Metabolomics guides rational development of a simplified cell culture medium for drug screening against <i>Trypanosoma brucei</i>

n vitro culture methods underpin many experimental approaches to biology and drug discovery. The modification of established cell culture methods to make them more biologically relevant or to optimize growth is traditionally a laborious task. Emerging metabolomic technology enables the rapid evaluation of intra- and extracellular metabolites and can be applied to the rational development of cell culture media. In this study, untargeted semiquantitative and targeted quantitative metabolomic analyses of fresh and spent media revealed the major nutritional requirements for the growth of bloodstream form &lt;i&gt;Trypanosoma brucei&lt;/i&gt;. The standard culture medium (HMI11) contained unnecessarily high concentrations of 32 nutrients that were subsequently removed to make the concentrations more closely resemble those normally found in blood. Our new medium, Creek's minimal medium (CMM), supports in vitro growth equivalent to that in HMI11 and causes no significant perturbation of metabolite levels for 94% of the detected metabolome (&#60;3-fold change; α = 0.05). Importantly, improved sensitivity was observed for drug activity studies in whole-cell phenotypic screenings and in the metabolomic mode of action assays. Four-hundred-fold 50% inhibitory concentration decreases were observed for pentamidine and methotrexate, suggesting inhibition of activity by nutrients present in HMI11. CMM is suitable for routine cell culture and offers important advantages for metabolomic studies and drug activity screening

Plasma lipids are dysregulated preceding diagnosis of preeclampsia or delivery of a growth restricted infantResearch in context

Summary: Background: Lipids serve as multifunctional metabolites that have important implications for the pregnant mother and developing fetus. Abnormalities in lipids have emerged as potential risk factors for pregnancy diseases, such as preeclampsia and fetal growth restriction. The aim of this study was to assess the potential of lipid metabolites for detection of late-onset preeclampsia and fetal growth restriction. Methods: We used a case-cohort of 144 maternal plasma samples at 36 weeks’ gestation from patients before the diagnosis of late-onset preeclampsia (n = 22), delivery of a fetal growth restricted infant (n = 55, defined as <5th birthweight centile), gestation-matched controls (n = 72). We performed liquid chromatography-tandem mass spectrometry (LC-QQQ) -based targeted lipidomics to identify 421 lipids, and fitted logistic regression models for each lipid, correcting for maternal age, BMI, smoking, and gestational diabetes. Findings: Phosphatidylinositol 32:1 (AUC = 0.81) and cholesterol ester 17:1 (AUC = 0.71) best predicted the risk of developing preeclampsia or delivering a fetal growth restricted infant, respectively. Five times repeated five-fold cross validation demonstrated the lipids alone did not out-perform existing protein biomarkers, soluble tyrosine kinase-1 (sFlt-1) and placental growth factor (PlGF) for the prediction of preeclampsia or fetal growth restriction. However, lipids combined with sFlt-1 and PlGF measurements improved disease prediction. Interpretation: This study successfully identified 421 lipids in maternal plasma collected at 36 weeks’ gestation from participants who later developed preeclampsia or delivered a fetal growth restricted infant. Our results suggest the predictive capacity of lipid measurements for gestational disorders holds the potential to improve non-invasive assessment of maternal and fetal health. Funding: This study was funded by a grant from National Health and Medical Research Council

Circadian and wake-dependent changes in human plasma polar metabolites during prolonged wakefulness:A preliminary analysis

Abstract Establishing circadian and wake-dependent changes in the human metabolome are critical for understanding and treating human diseases due to circadian misalignment or extended wake. Here, we assessed endogenous circadian rhythms and wake-dependent changes in plasma metabolites in 13 participants (4 females) studied during 40-hours of wakefulness. Four-hourly plasma samples were analyzed by hydrophilic interaction liquid chromatography (HILIC)-LC-MS for 1,740 metabolite signals. Group-averaged (relative to DLMO) and individual participant metabolite profiles were fitted with a combined cosinor and linear regression model. In group-level analyses, 22% of metabolites were rhythmic and 8% were linear, whereas in individual-level analyses, 14% of profiles were rhythmic and 4% were linear. We observed metabolites that were significant at the group-level but not significant in a single individual, and metabolites that were significant in approximately half of individuals but not group-significant. Of the group-rhythmic and group-linear metabolites, only 7% and 12% were also significantly rhythmic or linear, respectively, in ≥50% of participants. Owing to large inter-individual variation in rhythm timing and the magnitude and direction of linear change, acrophase and slope estimates also differed between group- and individual-level analyses. These preliminary findings have important implications for biomarker development and understanding of sleep and circadian regulation of metabolism

Accurate detection of acute sleep deprivation using a metabolomic biomarker—A machine learning approach

