 ± 1.7 µmol/L) and MLP (3.2 ± 1.1 µmol/L), between INACTIVE (3.3 ± 2.9 µmol/L) and ACTIVE (2.3 ± 1.1 µmol/L) days, or between men (3.0 ± 1.8 µmol/L), eumenorrheic women (3.0 ± 1.3 µmol/L) and contraceptive users (2.8 ± 1.4 µmol/L). Blood pressure was consistent across the menstrual cycle and pill days, but brachial systolic blood pressure was higher in males than females. There were no differences in brachial diastolic blood pressure or central blood pressure between the sexes. Repeated measures of TMAO, blood pressure, 17-ß-estradiol and progesterone were consistent in all populations. These findings suggest that the link between TMAO and blood pressure is limited in healthy young adults.

A comparison of advertised versus actual cannabidiol (CBD) content of oils, aqueous tinctures, e-liquids and drinks purchased in the UK.

BACKGROUND: Cannabidiol (CBD)-containing products are sold widely in consumer stores, but concerns have been raised regarding their quality, with notable discrepancies between advertised and actual CBD content. Information is limited regarding how different types of CBD products may differ in their deviation from advertised CBD concentrations. Therefore, CBD concentrations were quantified and compared in aqueous tinctures, oils, e-liquids and drinks. METHODS: Products (13 aqueous tinctures, 29 oils, 10 e-liquids and 11 drinks) were purchased online in the UK. CBD concentrations were quantified in aqueous tinctures, oils and e-liquids via high performance liquid chromatography (HPLC), and in drinks via gas chromatograhy-mass spectrometry (GC-MS). RESULTS: Measured concentrations fell -25.7 ± 17.3, -6.1 ± 7.8, -6.9 ± 4.6 and - 0.03 ± 0.06 mg/mL below advertised concentrations for aqueous tinctures, oils, e-liquids and drinks, respectively (medians ± interquartile ranges; p < .05). Oils deviated relatively less (-19.0 ± 14.5%) from advertised concentrations than e-liquids (-29.2 ± 10.2%), aqueous tinctures (-51.4 ± 41.4%) and drinks (-65.6 ± 36.5%; p < .01), whilst e-liquids deviated less than aqueous tinctures and drinks (p < .05), and deviation was not different between aqueous tinctures and drinks (p = .19). Only 5/63 (8%) products had measured concentrations within 10% of advertised concentrations. DISCUSSION: Similarly to previous studies, few products had measured CBD concentrations within 10% of advertised concentrations, with most falling below advertised concentrations. All individual product types deviated from advertised concentrations, with oils deviating least. These findings may be indicative of poor manufacturing standards, or that CBD undergoes degradation in consumer products. This reinforces concerns over quality of CBD-containing consumer products and may highlight the need for improved regulation of such products.

Mass spectrometry in the clinical laboratory. A short journey through the contribution to the scientific literature by CCLM.

Mass spectrometry (MS) has been a gold standard in the clinical laboratory for decades. Although historically refined to limited areas of study such as neonatal screening and steroid analysis, technological advancements in the field have resulted in MS becoming more powerful, versatile, and user-friendly than ever before. As such, the potential for the technique in clinical chemistry has exploded. The past two decades have seen advancements in biomarker detection for disease diagnostics, new methods for protein measurement, improved methodologies for reliable therapeutic drug monitoring, and novel technologies for automation and high throughput. Throughout this time, Clinical Chemistry and Laboratory Medicine has embraced the rapidly developing field of mass spectrometry, endeavoring to highlight the latest techniques and applications that have the potential to revolutionize clinical testing. This mini review will highlight a selection of these critical contributions to the field.

Mass Sportrometry: An annual look back at applications of mass spectrometry in sport and exercise science.

