Neuroproteomics and Systems Biology Approach to Identify Temporal Biomarker Changes Post Experimental Traumatic Brain Injury in Rats

Traumatic brain injury (TBI) represents a critical health problem of which diagnosis, management, and treatment remain challenging. TBI is a contributing factor in approximately one-third of all injury-related deaths in the United States. The Centers for Disease Control and Prevention estimate that 1.7 million people suffer a TBI in the United States annually. Efforts continue to focus on elucidating the complex molecular mechanisms underlying TBI pathophysiology and defining sensitive and specific biomarkers that can aid in improving patient management and care. Recently, the area of neuroproteomics-systems biology is proving to be a prominent tool in biomarker discovery for central nervous system injury and other neurological diseases. In this work, we employed the controlled cortical impact (CCI) model of experimental TBI in rat model to assess the temporal-global proteome changes after acute (1 day) and for the first time, subacute (7 days), post-injury time frame using the established cation-anion exchange chromatography-1D SDS gel electrophoresis LC-MS/MS platform for protein separation combined with discrete systems biology analyses to identify temporal biomarker changes related to this rat TBI model. Rather than focusing on any one individual molecular entity, we use

Cellular Metabolomics Profiles Associated With Drug Chemosensitivity in AML

BackgroundAcute myeloid leukemia (AML) is a hematological malignancy with a dismal prognosis. For over four decades, AML has primarily been treated by cytarabine combined with an anthracycline. Although a significant proportion of patients achieve remission with this regimen, roughly 40% of children and 70% of adults relapse. Over 90% of patients with resistant or relapsed AML die within 3 years. Thus, relapsed and resistant disease following treatment with standard therapy are the most common clinical failures that occur in treating this disease. In this study, we evaluated the relationship between AML cell line global metabolomes and variation in chemosensitivity.MethodsWe performed global metabolomics on seven AML cell lines with varying chemosensitivity to cytarabine and the anthracycline doxorubicin (MV4.11, KG-1, HL-60, Kasumi-1, AML-193, ME1, THP-1) using ultra-high performance liquid chromatography – mass spectrometry (UHPLC-MS). Univariate and multivariate analyses were performed on the metabolite peak intensity values from UHPLC-MS using MetaboAnalyst to identify cellular metabolites associated with drug chemosensitivity.ResultsA total of 1,624 metabolic features were detected across the leukemic cell lines. Of these, 187 were annotated to known metabolites. With respect to doxorubicin, we observed significantly greater abundance of a carboxylic acid (1-aminocyclopropane-1-carboxylate) and several amino acids in resistant cell lines. Pathway analysis found enrichment of several amino acid biosynthesis and metabolic pathways. For cytarabine resistance, nine annotated metabolites were significantly different in resistance vs. sensitive cell lines, including D-raffinose, guanosine, inosine, guanine, aldopentose, two xenobiotics (allopurinol and 4-hydroxy-L-phenylglycine) and glucosamine/mannosamine. Pathway analysis associated these metabolites with the purine metabolic pathway.ConclusionOverall, our results demonstrate that metabolomics differences contribute toward drug resistance. In addition, it could potentially identify predictive biomarkers for chemosensitivity to various anti-leukemic drugs. Our results provide opportunity to further explore these metabolites in patient samples for association with clinical response

Lipidomics and Redox Lipidomics Indicate Early Stage Alcohol-Induced Liver Damage.

Alcoholic fatty liver disease (AFLD) is characterized by lipid accumulation and inflammation and can progress to cirrhosis and cancer in the liver. AFLD diagnosis currently relies on histological analysis of liver biopsies. Early detection permits interventions that would prevent progression to cirrhosis or later stages of the disease. Herein, we have conducted the first comprehensive time-course study of lipids using novel state-of-the art lipidomics methods in plasma and liver in the early stages of a mouse model of AFLD, i.e., Lieber-DeCarli diet model. In ethanol-treated mice, changes in liver tissue included up-regulation of triglycerides (TGs) and oxidized TGs and down-regulation of phosphatidylcholine, lysophosphatidylcholine, and 20-22-carbon-containing lipid-mediator precursors. An increase in oxidized TGs preceded histological signs of early AFLD, i.e., steatosis, with these changes observed in both the liver and plasma. The major lipid classes dysregulated by ethanol play important roles in hepatic inflammation, steatosis, and oxidative damage. Conclusion: Alcohol consumption alters the liver lipidome before overt histological markers of early AFLD. This introduces the exciting possibility that specific lipids may serve as earlier biomarkers of AFLD than those currently being used

Metabolomics Insights into Chemical Convergence in Xanthomonas perforans and Metabolic Changes Following Treatment with the Small Molecule Carvacrol

