Circulatory levels of lysophosphatidylcholine species in obese adolescents: Findings from cross-sectional and prospective lipidomics analyses.

BACKGROUND AND AIMS: Obesity has reached epidemic proportions, emphasizing the importance of reliable biomarkers for detecting early metabolic alterations and enabling early preventative interventions. However, our understanding of the molecular mechanisms and specific lipid species associated with childhood obesity remains limited. Therefore, the aim of this study was to investigate plasma lipidomic signatures as potential biomarkers for adolescent obesity. METHODS AND RESULTS: A total of 103 individuals comprising overweight/obese (n = 46) and normal weight (n = 57) were randomly chosen from the baseline ORANGE (Obesity Reduction and Noncommunicable Disease Awareness through Group Education) cohort, having been followed up for a median of 7.1 years. Plasma lipidomic profiling was performed using the UHPLC-HRMS method. We used three different models adjusted for clinical covariates to analyze the data. Clustering methods were used to define metabotypes, which allowed for the stratification of subjects into subgroups with similar clinical and metabolic profiles. We observed that lysophosphatidylcholine (LPC) species like LPC.16.0, LPC.18.3, LPC.18.1, and LPC.20.3 were significantly (p < 0.05) associated with baseline and follow-up BMI in adolescent obesity. The association of LPC species with BMI remained consistently significant even after adjusting for potential confounders. Moreover, applying metabotyping using hierarchical clustering provided insights into the metabolic heterogeneity within the normal and obese groups, distinguishing metabolically healthy individuals from those with unhealthy metabolic profiles. CONCLUSION: The specific LPC levels were found to be altered and increased in childhood obesity, particularly during the follow-up. These findings suggest that LPC species hold promise as potential biomarkers of obesity in adolescents, including healthy and unhealthy metabolic profiles.

Rapid and Quantitative Analysis of Aflatoxin M1 From Milk Using Atmospheric Pressure-Matrix Assisted Laser Desorption/Ionization (AP-MALDI)-Triple Quadrupole Selected Reaction Monitoring.

BACKGROUND: Aflatoxin M1 (AFM1) is a carcinogenic hydroxylated metabolite commonly found in milk. It is relatively stable toward decontamination procedures posing a major health risk, and it requires an international regulatory mandate of detection at trace levels. OBJECTIVE: To develop a high-throughput, reliable, and compliant method for the identification of AFM1 in milk samples using atmospheric pressure-matrix assisted laser desorption/ionization (AP-MALDI) selected reaction monitoring (SRM) quantitation. METHOD: The milk sample was diluted in water and cleaned with immunoaffinity chromatography (IAC), followed by analysis using AP-MALDI hyphenated with a triple quadrupole mass spectrometer for SRM. RESULTS: A fast and reliable AP-MALDI SRM quantitative method was developed for the determination of AFM1 with analysis time of 1 min per sample. The diagnostic product ions of AFM1 at 273.1 u and 229.2 u were monitored during the SRM. The calibration curves yielded excellent linearity (R2 = 0.99) with good recoveries for quality control samples (97-106%). The ion ratios of the qualifier to quantifier displayed excellent RSD (1-7.8%) for n = 3. CONCLUSIONS: The developed method provided rapid quantification for AFM1. The fast AP-MALDI SRM method can allow analysis of AFM1 in a large number of milk samples. Given the time required for analysis, cost-effectiveness, and superior analytical performance, this method can be adopted in commercial food testing laboratories. HIGHLIGHTS: Aflatoxins (AF) are a major health risk. Speedy analysis of large sample sizes from food is a risk mitigation strategy but remains an unmet need. Quantitative, chromatography-free, and internal standard-free AP-MALDI SRM based analysis of AF is a high-throughput and cost-efficient alternative. Satisfactory performance was achieved for quantitative AP-MALDI SRM analysis of AFM1 in milk subsequent to a simple sample clean-up step.

Integrated genetic and metabolic landscapes predict vulnerabilities of temozolomide resistant glioblastoma cells.

