Biology subjects

Dickens, A. M.

Publications and source records attributed to Dickens, A. M..

13 recordsLinked to original sources

bioRxiv2026

Measurement of a panel of 21 steroids in a quantitative assay in human plasma, adipose tissue, and fecal samples using ultra-high-performance liquid chromatography-tandem mass spectrometry

Comprehensive detection of steroids, beyond the limited panels typically analyzed in clinical chemistry laboratories, has become increasingly important given their pivotal roles in diverse biological processes. However, steroid quantification poses several analytical challenges, including differences in ionization efficiency and structural similarities across the entire steroid metabolic network. To address these challenges, we developed a targeted ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay to analyze 21 steroids using reverse-phase chromatography combined with rapid polarity switching. Mass spectrometry (MS) analysis was performed in scheduled multiple reaction monitoring (sMRM) mode. Depending on the steroid and matrix, the validated lower limits of quantitation (LLOQ) ranged from 12.0 pM to 1216 pM in plasma and 41.1 pM to 384 pM in fecal sample homogenates. In adipose tissue, it was from 0.01 pmol/g to 9 pmol/g. Measured steroid concentrations obtained from the commercial control samples (MassTrak Steroid Serum QC Set 1 and the MassCheck(R) Steroid Panel 1 Serum Control) showed close agreement with the reference values. As a proof of concept, the method was successfully applied to 469 plasma samples in several projects, 15 adipose tissue samples, and 332 fecal samples, demonstrating its applicability to large-scale studies. In conclusion, the method enables sensitive, derivatization-free quantification of an expanded steroid panel in plasma and complex biological matrices, including adipose tissue and fecal samples, representing a significant advancement in comprehensive steroid profiling. Graphical abstractFor Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/737297v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@807582org.highwire.dtl.DTLVardef@1a2cbe9org.highwire.dtl.DTLVardef@f96f76org.highwire.dtl.DTLVardef@4b8c9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Feasibility of multimodal metabolic analysis for detecting early changes in acute neuroinflammation

Given the prevalence of metabolic perturbations in a variety of neurological and neurodegenerative diseases, understanding and monitoring brain metabolism is a key step in our advancement of therapies. The details of the citric acid cycle were established at the beginning of the last century but only recently have its metabolic intermediates been observed in vivo in the brain. In this study, we employed orthogonal analyses to investigate metabolic alterations in response to acute neuroinflammation in vivo, demonstrating a multi-technique approach that could be used for future studies. Hyperpolarized 13C-pyruvate spectroscopy revealed an early decline in pyruvate metabolism via pyruvate dehydrogenase (PDH), leading to reduced 13C-bicarbonate formation. This metabolic disruption occurred despite the absence of structural or perfusion changes on conventional MRI. Further analysis of polar metabolites in the ipsilateral hemisphere confirmed ongoing inflammatory processes. These findings highlight the potential of this dual technique approach to inform upon metabolic changes due to neuroinflammation. Combining methods to probe metabolism in invasive (metabolomics) and non-invasive (hyperpolarized MRI) manners, this represents a promising translational approach for real-time metabolic assessments in an area of the body, the brain, where studying processes such as metabolism has traditionally been challenging. This study has demonstrated the approach to monitor changes in metabolism in response to inflammation in the brain.

neuroscience↗

bioRxiv2025

Temporal Associations Between Human Milk Metabolites and the Infant Gut Microbiome and metabolome

IntroductionHuman milk is vital in establishing a healthy infant gut microbiome. Gut microbes and their metabolites are important for host development. However, our understanding of how milk metabolites are connected to the gut microbiome and metabolome in early-life remains limited. We aimed to investigate associations between the human milk metabolome and the gut microbiome in infancy. MethodsThe fecal and milk samples were collected at 2.5 (n=283), 6 (n=129) and 14 (n=65) months of age. Gut microbiota was analyzed with amplicon sequencing, the gut metabolome was analyzed with gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry, and milk metabolites were analyzed with proton nuclear magnetic resonance. ResultsBifidobacterium and other butyrate producers were associated with ethanolamine and methionine level in the milk, in a time-dependent manner. Elevated milk fucosylated oligosaccharide, LNFP-I and caprylate concentrations were positively associated with secondary bile acid levels following the introduction of solid foods. Additionally, acidic oligosaccharides 3-SL and 6-SL were positively associated with long-chain carbohydrates prior to solid food introduction, however negatively associated thereafter. ConclusionOur results highlight that milk metabolites are associated with biologically relevant gut microbial metabolites. Only limited associations were observed between milk composition and the microbiome, which differed between early and late infancy. This suggests milk metabolome may differentially influence microbial metabolism depending on dietary diversity and/or the maturity of the microbiome. ImportanceIts essential to raise awareness about breastfeeding and its variety of health benefits. Expanding our understanding of how human milk small molecules with potential bioactivity interact with the gut microbiome can help highlight these positive effects. Exploratory studies of human milk composition with gut microbial metabolites may support achieving this goal. Furthermore, understanding of biological effects of different components in human milk is important, and it can help to develop strategies to ensure optimal growth and development of an infant regardless of the breastfeeding status of the child.