Microbes are natural chemical factories and their metabolome comprise diverse arrays of chemicals. The genus Xanthomonas comprises some of the most important plant pathogens causing devastating yield losses globally and previous studies suggested that species in the genus are untapped chemical minefields. In this study, we applied an untargeted metabolomics approach to study the metabolome of a globally spread important xanthomonad, X. perforans. The pathogen is difficult to manage, but recent studies suggest that the small molecule carvacrol was efficient in disease control. Bacterial strains were treated with carvacrol, and samples were taken at time intervals (1 and 6 h). An untreated control was also included. There were five replicates for each sample and samples were prepared for metabolomics profiling using the standard procedure. Metabolomics profiling was carried out using a thermo Q-Exactive orbitrap mass spectrometer with Dionex ultra high-performance liquid chromatography (UHPLC) and an autosampler. Annotation of significant metabolites using the Metabolomics Standards Initiative level 2 identified an array of novel metabolites that were previously not reported in Xanthomonas perforans. These metabolites include methoxybrassinin and cyclobrassinone, which are known metabolites of brassicas; sarmentosin, a metabolite of the Passiflora-heliconiine butterfly system; and monatin, a naturally occurring sweetener found in Sclerochiton ilicifolius. To our knowledge, this is the first report of these metabolites in a microbial system. Other significant metabolites previously identified in non-Xanthomonas systems but reported in this study include maculosin; piperidine; β-carboline alkaloids, such as harman and derivatives; and several important medically relevant metabolites, such as valsartan, metharbital, pirbuterol, and ozagrel. This finding is consistent with convergent evolution found in reported biological systems. Analyses of the effect of carvacrol in time-series and associated pathways suggest that carvacrol has a global effect on the metabolome of X. perforans, showing marked changes in metabolites that are critical in energy biosynthesis and degradation pathways, amino acid pathways, nucleic acid pathways, as well as the newly identified metabolites whose pathways are unknown. This study provides the first insight into the X. perforans metabolome and additionally lays a metabolomics-guided foundation for characterization of novel metabolites and pathways in xanthomonad systems

Re-modeling of foliar membrane lipids in a seagrass allows for growth in phosphorus-deplete conditions.

In this study, we used liquid chromatography high-resolution tandem mass spectrometry to analyze the lipidome of turtlegrass (Thalassia testudinum) leaves with either extremely high phosphorus content or extremely low phosphorus content. Most species of phospholipids were significantly down-regulated in phosphorus-deplete leaves, whereas diacylglyceryltrimethylhomoserine (DGTS), triglycerides (TG), galactolipid digalactosyldiacylglycerol (DGDG), certain species of glucuronosyldiacylglycerols (GlcADG), and certain species of sulfoquinovosyl diacylglycerol (SQDG) were significantly upregulated, accounting for the change in phosphorus content, as well as structural differences in the leaves of plants growing across regions of varying elemental availability. These data suggest that seagrasses are able to modify the phosphorus content in leaf membranes dependent upon environmental availability

Insight into PreImplantation Factor (PIF*) Mechanism for Embryo Protection and Development: Target Oxidative Stress and Protein Misfolding (PDI and HSP) through Essential RIPK Binding Site

<div><p>Background</p><p>Endogenous PIF, upon which embryo development is dependent, is secreted only by viable mammalian embryos, and absent in non-viable ones. Synthetic PIF (sPIF) administration promotes singly cultured embryos development and protects against their demise caused by embryo-toxic serum. To identify and characterize critical sPIF-embryo protein interactions novel biochemical and bio-analytical methods were specifically devised.</p><p>Methods</p><p>FITC-PIF uptake/binding by cultured murine and equine embryos was examined and compared with scrambled FITC-PIF (control). Murine embryo (d10) lysates were fractionated by reversed-phase HPLC, fractions printed onto microarray slides and probed with Biotin-PIF, IDE and Kv1.3 antibodies, using fluorescence detection. sPIF-based affinity column was developed to extract and identify PIF-protein interactions from lysates using peptide mass spectrometry (LC/MS/MS). <i>In silico</i> evaluation examined binding of PIF to critical targets, using mutation analysis.</p><p>Results</p><p>PIF directly targets viable cultured embryos as compared with control peptide, which failed to bind. Multistep Biotin-PIF targets were confirmed by single-step PIF-affinity column based isolation. PIF binds protein disulfide isomerases a prolyl-4-hydroxylase β-subunit, (PDI, PDIA4, PDIA6-like) containing the antioxidant thioredoxin domain. PIF also binds protective heat shock proteins (70&90), co-chaperone, BAG-3. Remarkably, PIF targets a common RIPK site in PDI and HSP proteins. Further, single PIF amino acid mutation significantly reduced peptide-protein target bonding. PIF binds promiscuous tubulins, neuron backbones and ACTA-1,2 visceral proteins. Significant anti-IDE, while limited anti-Kv1.3b antibody-binding to Biotin-PIF positive lysates HPLC fractions were documented.</p><p>Conclusion</p><p>Collectively, data identifies PIF shared targets on PDI and HSP in the embryo. Such are known to play a critical role in protecting against oxidative stress and protein misfolding. PIF-affinity-column is a novel utilitarian method for small molecule targets direct identification. Data reveals and completes the understanding of mechanisms involved in PIF-induced autotrophic and protective effects on the embryo.</p></div