Metabolic reprogramming and its molecular underpinnings are critical to unravel the duality of cancer cell function and chemo-resistance. Here, we use a constraints-based integrated approach to delineate the interplay between metabolism and epigenetics, hardwired in the genome, to shape temozolomide (TMZ) resistance. Differential metabolism was identified in response to TMZ at varying concentrations in both the resistant neurospheroidal (NSP) and the susceptible (U87MG) glioblastoma cell-lines. The genetic basis of this metabolic adaptation was characterized by whole exome sequencing that identified mutations in signaling pathway regulators of growth and energy metabolism. Remarkably, our integrated approach identified rewiring in glycolysis, TCA cycle, malate aspartate shunt, and oxidative phosphorylation pathways. The differential killing of TMZ resistant NSP by Rotenone at low concentrations with an IC50 value of 5 nM, three orders of magnitude lower than for U87MG that exhibited an IC50 value of 1.8 mM was thus identified using our integrated systems-based approach.

Plausible diagnostic value of urinary isomeric dimethylarginine ratio for diabetic nephropathy.

Altered circulatory asymmetric and symmetric dimethylarginines have been independently reported in patients with end-stage renal failure suggesting their potential role as mediators and early biomarkers of nephropathy. These alterations can also be reflected in urine. Herein, we aimed to evaluate urinary asymmetric to symmetric dimethylarginine ratio (ASR) for early prediction of diabetic nephropathy (DN). In this cross-sectional study, individuals with impaired glucose tolerance (IGT), newly diagnosed diabetes (NDD), diabetic microalbuminuria (MIC), macroalbuminuria (MAC), and normal glucose tolerance (NGT) were recruited from Dr. Mohans' Diabetes Specialties centre, India. Urinary ASR was measured using a validated high-throughput MALDI-MS/MS method. Significantly lower ASR was observed in MIC (0.909) and MAC (0.741) in comparison to the NGT and NDD groups. On regression models, ASR was associated with MIC [OR: 0.256; 95% CI: 0.158-0.491] and MAC [OR 0.146; 95% CI: 0.071-0.292] controlled for all the available confounding factors. ROC analysis revealed ASR cut-point of 0.95 had C-statistic of 0.691 (95% CI: 0.627-0.755) to discriminate MIC from NDD with 72% sensitivity. Whereas, an ASR cut-point of 0.82 had C-statistic of 0.846 (95% CI: 0.800 - 0.893) had 91% sensitivity for identifying MAC. Our results suggest ASR as a potential early diagnostic biomarker for DN among the Asian Indians.

Superfluous glutamine synthetase activity in Chinese Hamster Ovary cells selected under glutamine limitation is growth limiting in glutamine-replete conditions and can be inhibited by serine.

Passaging and expansion of animal cells in lean maintenance medium could result in periods of limitation of some nutrients. Over time, such stresses could possibly result in selection of cells with metabolic changes and contribute to heterogeneity. Here, we investigate whether selection of Chinese Hamster Ovary (CHO) cells under glutamine limitation results in changes in growth under glutamine-replete conditions. In glutamine-limiting medium, compared to control cells passaged in glutamine-rich medium, the selected cells showed higher glutamine synthetase (GS) activity and attained a higher peak viable cell density (PVCD). Surprisingly, in glutamine-replete conditions, selected cells still showed a higher GS activity but a lower PVCD. We show that in glutamine-replete medium, PVCD of selected cells was restored on (a) inhibition of GS activity with methionine sulfoximine, (b) supplementation of aspartate-without affecting GS activity, and (c) supplementation of serine, which is reported to inhibit GS in vitro. Consistent with the reported effect of serine, inhibition of GS activity was observed upon serine supplementation along with reduced growth of cells under glutamine-limiting conditions. The latter observation is important for the design of glutamine-free culture medium and feed used for GS-CHO and GS-NS0. In summary, we show that CHO cells selected under glutamine limitation have superfluous GS activity in glutamine-replete medium, which negatively affects their PVCD. This may be due to its effect on availability of aspartate which was the limiting nutrient for the growth of selected cells in glutamine-replete conditions.

Quantitation of Neurotoxic Metabolites of the Kynurenine Pathway by Laser Desorption Ionization Mass Spectrometry (LDI-MS).