microbiology↗

bioRxiv2025

Metabolic effects and biotransformation of perfluorohexyloctane (F6H8) in human hepatocytes

Perfluorohexyloctane (F6H8) is a semifluorinated alkane recently approved for ophthalmic treatment of dry eye disease. Although considered locally safe for topical use, its structural similarity to persistent per- and polyfluoroalkyl substances (PFAS) raises concerns about systemic accumulation and long-term toxicity. To investigate potential hepatic effects, we examined the metabolic impact of F6H8 exposure in human HepaRG hepatocytes across a broad concentration range representing short- and long-term exposure scenarios. Untargeted metabolic profiling by ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-OTOFMS) was performed on intracellular extracts and extracellular media. F6H8 induced pronounced, concentration-dependent metabolic alterations, many of which exhibited non-monotonic responses. Low concentrations primarily affected amino acid, fatty acid, and lipid metabolism, while central carbon metabolism was disrupted only at the highest exposures. Notably, a putative biotransformation product, perfluorohexyloctanoic acid, was detected, suggesting metabolic persistence and conversion to a PFAS-like structure. These findings indicate that F6H8 elicits broad metabolic reprogramming and may not be metabolically inert as previously assumed. Given its clinical use and structural similarity to persistent fluorochemicals, the results highlight the need for comprehensive, long-term safety assessment of F6H8 and related semifluorinated alkanes.

pharmacology and toxicology↗

bioRxiv2025

Faecal transplantation from exuberant toddlers increases exploratory behaviour in rats

BackgroundBehavioural phenotypes have previously been transferred via faecal microbiota transplantation (FMT) from patients with psychiatric disorders to rodents. Studies indicate that the gut microbiota composition may be linked to certain temperament traits, defined as biologically-based differences in emotional reactivity and self-regulation. Here, we aimed to determine if the gut microbiota plays a role in temperament using an FMT approach. We focused on the temperament traits of exuberance, defined as positive reactivity, decreased behavioural inhibition, and high behavioural approach tendencies. MethodsFaeces from 2.5-year-old toddlers from FinnBrain Birth Cohort Study with high exuberance/approach or high behavioural inhibition in the LabTAB bubbles-episode was transferred to juvenile male Sprague Dawley rats (age 22/23 days). Behaviour of the rat recipients (n=53) was assessed using the novel non-social arena, novel social arena, hole board test for exploratory behaviour, social approach-avoidance test, and forced swim test. The faecal pellets collected from the rodents were analyzed with 16s rRNA sequencing and faecal samples from the sample of toddlers (which included the donors, n=176) were analysed using short-read metagenomic sequencing. The striatum and prefrontal cortex from the rodents brains were analysed post-mortem using RNAseq. ResultsMicrobiome from toddlers with high exuberance traits induced increased exploratory behaviour compared to vehicle-controls and rats receiving faeces from inhibited toddlers. Locomotor activity, social, and depressive-like behaviour remained unaffected. We noted a downregulation of the dopamine synapse pathway within the striatum of the rats that received faeces from the inhibited trait donors compared with vehicle-controls. Faecal microbiota of rats receiving faeces from the same donor resembled more each other than rats from a different cage. Clostridium species AM29 11AC in toddler microbiome was positively related to exuberance, but there were no cross-sectional associations between faecal metabolites in the human sample. ConclusionsFMT from exuberant toddlers lead to altered exploratory-related behaviour in rats.