Research in sport and exercise science (SES) is reliant on robust analyses of biomarker measurements to assist with the interpretation of physiological outcomes. Mass spectrometry (MS) is an analytical approach capable of highly sensitive, specific, precise, and accurate analyses of a range of biomolecules, many of which are of interest in SES including, but not limited to, endogenous metabolites, exogenously administered compounds (e.g. supplements), mineral ions, and circulating/tissue proteins. This annual review provides a summary of the applications of MS across studies investigating aspects related to sport or exercise in manuscripts published, or currently in press, in 2022. In total, 93 publications are included and categorized according to their methodologies including targeted analyses, metabolomics, lipidomics, proteomics, and isotope ratio/elemental MS. The advantageous analytical opportunities afforded by MS technologies are discussed across a selection of relevant articles. In addition, considerations for the future of MS in SES, including the need to improve the reporting of assay characteristics and validation data, are discussed, alongside the recommendation for selected current methods to be superseded by MS-based approaches where appropriate. The review identifies that a targeted, mostly quantitative, approach is the most commonly applied MS approach within SES, although there has also been a keen interest in the use of 'omics' to perform hypothesis-generating research studies. Nonetheless, MS is not commonplace in SES at this time, but its use to expand, and possibly improve, the analytical options should be continually considered to exploit the benefits of analytical chemistry in exercise/sports-based research. Overall, it is exciting to see the gradually increasing adoption of MS in SES and it is expected that the number, and quality, of MS-based assays in SES will increase over time, with the potential for 2023 to further establish this technique within the field.

Applications of ambient ionization mass spectrometry in 2022: An annual review.

The development of ambient ionization mass spectrometry (AIMS) has transformed analytical science, providing the means of performing rapid analysis of samples in their native state, both in and out of the laboratory. The capacity to eliminate sample preparation and pre-MS separation techniques, leading to true real-time analysis, has led to AIMS naturally gaining a broad interest across the scientific community. Since the introduction of the first AIMS techniques in the mid-2000s, the field has exploded with dozens of novel ion sources, an array of intriguing applications, and an evident growing interest across diverse areas of study. As the field continues to surge forward each year, ambient ionization techniques are increasingly becoming commonplace in laboratories around the world. This annual review provides an overview of AIMS techniques and applications throughout 2022, with a specific focus on some of the major fields of research, including forensic science, disease diagnostics, pharmaceuticals and food sciences. New techniques and methods are introduced, demonstrating the unwavering drive of the analytical community to further advance this exciting field and push the boundaries of what analytical chemistry can achieve.

Endothelial Dysfunction and Heart Failure with Preserved Ejection Fraction-An Updated Review of the Literature.

Heart failure (HF) is a clinical syndrome consisting of typical symptoms and signs due to structural and/or functional abnormalities of the heart, resulting in elevated intracardiac pressures and/or inadequate cardiac output. The vascular system plays a crucial role in the development and progression of HF regardless of ejection fraction, with endothelial dysfunction (ED) as one of the principal features of HF. The main ED manifestations (i.e., impaired endothelium-dependent vasodilation, increased oxidative stress, chronic inflammation, leukocyte adhesion, and endothelial cell senescence) affect the systemic and pulmonary haemodynamic and the renal and coronary circulation. The present review is aimed to discuss the contribution of ED to HF pathophysiology-in particular, HF with preserved ejection fraction-ED role in HF patients, and the possible effects of pharmacological and non-pharmacological approaches. For this purpose, relevant data from a literature search (PubMed, Scopus, EMBASE, and Medline) were reviewed. As a result, ED, assessed via venous occlusion plethysmography or flow-mediated dilation, was shown to be independently associated with poor outcomes in HF patients (e.g., mortality, cardiovascular events, and hospitalization due to worsening HF). In addition, SGLT2 inhibitors, endothelin antagonists, endothelial nitric oxide synthase cofactors, antioxidants, and exercise training were shown to positively modulate ED in HF. Despite the need for future research to better clarify the role of the vascular endothelium in HF, ED represents an interesting and promising potential therapeutic target.

Effects of Exercise on Heart Failure with Preserved Ejection Fraction: An Updated Review of Literature.