The metabolites of the mammalian kynurenine (KYN) pathway are generated from a branch of tryptophan metabolic pathway. The latter generates 3-hydroxykynurenine (3-HK), kynurenic acid (KYNA), quinolinic acid (QUIN), and picolinic acid (PIC) which are all strongly neuroactive, often with dramatically contrasting functional outcomes. Whereas KYNA and PIC are neuroprotective, 3-HK and QUIN are potently neurotoxic and attributed in major neurodegenerative diseases like schizophrenia, Alzheimer's disease, Huntington's disease, bipolar disorder, and depression. It is increasingly evident that the ratio(s) between the neurotoxic and neuroprotective metabolites may help predict the manifestations of disease vs. health. Therefore high-throughput platforms for determining the relative levels of these kynurenine metabolites in biofluids offer considerable potential. Current analytical tools for studying KYN pathway include assays of branching enzymes, PCR, immunoanalysis, and LCMS. None of these offer high-throughput, cost-effective analyses suited for clinical or drug-screening applications. In this report a laser desorption ionization mass spectrometry (LDI-MS) method is described using SBA-15 mesoporous silica. The system allows fast, high-resolution quantitation of neurotoxic kynurenines using targeted metabolomics on conventional MALDI platforms.

Integrative analysis of rewired central metabolism in temozolomide resistant cells.

An authenticated U87MG clonal glioblastoma cell line was investigated to identify a sub-population of neurospheroidal (NSP) cells within the main epithelial population (U87MG). The NSP cells sorted using Fluorescence Assisted Cell Sorting (FACS) showed varied morphology, 30% lower growth rates, 40% higher IC50 values for temozolomide drug and could differentiate into the glial cell type (NDx). Metabolite profiling using HR-LCMS identified glucose, glutamine and serine in both populations and tryptophan only in U87MG as growth limiting substrates. Glycine, alanine, glutamate and proline were secreted by U87MG, however proline and glycine were re-utilized in NSP. Exo-metabolite profiling and phenotypic microarrays identified differential metabolism of primary carbon sources glucose and derived pyruvate for U87MG; glutamine and derived glutamate metabolism in NSP. Differential mRNA abundance of AKT1, PTEN, PIK3CA controlling metabolism, drug efflux, nutrient transport and epigenetic control MDM2 are potentially critical in shaping DNA methylation effects of temozolomide. Our study provides a new insight into the combined effect of these factors leading to temozolomide resistance in NSP.

A scalable metabolite supplementation strategy against antibiotic resistant pathogen Chromobacterium violaceum induced by NAD+/NADH+ imbalance.

BACKGROUND: The leading edge of the global problem of antibiotic resistance necessitates novel therapeutic strategies. This study develops a novel systems biology driven approach for killing antibiotic resistant pathogens using benign metabolites. RESULTS: Controlled laboratory evolutions established chloramphenicol and streptomycin resistant pathogens of Chromobacterium. These resistant pathogens showed higher growth rates and required higher lethal doses of antibiotic. Growth and viability testing identified malate, maleate, succinate, pyruvate and oxoadipate as resensitising agents for antibiotic therapy. Resistant genes were catalogued through whole genome sequencing. Intracellular metabolomic profiling identified violacein as a potential biomarker for resistance. The temporal variance of metabolites captured the linearized dynamics around the steady state and correlated to growth rate. A constraints-based flux balance model of the core metabolism was used to predict the metabolic basis of antibiotic susceptibility and resistance. CONCLUSIONS: The model predicts electron imbalance and skewed NAD/NADH ratios as a result of antibiotics - chloramphenicol and streptomycin. The resistant pathogen rewired its metabolic networks to compensate for disruption of redox homeostasis. We foresee the utility of such scalable workflows in identifying metabolites for clinical isolates as inevitable solutions to mitigate antibiotic resistance.

Determination of Triazines and Triazoles in Grapes Using Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization High-Resolution Mass Spectrometry.

