Project description:A study to investigate the effect of small molecule lipid inducing compounds that leads to hyper-accumulation of lipids in N replete cells of Chlamydomonas reinhardtii. These compounds were identified through a high throughput screening designed for that purpose. The highthrouhput screen (HTS) evaluated 43,783 compounds and identified 367 primary hits. These 367 hits were further retested using a 8-point dilution series (from 0.25 to 30 uM) and verified the activity of 243 compounds that induce the hyper lipid accumulating phenotype in algae. Once the hit compounds were identified and confirmed, we then performed extensive chemoinformatics analysis to look for common scaffolds and identified several common substructures. We then selected 15 top performing compounds from 5 diverse structural groups and tested for biochemical parameters such as growth, lipid accumulating capacity, effect on photosynthetic rates, respiration rates, oxygen consumption rates, analysis of different lipid species to identify and quantify fatty acid species using GC-MS, transcriptome analysis using next generation sequencing (RNASeq), untargeted metabolomics using LC-MS, lipidome analysis using FT-ICR MS. To understand the global changes in the proteome, 2 structurally different compounds were selected and compared to the control without compound treatment.
Project description:There are many theories as to the mode of action of miltefosine against Leishmania including alterations to the membrane lipid content, induction of apoptosis and modulation of macrophage responses. Here we perform untargeted metabolomics to elucidate the metabolic changes involved in miltefosine action. Over 800 metabolites were detected, 10% of which were significantly altered after 3.75 h. Many of the changes related to an increase in alkane fragment and sugar release. Fragment release is synchronised with reactive oxygen species production, but native membrane phospholipids remain intact. Signs of DNA damage were also detected as were changes to the levels of some thiols and polyamines. After 5 h of miltefosine treatment the cells showed depleted levels of most metabolites, indicating that the cells' outer membrane integrity had become compromised and internal metabolites were escaping upon cell death. In miltefosine resistant cells, the drug was not internalised and the changes to the internal metabolite levels were not seen. In contrast, cells resistant to antimony (SbIII) had similar corresponding alterations to the levels of internal metabolites as wild-type cells. A detailed knowledge of the mode of action of miltefosine will be important to inform the design of combination therapies to combat leishmaniasis, something that the research community should be prioritising in the coming years.
Project description:Metabolomics has been increasingly applied to biomarker discovery, as untargeted metabolic profiling represents a powerful exploratory tool for identifying causal links between biomarkers and disease phenotypes. In the present work, we used untargeted metabolomics to investigate plasma specimens of rats, dogs, and mice treated with small-molecule drugs designed for improved glycemic control of type 2 diabetes mellitus patients via activation of GPR40. The in vivo pharmacology of GPR40 is not yet fully understood. Compounds targeting this receptor have been found to induce drug-induced liver injury (DILI). Metabolomic analysis facilitating an integrated UPLC-TWIMS-HRMS platform was used to detect metabolic differences between treated and non-treated animals within two 4-week toxicity studies in rat and dog, and one 2-week toxicity study in mouse. Multivariate statistics of untargeted metabolomics data subsequently revealed the presence of several significantly upregulated endogenous compounds in the treated animals whose plasma level is known to be affected during DILI. A specific bile acid metabolite useful as endogenous probe for drug-drug interaction studies was identified (chenodeoxycholic acid-24 glucuronide), as well as a metabolic precursor indicative of acidic bile acid biosynthesis (7α-hydroxy-3-oxo-4-cholestenoic acid). These results correlate with typical liver toxicity parameters on the individual level.
Project description:BackgroundThe pathobiological mechanisms associated with perinatal asphyxia and hypoxic-ischemic encephalopathy are complex and poorly understood. The metabolic effects of therapeutic hypothermia have been partially explored.MethodsWe conducted a single-center longitudinal study to investigate the metabolic effects of perinatal asphyxia and hypoxic-ischemic encephalopathy on the urinary metabolome of a group of 12 asphyctic infants over time compared to 22 matched healthy newborns, using untargeted metabolomics based on mass spectrometry.FindingsOver-representation pathway analysis identified the steroidogenesis pathway as being significantly disrupted, with reduced steroid levels in the first three days of life despite treatment with hypothermia. Comparison with matched healthy newborns showed that the urinary steroid content was lower in asphyctic infants before hypothermia. The lysine degradation and carnitine synthesis pathways were also significantly affected.InterpretationSteroidogenesis is significantly disrupted in asphyctic infants compared to healthy newborns. Given how neurosteroids are involved in neuromodulation and neuroprotection, translational research is warranted on the potential role of neurosteroid-based intervention in asphyctic infants.FundingNone.
Project description:Thermal processes are widely used in small molecule chemical analysis and metabolomics for derivatization, vaporization, chromatography, and ionization, especially in gas chromatography mass spectrometry (GC/MS). In this study the effect of heating was examined on a set of 64 small molecule standards and, separately, on human plasma metabolite extracts. The samples, either derivatized or underivatized, were heated at three different temperatures (60, 100, and 250 °C) at different exposure times (30 s, 60 s, and 300 s). All the samples were analyzed by liquid chromatography coupled to electrospray ionization mass spectrometry (LC/MS) and the data processed by XCMS Online ( xcmsonline.scripps.edu ). The results showed that heating at an elevated temperature of 100 °C had an appreciable effect on both the underivatized and derivatized molecules, and heating at 250 °C created substantial changes in the profile. For example, over 40% of the molecular peaks were altered in the plasma metabolite analysis after heating (250 °C, 300s) with a significant formation of degradation and transformation products. The analysis of 64 small molecule standards validated the temperature-induced changes observed on the plasma metabolites, where most of the small molecules degraded at elevated temperatures even after minimal exposure times (30 s). For example, tri- and diorganophosphates (e.g., adenosine triphosphate and adenosine diphosphate) were readily degraded into a mono-organophosphate (e.g., adenosine monophosphate) during heating. Nucleosides and nucleotides (e.g., inosine and inosine monophosphate) were also found to be transformed into purine derivatives (e.g., hypoxanthine). A newly formed transformation product, oleoyl ethyl amide, was identified in both the underivatized and derivatized forms of the plasma extracts and small molecule standard mixture, and was likely generated from oleic acid. Overall these analyses show that small molecules and metabolites undergo significant time-sensitive alterations when exposed to elevated temperatures, especially those conditions that mimic sample preparation and analysis in GC/MS experiments.