Heart failure with preserved ejection fraction (HFpEF) represents the most common HF phenotype of patients aged > 65 years, with an incidence and a prevalence that are constantly growing. The HFpEF cardinal symptom is exercise intolerance (EI), defined as the impaired ability to perform physical activity and to reach the predicted age-related level of exercise duration in the absence of symptoms­such as fatigue or dyspnea­and is associated with a poor quality of life, a higher number of hospitalizations, and poor outcomes. The evidence of the protective effect between exercise and adverse cardiovascular outcomes is numerous and long-established. Regular exercise is known to reduce cardiovascular events and overall mortality both in apparently healthy individuals and in patients with established cardiovascular disease, representing a cornerstone in the prevention and treatment of many cardio-metabolic conditions. Several studies have investigated the role of exercise in HFpEF patients. The present review aims to dwell upon the effects of exercise on HFpEF. For this purpose, the relevant data from a literature search (PubMed, EMBASE, and Medline) were reviewed. The analysis of these studies underlines the fact that exercise training programs improve the cardiorespiratory performance of HFpEF patients in terms of the increase in peak oxygen uptake, the 6 min walk test distance, and the ventilatory threshold; on the other hand, diastolic or systolic functions are generally unchanged or only partially modified by exercise, suggesting that multiple mechanisms contribute to the improvement of exercise tolerance in HFpEF patients. In conclusion, considering that exercise training programs are able to improve the cardiorespiratory performance of HFpEF patients, the prescription of exercise training programs should be encouraged in stable HFpEF patients, and further research is needed to better elucidate the pathophysiological mechanisms underpinning the beneficial effects described.

Simple, high-throughput measurement of gut-derived short-chain fatty acids in clinically relevant biofluids using gas chromatography-mass spectrometry.

Introduction: The quantitative measurement of circulating gut bacteria-derived metabolites has increased in recent years due to their associations with health and disease. While much of the previous attention has been placed on metabolites considered as deleterious to health, a shift to the investigation of short-chain fatty acids (SCFAs) as potential health promotors has been observed. Objectives: To develop a simple, high-throughput and quantitative assay to measure gut-derived SCFAs in clinically relevant biofluids using gas chromatography-mass spectrometry (GC-MS). Methods: A short (7.5 min) GC-MS assay was optimized for measurement of seven straight- and branched-chain SCFAs and their deuterated isotopes using a wax-based column for analysis without prior derivatization. The assay was validated using routine criteria to assess precision, accuracy, matrix effects, recovery, and extraction reproducibility. Assay applicability was tested in cohorts of healthy individuals and kidney disease patients. Results: The assay was demonstrated to be precise, accurate and reproducible with acceptable levels of matrix effect and analyte recovery. Lower limits of detection and quantitation were in the low ng/mL range. An investigation into different blood collection tube chemistries demonstrated that lithium heparin plasma and serum clotting activator tubes are recommended for use in future cross-study comparisons. Kidney disease patient analyses demonstrated variable differences across SCFAs when comparing hemodialysis to earlier stages of chronic kidney disease, demonstrating the suitability of the assay for translation to clinical analyses. Conclusion: The assay has been validated and identified as reliable for use in larger-scale studies for the analysis of SCFAs in human plasma and serum.

The Impact of a Graded Maximal Exercise Protocol on Exhaled Volatile Organic Compounds: A Pilot Study.

Exhaled volatile organic compounds (VOCs) are of interest due to their minimally invasive sampling procedure. Previous studies have investigated the impact of exercise, with evidence suggesting that breath VOCs reflect exercise-induced metabolic activity. However, these studies have yet to investigate the impact of maximal exercise to exhaustion on breath VOCs, which was the main aim of this study. Two-litre breath samples were collected onto thermal desorption tubes using a portable breath collection unit. Samples were collected pre-exercise, and at 10 and 60 min following a maximal exercise test (VO2MAX). Breath VOCs were analysed by thermal desorption-gas chromatography-mass spectrometry using a non-targeted approach. Data showed a tendency for reduced isoprene in samples at 10 min post-exercise, with a return to baseline by 60 min. However, inter-individual variation meant differences between baseline and 10 min could not be confirmed, although the 10 and 60 min timepoints were different (p = 0.041). In addition, baseline samples showed a tendency for both acetone and isoprene to be reduced in those with higher absolute VO2MAX scores (mL(O2)/min), although with restricted statistical power. Baseline samples could not differentiate between relative VO2MAX scores (mL(O2)/kg/min). In conclusion, these data support that isoprene levels are dynamic in response to exercise.

Applications of ambient ionization mass spectrometry in 2021: An annual review.