A chromatography-free atmospheric pressure matrix-assisted laser desorption/ionization high-resolution mass spectrometry (AP-MALDI HRMS) method is described for the simultaneous and quantitative detection of triazines and triazoles in grapes. The analytes were detected reproducibly with high mass accuracy (mass error within 5 ppm) and further confirmed by collision-induced dissociation fragmentation in tandem MS. The LODs and LOQs for all the analytes were found to be in the nanogram per gram level (15-20 ng/g LOQ). Internal standard-normalized high-resolution accurate mass-extracted (HR-AM) peak intensities of the detected ions were used to generate the concentration response curves. Linearity (with R2 values around 0.99) was obtained for these curves within a concentration range of 20-200 ng/g of the individual analytes. The accuracy and precision of the method were further established using QC samples. Validation and performance comparison of the AP-MALDI HRMS method with an existing standard method using LC with triple quadrupole MS was carried out (evaluating sensitivity, accuracy, precision, and analysis time) using 20 table-grape field samples after QuEChERS extraction.

CHO Cells adapted to inorganic phosphate limitation show higher growth and higher pyruvate carboxylase flux in phosphate replete conditions.

Inorganic phosphate (Pi ) is an essential ion involved in diverse cellular processes including metabolism. Changes in cellular metabolism upon long term adaptation to Pi limitation have been reported in E. coli. Given the essential role of Pi , adaptation to Pi limitation may also result in metabolic changes in animal cells. In this study, we have adapted CHO cells producing recombinant IgG to limiting Pi conditions for 75 days. Not surprisingly, adapted cells showed better survival under Pi limitation. Here, we report the finding that such cells also showed better growth characteristics compared to control in batch culture replete with Pi (higher peak density and integral viable cell density), accompanied by a lower specific oxygen uptake rate and cytochrome oxidase activity towards the end of exponential phase. Surprisingly, the adapted cells grew to a lower peak density under glucose limitation. This suggests long term Pi limitation may lead to selection for an altered metabolism with higher dependence on glucose availability for biomass assimilation compared to control. Steady state U-13 C glucose labeling experiments suggest that adapted cells have a higher pyruvate carboxylase flux. Consistent with this observation, supplementation with aspartate abolished the peak density difference whereas supplementation with serine did not abolish the difference. This supports the hypothesis that cell growth in the adapted culture might be higher due to a higher pyruvate carboxylase flux. Decreased fitness under carbon limitation and mutations in the sucABCD operon has been previously reported in E. coli upon long term adaptation to Pi limitation, suggestive of a similarity in cellular response among such diverse species. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:749-758, 2017.

Enhanced permeation, leaf retention, and plant protease inhibitor activity with bicontinuous microemulsions.

Bicontinuous microemulsions (BCMEs) have excellent solubulizing properties along with low interfacial tension and aqueous content that can be controlled. In this work, water soluble plant protease inhibitor (PI), well characterized for its activity against insect pests, was incorporated into a BCME system and explored for permeation on hydrophobic leaf surfaces and protease inhibition activity. The bicontinuous nature of the microemulsion containing water:2-propanol:1-butanol (55:35:10 w/w) was characterized using conductivity and self-diffusion coefficient measurements. The PI was soluble in the water-rich bicontinuous domains, stable in the microemulsions, and protease inhibition activity was retained for a prolonged duration. The microemulsions ensured greater wettability and a wider spread of the PI on hydrophobic leaf surfaces as revealed by contact angle measurements. Significantly, trypsin inhibition activity assays of the PI recovered from the leaves after delivery from the microemulsion indicated a significant increase in the PI retention on the leaf. This BCME enabled greater leaf permeation and retention of the PI can be attributed to a temporary disruption of the waxy leaf surface followed by self-repair without causing any long term damage to the plant.

Lanthanide phytanates: liquid-crystalline phase behavior, colloidal particle dispersions, and potential as medical imaging agents.

Lanthanide salts of phytanic acid, an isoprenoid-type amphiphile, have been synthesized and characterized. Elemental analysis and FTIR spectroscopy were used to confirm the formed product and showed that three phytanate anions are complexed with one lanthanide cation. The physicochemical properties of the lanthanide phytanates were investigated using DSC, XRD, SAXS, and cross-polarized optical microscopy. Several of the hydrated salts form a liquid-crystalline hexagonal columnar mesophase at room temperature, and samarium(III) phytanate forms this phase even in the absence of water. Select lanthanide phytanates were dispersed in water, and cryo-TEM images indicate that some structure has been retained in the dispersed phase. NMR relaxivity measurements were conducted on these systems. It has been shown that a particulate dispersion of gadolinium(III) phytanate displays proton relaxivity values comparable to those of a commercial contrast agent for magnetic resonance imaging and a colloidal dispersion of europium(III) phytanate exhibits the characteristics of a fluorescence imaging agent.