microbiology↗

bioRxiv2025

Microbial lipid shifts in a multi-stage simulated gut

Food residues that bypass human digestion are further digested by gut microbes, leading to the production of diverse metabolites, including lipids. To investigate how lipids are affected during this transition, we used a colon simulator with four distinct vessels that mimics the proximal to distal part of the human colon. We observed dynamic shifts in a diverse array of microbially derived lipid molecules in the simulated intestinal chyme, including bile acids and N-acyl amides with short and odd-chain lipids. Histamine-linked N-acyl lipids increased from the proximal to the distal colon vessels (pH 5.5 - 7.0), whereas putrescine-linked, initially abundant in the media, decreased across the colon vessels. We uncovered dynamic associations between in vitro-derived short-chain N-acyl lipids and major lipid species such as cholesterol esters, phosphatidylethanolamines, ceramides, and sphingomyelins. To determine the broader relevance of these findings, we applied a reverse metabolomics approach and examined lipid profiles in human small intestine and fecal samples from public datasets. This validated the colon simulator as a model for studying diet-derived and microbially transformed metabolites with relevance to human and animal health and could perhaps be used as a strategy to discover microbial metabolites.

biochemistry↗

bioRxiv2025

Biomolecular tracking by FIRESCAPE reveals distinct modes of clearance, damage induction and cellular uptake for extracellular histone H3

In attempting to observe the behaviour of a protein of interest in vivo, akin to other observer effects, current techniques require modifications or interventions that inherently alter the protein or its host, leaving one uncertain as to whether natural behaviour remains unperturbed. The study of chromatin has mostly been restricted to defining its function in the nucleus, where histone proteins fulfil vital roles in packaging genomic DNA and regulating transcription. However, chromatin components can be released into the extracellular space, either intentionally via cellular secretion or during disease-induced cell death. These extracellular chromatin components, depending on the context, can consist of: free histones, free DNA, intact nucleosomes (histone octamers wrapped by DNA), or heterogeneous, higher order structures such as neutrophil extracellular traps (NETs). They have been associated with diverse pathologies such as inflammation, cancer, and sepsis, and distinct toxic effects. However, there is a widely acknowledged lack of methods that distinguish between them and between their unique functions. Here, we now address protein observer effects to explore the fate and function of extracellular free histones by utilizing FIRESCAPE ([18F]-Fluorine Isotopic Radiolabeling Enabling Scanning of Clearance After Proteolytic Events), a novel radiolabeling concept that leverages the unique, high-sensitivity properties of the radioisotope fluorine-18, 18F, and residue-specific protein editing chemistry. By installing close and then true 18F-containing protein sidechain mimics site-specifically, FIRESCAPE enables the hierarchical in vivo scanning of the half-lives, proteolytic susceptibility and clearance of single residues in a protein of interest, and at microdoses far below toxic levels (low nanomole). These radioequivalent proteins bearing near-zero-size, zero-background labels now precisely reveal the strikingly distinct distribution, half-lives, damage-inducing abilities and accumulation of free extracellular histones in cellulo and in vivo compared to intact nucleosomes. Free extracellular histone H3 is rapidly cleared from circulation, mediated first by proteolysis of the histone tail. By contrast, direct injection of free histones vs nucleosomes into tissue that is unprotected by such proteolysis (brain), provokes a starkly different response; free histones exhibited limited diffusion and swiftly promoted damage both in cell culture and in vivo, whilst intact nucleosomes were essentially passive and benign. Remarkably, synthetic extracellular histone H3 was observed to enter cells and integrate into chromatin, indistinguishable from native H3 in both localization and post-translational modification (PTM) accumulation, yet paired with cellular and tissue damage. The exploratory studies described here now provide much needed clarity to the distinct fates and effects of extracellular histones vs nucleosomes, in particular the strongly damaging effects of free histones, their rapid uptake into cells, and an associated histone-specific proteolysis pathway via the removal of the histone tail.

biochemistry↗

bioRxiv2025

Isostere 18F-protein post-translational editing enables dynamic tracking of neurodegeneration biomarkers