Sleep deprivation enhances risk for serious injury and fatality on the roads and in workplaces. To facilitate future management of these risks through advanced detection, we developed and validated a metabolomic biomarker of sleep deprivation in healthy, young participants, across three experiments. Bi-hourly plasma samples from 2 × 40-hour extended wake protocols (for train/test models) and 1 × 40-hour protocol with an 8-hour overnight sleep interval were analyzed by untargeted liquid chromatography–mass spectrometry. Using a knowledge-based machine learning approach, five consistently important variables were used to build predictive models. Sleep deprivation (24 to 38 hours awake) was predicted accurately in classification models [versus well-rested (0 to 16 hours)] (accuracy = 94.7%/AUC 99.2%, 79.3%/AUC 89.1%) and to a lesser extent in regression (R2 = 86.1 and 47.8%) models for within- and between-participant models, respectively. Metabolites were identified for replicability/future deployment. This approach for detecting acute sleep deprivation offers potential to reduce accidents through “fitness for duty” or “post-accident analysis” assessments

Efficacy of antimicrobial polymer coatings in an animal model of bacterial infection associated with foreign body implants

Objectives: To assess support discs, comprising polyethylene terephthalate (PET), coated with different polymer/levofloxacin combinations for antimicrobial activity in an animal model of infection, in order to explore the use of specific polymer coatings incorporating levofloxacin as a means of reducing device-related infections. Methods: Aliphatic polyester-polyurethanes containing different ratios of poly(lactic acid) diol and poly(caprolactone) diol were prepared, blended with levofloxacin and then used to coat support discs. The in vitro levofloxacin release profiles from these discs were measured in aqueous solution. Mice were surgically implanted with the coated discs placed subcutaneously and infection was initiated by injection of 106 cfu of Staphylococcus aureus into the subcutaneous pocket containing the implant. After 5, 10, 20 and 30 days, the discs were removed, and the number of bacteria adhering to the implant and the residual antimicrobial activity of the discs were determined. Results: In vitro, the release of levofloxacin from the coated discs occurred at a constant rate and then reached a plateau at different timepoints, depending on the polymer preparation used. In vivo, none of the discs coated with polymer blends containing levofloxacin was colonized by S. aureus, whereas 94% of the discs coated with polymer alone were infected. All discs coated with levofloxacin-blended polymers displayed residual antimicrobial activity for at least 20 days post-implantation. Conclusions: Bioerodable polyester-polyurethane polymer coatings containing levofloxacin can prevent bacterial colonization of implants in an intra-operative model of device-related infections.</p

Matrix Selection for the Visualization of Small Molecules and Lipids in Brain Tumors Using Untargeted MALDI-TOF Mass Spectrometry Imaging

Matrix-assisted laser desorption/ionization mass spectrometry imaging allows for the study of metabolic activity in the tumor microenvironment of brain cancers. The detectable metabolites within these tumors are contingent upon the choice of matrix, deposition technique, and polarity setting. In this study, we compared the performance of three different matrices, two deposition techniques, and the use of positive and negative polarity in two different brain cancer types and across two species. Optimal combinations were confirmed by a comparative analysis of lipid and small-molecule abundance by using liquid chromatography–mass spectrometry and RNA sequencing to assess differential metabolites and enzymes between normal and tumor regions. Our findings indicate that in the tumor-bearing brain, the recrystallized α-cyano-4-hydroxycinnamic acid matrix with positive polarity offered superior performance for both detected metabolites and consistency with other techniques. Beyond these implications for brain cancer, our work establishes a workflow to identify optimal matrices for spatial metabolomics studies

Minimal expression of dysferlin prevents development of dysferlinopathy in dysferlin exon 40a knockout mice

Abstract Dysferlin is a Ca2+-activated lipid binding protein implicated in muscle membrane repair. Recessive variants in DYSF result in dysferlinopathy, a progressive muscular dystrophy. We showed previously that calpain cleavage within a motif encoded by alternatively spliced exon 40a releases a 72 kDa C-terminal minidysferlin recruited to injured sarcolemma. Herein we use CRISPR/Cas9 gene editing to knock out murine Dysf exon 40a, to specifically assess its role in membrane repair and development of dysferlinopathy. We created three Dysf exon 40a knockout (40aKO) mouse lines that each express different levels of dysferlin protein ranging from ~ 90%, ~ 50% and ~ 10–20% levels of wild-type. Histopathological analysis of skeletal muscles from all 12-month-old 40aKO lines showed virtual absence of dystrophic features and normal membrane repair capacity for all three 40aKO lines, as compared with dysferlin-null BLAJ mice. Further, lipidomic and proteomic analyses on 18wk old quadriceps show all three 40aKO lines are spared the profound lipidomic/proteomic imbalance that characterises dysferlin-deficient BLAJ muscles. Collective results indicate that membrane repair does not depend upon calpain cleavage within exon 40a and that ~ 10–20% of WT dysferlin protein expression is sufficient to maintain the muscle lipidome, proteome and membrane repair capacity to crucially prevent development of dysferlinopathy

Microbe-Metabolite Associations Linked to the Rebounding Murine Gut Microbiome Postcolonization with Vancomycin-Resistant Enterococcus faecium.