Phosphopeptide analysis by directly coupling two-dimensional planar electrochromatography/thin-layer chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A novel strategy is presented for the fractionation of complex peptide mixtures using two-dimensional planar electrochromatography/thin-layer chromatography (2D PEC/TLC). Phosphopeptides migrate more slowly in the first dimension, based upon their anionic phosphate residues, and certain predominantly acidic phosphopeptides even migrate in the opposite direction, relative to the bulk of the peptides. Phosphopeptides are further distinguished based upon hydrophilicity in the second dimension. This permits a restricted region of the plate to be directly interrogated for the presence of phosphopeptides by mass spectrometry (MS). Phosphopeptide analysis from the plates by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-MS and tandem MS enabled peptide sequencing and identification.

Molecular dynamics simulations of multilayer films of polyelectrolytes and nanoparticles.

We performed molecular dynamics simulations of multilayer assemblies of flexible polyelectrolytes and nanoparticles. The film was constructed by sequential adsorption of oppositely charged polymers and nanoparticles in layer-by-layer fashion from dilute solutions. We have studied multilayer films assembled from oppositely charged polyelectrolytes, oppositely charged nanoparticles, and mixed films containing both nanoparticles and polyelectrolytes. For all studied systems, the multilayer assembly proceeds through surface overcharging after completion of each deposition step. There is almost linear growth in the surface coverage and film thickness. The multilayer films assembled from nanoparticles show better layer stratification but at the same time have higher film roughness than those assembled from flexible polyelectrolytes.

Molecular dynamics simulations of polyelectrolyte multilayering on a charged particle.

Molecular dynamics simulations of polyelectrolyte multilayering on a charged spherical particle revealed that the sequential adsorption of oppositely charged flexible polyelectrolytes proceeds with surface charge reversal and highlighted electrostatic interactions as the major driving force of layer deposition. Far from being completely immobilized, multilayers feature a constant surge of chain intermixing during the deposition process, consistent with experimental observations of extensive interlayer mixing in these films. The formation of multilayers as well as the extent of layer intermixing depends on the degree of polymerization of the polyelectrolyte chains and the fraction of charge on its backbone. The presence of ionic pairs between oppositely charged macromolecules forming layers seems to play an important role in stabilizing the multilayer film.

Molecular dynamics simulations of electrostatic layer-by-layer self-assembly.

Electrostatic assembly of multilayered thin films through sequential adsorption of polyions in layer-by-layer fashion utilizes the strong electrostatic attraction between oppositely charged molecules. We perform molecular dynamics simulations of multilayers of flexible polyelectrolytes around a charged spherical particle. Our simulations establish that the charge reversal after each deposition step is a crucial factor for the steady layer growth. The multilayers appear to be nonequilibrium structures.

Ultrathin layered myoglobin-polyion films functional and stable at acidic pH values.

Cross-linking of myoglobin (Mb) promoted by 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide within films of polystyrene sulfonate after layer-by-layer self-assembly provided remarkable stabilization. Cross-linking greatly improved adhesion of the films to fused silica slides and allowed extensive optical studies over a wide pH range. Circular dichroism and visible absorbance spectra showed that Mb retained its native conformation when films were placed in solutions of pH as low as 2 and up to pH 11. Linear dichroism revealed an average orientation of the Mb iron heme cofactors of 58 degrees to the film normal. High concentrations of urea did denature the protein in the films, however. At pH 1, Mb in solution is fully unfolded but retained considerable alpha-helical content in the cross-linked films. Both the polyion film environment and cross-linking seem to play roles in stabilizing protein secondary structure and function at low pH. Cross-linked myoglobin-polyion films on pyrolytic graphite electrodes were used in strongly acidic solutions for the electrochemical catalytic reduction of trichloracetic acid, hydrogen peroxide, and oxygen. The pH-dependent catalytic reduction of trichloracetic acid was faster in 0.1 M HCl than in the medium pH range.