Ambient ionization mass spectrometry (AIMS) has revolutionized the field of analytical chemistry, enabling the rapid, direct analysis of samples in their native state. Since the inception of AIMS almost 20 years ago, the analytical community has driven the further development of this suite of techniques, motivated by the plentiful advantages offered in addition to traditional mass spectrometry. Workflows can be simplified through the elimination of sample preparation, analysis times can be significantly reduced and analysis remote from the traditional laboratory space has become a real possibility. As such, the interest in AIMS has rapidly spread through analytical communities worldwide, and AIMS techniques are increasingly being integrated with standard laboratory operations. This annual review covers applications of AIMS techniques throughout 2021, with a specific focus on AIMS applications in a number of key fields of research including disease diagnostics, forensics and security, food safety testing and environmental sciences. While some new techniques are introduced, the focus in AIMS research is increasingly shifting from the development of novel techniques toward efforts to improve existing AIMS techniques, particularly in terms of reproducibility, quantification and ease-of-use.

A monolithic single-chip point-of-care platform for metabolomic prostate cancer detection.

There is a global unmet need for rapid and cost-effective prognostic and diagnostic tools that can be used at the bedside or in the doctor's office to reduce the impact of serious disease. Many cancers are diagnosed late, leading to costly treatment and reduced life expectancy. With prostate cancer, the absence of a reliable test has inhibited the adoption of screening programs. We report a microelectronic point-of-care metabolite biomarker measurement platform and use it for prostate cancer detection. The platform, using an array of photodetectors configured to operate with targeted, multiplexed, colorimetric assays confined in monolithically integrated passive microfluidic channels, completes a combined assay of 4 metabolites in a drop of human plasma in under 2 min. A preliminary clinical study using l-amino acids, glutamate, choline, and sarcosine was used to train a cross-validated random forest algorithm. The system demonstrated sensitivity to prostate cancer of 94% with a specificity of 70% and an area under the curve of 0.78. The technology can implement many similar assay panels and hence has the potential to revolutionize low-cost, rapid, point-of-care testing.

The Athlete and Gut Microbiome: Short-chain Fatty Acids as Potential Ergogenic Aids for Exercise and Training.

Short-chain fatty acids (SCFAs) are metabolites produced in the gut via microbial fermentation of dietary fibers referred to as microbiota-accessible carbohydrates (MACs). Acetate, propionate, and butyrate have been observed to regulate host dietary nutrient metabolism, energy balance, and local and systemic immune functions. In vitro and in vivo experiments have shown links between the presence of bacteria-derived SCFAs and host health through the blunting of inflammatory processes, as well as purported protection from the development of illness associated with respiratory infections. This bank of evidence suggests that SCFAs could be beneficial to enhance the athlete's immunity, as well as act to improve exercise recovery via anti-inflammatory activity and to provide additional energy substrates for exercise performance. However, the mechanistic basis and applied evidence for these relationships in humans have yet to be fully established. In this narrative review, we explore the existing knowledge of SCFA synthesis and the functional importance of the gut microbiome composition to induce SCFA production. Further, changes in gut microbiota associated with exercise and various dietary MACs are described. Finally, we provide suggestions for future research and practical applications, including how these metabolites could be manipulated through dietary fiber intake to optimize immunity and energy metabolism.

Epigenetic reprogramming enhances the therapeutic efficacy of osteoblast-derived extracellular vesicles to promote human bone marrow stem cell osteogenic differentiation.