The neurofilament light chain protein (NfL) is a suggested general marker for neuronal loss. Its release from brain parenchyma into cerebral spinal fluid, and presumed detection in blood has seen it established as a first blood-based marker of disease activity and drug efficacy in multiple sclerosis (MS) and in the presymptomatic diagnosis and assessment of disease course for other neurodegenerative disorders.1 However, the lack of characterisation of its behaviour in circulation, largely due to its antibody-dependent measurement, have hampered the biological interpretation of these measurements, especially after acute injury such as in MS relapse or head trauma.2 Here, we describe a strategy for exploiting positron emission tomography (PET) imaging using isosteric protein mimics following the installation of a fluorine-18 label that is benign enough to provide sensitive, real-time information on the dynamics and trafficking of NfL protein. This circumvents the limits of current methods that integrate 18F into proteins through the bio-conjugation of bulky, unnatural groups, which we show perturb NfLs assembly and functional properties from those in the natural state. We use a visible-light-driven reaction to access radioactive isostere proteins that are unperturbed and so closely resemble their native form. In this way, generation of [18F]fluoroalkyl radicals that can be rapidly reacted at pre-defined sites on proteins creates mimics of proteinogenic side chains bearing near-zero-size labels to probe proteins in functionally true form. These prosthetic-free, protein radiotracers can be generated in excellent radiochemical yield (up to 67%) via a semi-automated protocol in just 15 mins. High associated molar activities (precursor up to 102 GBq mol-1) allowed high sensitivity dynamic observations in blood, brain and cerebrospinal fluid, enabling even the first unambiguous observations of spinal flow kinetics using proteins. These dynamics, including the high rate of spinal flow (on the order of mm per min) and drainage of NfL from CSF into sacral lymph nodes, now provides evidence that the slow fall rate of antibody-detected markers that is observed after acute neural insults is not due to a long half-life, but rather reflects sustained neuronal loss. This discovery will now help to better correlate clinical and radiological features of disease with NfL blood levels. Our methodology now demonstrates the broad potential of a near-zero-size labelling method for the functional study of proteins in whole organisms without interfering with their biological activity and native assembly.

synthetic biology↗

bioRxiv2024

Fecal microbiota and metabolite composition associates with stool consistency in young children

Transit time, fluid intake, diet, and overall gut health influence stool consistency. However, the relationships between the gut metabolome (microbial metabolites) and stool consistency in infants and young children remain poorly understood. Here, we analyzed the metabolome and microbiota of 618 stool samples from children aged 2.5 months (n = 360), 6 months (n = 229), 14 months (n = 274), and 30 months (n = 169) from the FinnBrain Birth Cohort Study, and related these data to stool water content and parent-reported stool consistency. Breastfeeding showed the strongest association with both stool consistency and fecal water content. Concentrations of newly identified microbial bile acid amidates were associated with constipation and stool water content, while bile salt hydrolase, an enzyme involved in bile acid deconjugation and conjugation was predicted to be negatively associated with stool water content. In addition, short-chain fatty acids, particularly acetate, were positively associated with stool water content, whereas branched-chain short-chain fatty acids showed negative associations. These findings suggest that longer gut transit time permits more extensive microbial metabolism, including the transformation of bile acid amidates. We also found that microbial taxonomic richness, diversity, and community composition were primarily associated with stool water content, with only weak associations with stool consistency. Overall, our results highlight the importance of documenting stool consistency in fecal metabolomics and microbiome research, and provide new insights into how breastfeeding, microbial metabolism, and gut transit time shape early-life gut development.

systems biology↗

bioRxiv2023

Comparative metabolomics and microbiome analysis of Ethanol vs. OMNImet/gene GUT fecal stabilization

Metabolites from feces provide important insights into the functionality of the gut microbiome. As immediate freezing is not always feasible in gut microbiome studies, there is a need for sampling protocols that provide stability of the fecal metabolome and microbiome at room temperature (RT). For this purpose, we investigated the stability of various metabolites and the microbiome (16S ribosomal RNA) in feces collected in 95% ethanol (EtOH) or OMNImet(R)*GUT/ OMNIgene(R)*GUT. To simulate in field-collection scenarios, the samples were stored at different temperatures at varying durations (24h +4{degrees}C, 24h RT, 36h RT, 48h RT, and 7 days RT), and compared to aliquots immediately frozen at -80{degrees}C. We applied several targeted and untargeted metabolomics platforms to measure lipids, polar untargeted metabolites, endocannabinoids, short chain fatty acids (SCFAs), and bile acids (BAs). We found that SCFAs in the non-stabilized samples increased over time, while a stable profile was recorded in sample aliquots stored in 95% EtOH and OMNImet(R)*GUT. When comparing the metabolite levels between fecal aliquots stored at room temperature and at +4{degrees}C, we detected several changes in microbial metabolites, including multiple BAs and SCFAs. Taken together, we found that storing fecal samples at room temperature and stabilizing them in 95% EtOH yielded metabolomic results comparable to flash freezing. We also found that overall composition of the gut microbiome did not vary significantly between different storage types. However, there were notable differences observed in alpha diversity. Taken together, the stability of the metabolome and microbiome in 95 % EtOH provided similar results as the validated commercial collection kits OMNImet(R)*GUT and OMNIgene(R)*GUT, respectively. IMPORTANCEThe analysis of the gut metabolome and microbiome requires the separate collection of fecal specimens using conventional methods or commercial kits. However, these approaches can potentially introduce sampling errors and biases. In addition, the logistical requirements of studying large human cohorts have driven the need for home collection and transport of human fecal specimens at room temperature. By adopting a unified sampling approach at room temperature, we can enhance sampling convenience and practicality, leading to a more precise and comprehensive understanding of gut microbial function. However, the development and applications of such unified sampling systems still face limitations. The results presented in this study aim to address this knowledge gap by investigating the stability of metabolites and the microbiome (16S ribosomal RNA) from fecal samples collected using 95% EtOH, in comparison to well-established commercial collection kits for fecal metabolome (OMNImet(R)*GUT) and microbiome (OMNIgene(R) *GUT) profiling. Additionally, we perform a comparative analysis of various platforms and metabolomic coverage using matrices containing ethanol, evaluating aspects of sensitivity, robustness, and throughput.