Project description:Small molecules that induce protein-protein associations represent powerful tools to modulate cell circuitry. We sought to develop a platform for the direct discovery of compounds able to induce association of any two preselected proteins, using the E3 ligase von Hippel-Lindau (VHL) and bromodomains as test systems. Leveraging the screening power of DNA-encoded libraries (DELs), we synthesized ~1 million DNA-encoded compounds that possess a VHL-targeting ligand, a variety of connectors and a diversity element generated by split-and-pool combinatorial chemistry. By screening our DEL against bromodomains in the presence and absence of VHL, we could identify VHL-bound molecules that simultaneously bind bromodomains. For highly barcode-enriched library members, ternary complex formation leading to bromodomain degradation was confirmed in cells. Furthermore, a ternary complex crystal structure was obtained for our most enriched library member with BRD4BD1 and a VHL complex. Our work provides a foundation for adapting DEL screening to the discovery of proximity-inducing small molecules.
Project description:Acute leukemias (AL) are aggressive neoplasms with high mortality rates. Metabolomics and oxidative status have emerged as important tools to identify new biomarkers with clinical utility. To identify the metabolic differences between healthy individuals (HI) and patients with AL, a multiplatform untargeted metabolomic and lipidomic approach was conducted using liquid and gas chromatography coupled with quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS or GC-QTOF-MS). Additionally, the total antioxidant capacity (TAC) was measured. A total of 20 peripheral blood plasma samples were obtained from patients with AL and 18 samples from HI. Our analysis revealed 135 differentially altered metabolites in the patients belonging to 12 chemical classes; likewise, the metabolic pathways of glycerolipids and sphingolipids were the most affected in the patients. A decrease in the TAC of the patients with respect to the HI was evident. This study conducted with a cohort of Colombian patients is consistent with observations from other research studies that suggest dysregulation of lipid compounds. Furthermore, metabolic differences between patients and HI appear to be independent of lifestyle, race, or geographic location, providing valuable information for future advancements in understanding the disease and developing more global therapies.
Project description:PurposeExposure of the general population to ionizing radiation has increased in the past decades, primarily due to long distance travel and medical procedures. On the other hand, accidental exposures, nuclear accidents, and elevated threats of terrorism with the potential detonation of a radiological dispersal device or improvised nuclear device in a major city, all have led to increased needs for rapid biodosimetry and assessment of exposure to different radiation qualities and scenarios. Metabolomics, the qualitative and quantitative assessment of small molecules in a given biological specimen, has emerged as a promising technology to allow for rapid determination of an individual's exposure level and metabolic phenotype. Advancements in mass spectrometry techniques have led to untargeted (discovery phase, global assessment) and targeted (quantitative phase) methods not only to identify biomarkers of radiation exposure, but also to assess general perturbations of metabolism with potential long-term consequences, such as cancer, cardiovascular, and pulmonary disease.ConclusionsMetabolomics of radiation exposure has provided a highly informative snapshot of metabolic dysregulation. Biomarkers in easily accessible biofluids and biospecimens (urine, blood, saliva, sebum, fecal material) from mouse, rat, and minipig models, to non-human primates and humans have provided the basis for determination of a radiation signature to assess the need for medical intervention. Here we provide a comprehensive description of the current status of radiation metabolomic studies for the purpose of rapid high-throughput radiation biodosimetry in easily accessible biofluids and discuss future directions of radiation metabolomics research.
Project description:Diagnosis of ovarian cancer is difficult due to the lack of clinical symptoms and effective screening algorithms. In this study, we aim to develop models for ovarian cancer diagnosis by detecting metabolites in urine and plasma samples. Ultra-high-performance liquid chromatography and quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) in positive ion mode was used for metabolome quantification in 235 urine samples and 331 plasma samples. Then, Urine and plasma metabolomic profiles were analyzed by univariate and multivariate statistics. Four groups of samples: normal control, benign, borderline and malignant ovarian tumors were enrolled in this study. A total of 1330 features and 1302 features were detected from urine and plasma samples respectively. Based on two urine putative metabolites, five plasma putative metabolites and five urine putative metabolites, three models for distinguishing normal-ovarian tumors, benign-malignant (borderline + malignant) and borderline-malignant ovarian tumors were developed respectively. The AUC (Area Under Curve) values were 0.987, 0876 and 0.943 in discovery set and 0.984, 0.896 and 0.836 in validation set for three models. Specially, the diagnostic model based on 5 plasma putative metabolites had better early-stage diagnosis performance than CA125 alone. The AUC values of the model were 0.847 and 0.988 in discovery and validation set respectively. Our results showed that normal and ovarian tumors have unique metabolic signature in urine and plasma samples, which shed light on the ovarian cancer diagnosis and classification.