Vancomycin-resistant Enterococcus faecium (VREfm) is an emerging antibiotic-resistant pathogen. Strain-level investigations are beginning to reveal the molecular mechanisms used by VREfm to colonize regions of the human bowel. However, the role of commensal bacteria during VREfm colonization, in particular following antibiotic treatment, remains largely unknown. We employed amplicon 16S rRNA gene sequencing and metabolomics in a murine model system to try and investigate functional roles of the gut microbiome during VREfm colonization. First-order taxonomic shifts between Bacteroidetes and Tenericutes within the gut microbial community composition were detected both in response to pretreatment using ceftriaxone and to subsequent VREfm challenge. Using neural networking approaches to find cooccurrence profiles of bacteria and metabolites, we detected key metabolome features associated with butyric acid during and after VREfm colonization. These metabolite features were associated with Bacteroides, indicative of a transition toward a preantibiotic naive microbiome. This study shows the impacts of antibiotics on the gut ecosystem and the progression of the microbiome in response to colonization with VREfm. Our results offer insights toward identifying potential nonantibiotic alternatives to eliminate VREfm through metabolic reengineering to preferentially select for BacteroidesIMPORTANCE This study demonstrates the importance and power of linking bacterial composition profiling with metabolomics to find the interactions between commensal gut bacteria and a specific pathogen. Knowledge from this research will inform gut microbiome engineering strategies, with the aim of translating observations from animal models to human-relevant therapeutic applications

The placental lipidome of maternal antenatal depression predicts socio-emotional problems in the offspring

While maternal mental health strongly influences neurodevelopment and health in the offspring, little is known about the determinants of inter-individual variation in the mental health of mothers. Likewise, the in utero biological pathways by which variation in maternal mental health affects offspring development remain to be defined. Previous studies implicate lipids, consistent with a known influence on cognitive and emotional function, but the relevance for maternal mental health and offspring neurodevelopment is unclear. This study characterizes the placental and circulatory lipids in antenatal depression, as well as socio-emotional outcomes in the offspring. Targeted liquid chromatography-mass spectrometry covering 470 lipid species was performed on placenta from 186 women with low (n = 70) or high (n = 116) levels of antenatal depressive symptoms assessed using the Edinburgh Postnatal Depression Scale at 26 weeks’ gestation. Child socio-emotional outcomes were assessed from the Child Behavior Check List (CBCL) at 48 months. Seventeen placental lipid species showed an inverse association with antenatal EPDS scores. Specifically, lower levels of phospholipids containing LC-PUFAs: omega-3 docosapentaenoic acid (DPA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and omega-6 arachidonic acid (AA) were significantly associated with depressive symptoms. Additional measurement of LC-PUFA in antenatal plasma samples at mid-gestation confirmed the reduced circulation of these specific fatty acids in mothers. Reduced concentration of the placental phospholipids also predicted poorer socio-emotional outcomes in the offspring. This study provides new insights into the role of the materno-fetal lipid cross-talk as a mechanism linking maternal mental health to that of the offspring. These findings show the potential utility of nutritional approaches among pregnant women with depressive symptoms to reduce offspring risk for later socio-emotional problems

The placental lipidome of maternal antenatal depression predicts socio-emotional problems in the offspring

While maternal mental health strongly influences neurodevelopment and health in the offspring, little is known about the determinants of inter-individual variation in the mental health of mothers. Likewise, the in utero biological pathways by which variation in maternal mental health affects offspring development remain to be defined. Previous studies implicate lipids, consistent with a known influence on cognitive and emotional function, but the relevance for maternal mental health and offspring neurodevelopment is unclear. This study characterizes the placental and circulatory lipids in antenatal depression, as well as socio-emotional outcomes in the offspring. Targeted liquid chromatography-mass spectrometry covering 470 lipid species was performed on placenta from 186 women with low (n = 70) or high (n = 116) levels of antenatal depressive symptoms assessed using the Edinburgh Postnatal Depression Scale at 26 weeks’ gestation. Child socio-emotional outcomes were assessed from the Child Behavior Check List (CBCL) at 48 months. Seventeen placental lipid species showed an inverse association with antenatal EPDS scores. Specifically, lower levels of phospholipids containing LC-PUFAs: omega-3 docosapentaenoic acid (DPA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and omega-6 arachidonic acid (AA) were significantly associated with depressive symptoms. Additional measurement of LC-PUFA in antenatal plasma samples at mid-gestation confirmed the reduced circulation of these specific fatty acids in mothers. Reduced concentration of the placental phospholipids also predicted poorer socio-emotional outcomes in the offspring. This study provides new insights into the role of the materno-fetal lipid cross-talk as a mechanism linking maternal mental health to that of the offspring. These findings show the potential utility of nutritional approaches among pregnant women with depressive symptoms to reduce offspring risk for later socio-emotional problems