biochemistry↗

bioRxiv2022

Dynamics of lipidome in a colon simulator

Current evidence suggests that gut microbiome derived lipids play crucial role in the regulation of host lipid metabolism. However, not much is known about the dynamics of gut microbial lipids within the distinct gut biogeographic. Here we employed targeted and untargeted lipidomics in the in vitro derived feces. Simulated intestinal chyme was collected from in vitro gut vessels (V1-V4), representing proximal to distal parts of the colon after 24 and 48 h with/without PDX treatment. In total 44 simulated chyme samples were collected from the in vitro colon simulator. Factor analysis showed that vessel and time had the strongest impact on the simulated intestinal chyme lipid profiles. We found that levels of phosphatidylcholines, sphingomyelins, triacylglycerols and endocannabinoids were altered in at least one vessel (V1-V4) during simulation. We also found that concentrations of triacylglycerols, diacylglycerols and endocannabinoids changed with time (24 vs. 48 h simulation). Together, we found that the simulated intestinal chyme revealed a wide range of lipids that remained altered in different compartments of the human colon model over time.

biochemistry↗

bioRxiv2021

Quantitative analysis and genome-scale modeling of human CD4+ T-cell differentiation reveals subset-specific regulation of glycosphingolipid pathways

T-cells are sentinels of adaptive cell-mediated immune responses. T-cell activation, proliferation and differentiation involves metabolic reprogramming involving the interplay of genes, proteins and metabolites. Here, we aim to understand the metabolic pathways involved in the activation and functional differentiation of human CD4+ T-cell subsets (Th1, Th2, Th17 and iTregs). We combined genome-scale metabolic modeling, gene expression data, targeted and non-targeted lipidomics experiments, together with in vitro gene knockdown experiments and showed that human CD4+ T-cells undergo specific metabolic changes during activation and functional differentiation. In addition, we identified and confirmed the importance of ceramide and glycosphingolipid synthesis pathways in Th17 differentiation and effector functions. Finally, through in vitro gene knockdown experiments, we substantiated the requirement of serine palmitoyl transferase (SPT), a de novo sphingolipid pathway in the expression of proinflammatory cytokine (IL17A and IL17F) by Th17 cells. Our findings may provide a comprehensive resource for identifying CD4+ T-cell-specific targets for their selective manipulation under disease conditions, particularly, diseases characterized by an imbalance of Treg / Th17 cells. Our data also suggest a role for elevated levels of ceramides in conditions comorbid with these diseases, e.g., obesity and insulin resistance.

immunology↗

bioRxiv2019

Metabolic Alterations in Human Peripheral Blood Mononuclear Cells Associate with Progression to Islet Autoimmunity and Type 1 Diabetes

Previous metabolomics studies suggest that type 1 diabetes (T1D) is preceded by specific metabolic disturbances. Here we asked whether distinct metabolic patterns occur in peripheral blood mononuclear cells (PBMCs) of children later developing pancreatic {beta}-cell autoimmunity or overt T1D. In a longitudinal cohort setting, PBMC metabolomic analysis was applied in children who either (1) progressed to T1D (PT1D, n=34), (2) seroconverted to [≥]1 islet autoantibody without progressing to T1D (P1Ab, n=27), or (3) remained autoantibody negative during follow-up (CTRL, n=10). During the first year of life, levels of most lipids and polar metabolites were lower in PT1D and P1Ab, versus CTRLs. Pathway overrepresentation analysis suggested alanine, aspartate, glutamate, glycerophospholipid and sphingolipid metabolism were overrepresented in PT1D. Genome-scale metabolic models of PBMCs in T1D progression were developed using available transcriptomics data and constrained with metabolomics data from our study. Metabolic modeling confirmed altered ceramide pathways as specifically associated with T1D progression.

molecular biology↗