Extracellular vesicles (EVs) are emerging in tissue engineering as promising acellular tools, circumventing many of the limitations associated with cell-based therapies. Epigenetic regulation through histone deacetylase (HDAC) inhibition has been shown to increase differentiation capacity. Therefore, this study aimed to investigate the potential of augmenting osteoblast epigenetic functionality using the HDAC inhibitor Trichostatin A (TSA) to enhance the therapeutic efficacy of osteoblast-derived EVs for bone regeneration. TSA was found to substantially alter osteoblast epigenetic function through reduced HDAC activity and increased histone acetylation. Treatment with TSA also significantly enhanced osteoblast alkaline phosphatase activity (1.35-fold), collagen production (2.8-fold) and calcium deposition (1.55-fold) during osteogenic culture (P ≤ 0.001). EVs derived from TSA-treated osteoblasts (TSA-EVs) exhibited reduced particle size (1-05-fold) (P > 0.05), concentration (1.4-fold) (P > 0.05) and protein content (1.16-fold) (P ≤ 0.001) when compared to untreated EVs. TSA-EVs significantly enhanced the proliferation (1.13-fold) and migration (1.3-fold) of human bone marrow stem cells (hBMSCs) when compared to untreated EVs (P ≤ 0.05). Moreover, TSA-EVs upregulated hBMSCs osteoblast-related gene and protein expression (ALP, Col1a, BSP1 and OCN) when compared to cells cultured with untreated EVs. Importantly, TSA-EVs elicited a time-dose dependent increase in hBMSCs extracellular matrix mineralisation. MicroRNA profiling revealed a set of differentially expressed microRNAs from TSA-EVs, which were osteogenic-related. Target prediction demonstrated these microRNAs were involved in regulating pathways such as 'endocytosis' and 'Wnt signalling pathway'. Moreover, proteomics analysis identified the enrichment of proteins involved in transcriptional regulation within TSA-EVs. Taken together, our findings suggest that altering osteoblasts' epigenome accelerates their mineralisation and promotes the osteoinductive potency of secreted EVs partly due to the delivery of pro-osteogenic microRNAs and transcriptional regulating proteins. As such, for the first time we demonstrate the potential to harness epigenetic regulation as a novel engineering approach to enhance EVs therapeutic efficacy for bone repair.

Metabolomic analysis reveals reliance on secondary plant metabolites to facilitate carnivory in the Cape sundew, Drosera capensis.

BACKGROUND AND AIMS: Secondary metabolites are integral to multiple key plant processes (growth regulation, pollinator attraction and interactions with conspecifics, competitors and symbionts) yet their role in plant adaptation remains an underexplored area of research. Carnivorous plants use secondary metabolites to acquire nutrients from prey, but the extent of the role of secondary metabolites in plant carnivory is not known. We aimed to determine the extent of the role of secondary metabolites in facilitating carnivory of the Cape sundew, Drosera capensis. METHODS: We conducted metabolomic analysis of 72 plants in a time-series experiment before and after simulated prey capture. We used ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and the retention time index to identify compounds in the leaf trap tissue that changed up to 72 h following simulated prey capture. We identified associated metabolic pathways, and cross-compared these compounds with metabolites previously known to be involved in carnivorous plants across taxa. KEY RESULTS: For the first time in a carnivorous plant, we have profiled the whole-leaf metabolome response to prey capture. Reliance on secondary plant metabolites was higher than previously thought - 2383 out of 3257 compounds in fed leaves had statistically significant concentration changes in comparison with unfed controls. Of these, ~34 compounds are also associated with carnivory in other species; 11 are unique to Nepenthales. At least 20 compounds had 10-fold changes in concentration, 12 of which had 30-fold changes and are typically associated with defence or attraction in non-carnivorous plants. CONCLUSIONS: Secondary plant metabolites are utilized in plant carnivory to an extent greater than previously thought - we found a whole-metabolome response to prey capture. Plant carnivory, at the metabolic level, likely evolved from at least two distinct functions: attraction and defence. Findings of this study support the hypothesis that secondary metabolites play an important role in plant diversification and adaptation to new environments.

A call for the standardised reporting of factors affecting the exogenous loading of extracellular vesicles with therapeutic cargos.

Extracellular vesicles (EVs) are complex nanoparticles required for the intercellular transfer of diverse biological cargoes. Unlike synthetic nanoparticles, EVs may provide a natural platform for the enhanced targeting and functional transfer of therapeutics across complex and often impenetrable biological boundaries (e.g. the blood-brain barrier or the matrix of densely organised tumours). Consequently, there is considerable interest in utilising EVs as advanced drug delivery systems for the treatment of a range of challenging pathologies. Within the past decade, efforts have focused on providing standard minimal requirements for conducting basic EV research. However, no standard reporting framework has been established governing the therapeutic loading of EVs for drug delivery applications. The purpose of this review is to critically evaluate progress in the field, providing an initial set of guidelines that can be applied as a benchmark to enhance reproducibility and increase the likelihood of translational outcomes.