Dataset for analysis of metabolic pathways and their reversibility associated with anti-proliferative effect of metformin in liver cancer cells.

Despite epidemiological indications, utility of metformin in liver cancer remains debated and the understanding of the mechanism underlying its anti-cancer effects remains incomplete. Particularly, whether it operates via similar mechanism under glucose-sufficient and glucose- deficient environments or whether these effects are reversible remains unexplored. This metabolomic dataset was collected from liver cancer (HepG2) cells treated with metformin or placebo over a period of 3 h to 48 h as well as from cells recovering after metformin withdrawal. Cells were exposed to placebo or 2.5 mM metformin with or without glucose (5 mM) supplementation. The cells were harvested at 3, 6, 12, 24, and 48 h post-treatment. Cells were also harvested after 24 h of treatment under one of these conditions followed by reversal of glucose and/or metformin exposure status for 48 h. Metabolites from six biological replicates of each experimental group were extracted using chilled monophasic metabolite extraction solvent (Water: Acetonitrile: Isopropanol= 2:3:3) containing homovanillic acid as an internal standard. Samples were derivatized using MOX reagent followed by MSTFA. Untargeted metabolomic profiling of derivatized samples were performed using an Agilent 7890B gas chromatograph coupled to a 5977B single quadrupole mass spectrometer. Analytes were injected through a splitless liner and separated on a HP-5MS ultra-inert column using ultrapure helium as the carrier gas. Peak alignment, annotation, and integration were done using Agilent MassHunter Quantitative analysis software. Multivariate analysis was performed using MetaboAnalyst 5.0. These experiments were performed to unravel the longitudinal evolution of cellular metabolome in response to metformin treatment, its glucose dependence, as well as to examine the reversibility of these changes. The dataset can help to identify glucose-independent pathways involved in anti-cancer effect of metformin. The dataset can be used to design experiments to develop novel therapeutic combinations synergistically acting with metformin to cripple the metabolic fitness of cancer cells. It can also help to develop experiments to test the effect of metformin withdrawal in liver cancer.

Protocol for analyzing energy metabolic pathway dependency in human liver cancer cell lines.

Cellular energy metabolism analysis is complex, expensive, and indirect. We present a protocol to analyze relative contribution of metabolic pathways to ATP production by directly measuring ATP levels. We describe steps for cell counting and seeding in 96-well plate, treating with metformin, and systematic inhibition with metabolic inhibitors. We then detail procedures for a viability and ATP assay and calculating energy metabolism dependency. This high-throughput and accessible protocol works with any cell line and allows for flexible perturbation studies.

Identification of glucose-independent and reversible metabolic pathways associated with anti-proliferative effect of metformin in liver cancer cells.

INTRODUCTION: Despite the ability of cancer cells to survive glucose deprivation, most studies on anti-cancer effect of metformin explored its impact on glucose metabolism. No study ever examined whether its anti-cancer effect is reversible. Existing evidences warrant understanding of glucose-independent non-cytotoxic anti-proliferative effect of metformin to rationalize its role in liver cancer. OBJECTIVES: Characterization of glucose-independent anti-proliferative metabolic effects of metformin as well as analysis of their reversibility in liver cancer cells. METHODOLOGY: The dose-dependent effects of metformin on HepG2 cells were examined in presence and absence of glucose. The longitudinal evolution of metabolome was analyzed along with gene and protein expression as well as their correlations with and reversibility of cellular phenotype and metabolic signatures. RESULTS: Metformin concentrations up to 2.5 mM were found to be anti-proliferative irrespective of presence of glucose without significant increase in cytotoxicity. Apart from mitochondrial impairment, derangement of fatty acid desaturation, one-carbon, glutathione, and polyamine metabolism were associated with metformin treatment irrespective of glucose supplementation. Depletion of pantothenic acid, downregulation of essential amino acid uptake and metabolism alongside purine salvage were identified as novel glucose-independent effects of metformin. These were significantly correlated with cMyc expression and reduction in proliferation. Rescue experiments established reversibility upon metformin withdrawal and tight association between proliferation, metabotype, and cMyc expression. CONCLUSIONS: The derangement of multiple glucose-independent metabolic pathways, which are often upregulated in therapy-resistant cancer, and concomitant cMyc downregulation coordinately contribute to the anti-proliferative effect of metformin in liver cancer cells. These are reversible and may influence its therapeutic utility.

Effect of short-term use of FFP2 (N95) masks on the salivary metabolome of young healthy volunteers: a pilot study.

The use of face masks has become an integral part of public life in the post-pandemic era. However, the understanding of the effect of wearing masks on physiology remains incomplete and is required for informing public health policies. For the first time, we report the effects of wearing FFP2 masks on the metabolic composition of saliva, a proximal matrix to breath, along with cardiopulmonary parameters. Un-induced saliva was collected from young (31.2 ± 6.3 years) healthy volunteers (n = 10) before and after wearing FFP2 (N95) masks for 30 minutes and analyzed using GCMS. The results showed that such short-term mask use did not cause any significant change in heart rate, pulse rate or SpO2. Three independent data normalization approaches were used to analyze the changes in metabolomic signature. The individuality of the overall salivary metabotype was found to be unaffected by mask use. However, a trend of an increase in the salivary abundance of L-fucose, 5-aminovaleric acid, putrescine and phloretic acid was indicated irrespective of the method of data normalization. Quantitative analysis confirmed increases in concentrations of these metabolites in saliva of paired samples amid high inter-individual variability. The results showed that while there was no significant change in measured physiological parameters and individual salivary metabotypes, mask use was associated with correlated changes in these metabolites plausibly originating from altered microbial metabolic activity. These results might also explain the change in odour perception reported to be associated with mask use. Potential implications of these changes on mucosal health and immunity warrants further investigation to evolve more prudent mask use policies.

Thiourea mediated ROS-metabolites reprogramming restores root system architecture under arsenic stress in rice.

Arsenic (As) is a ubiquitous carcinogenic metalloid that enters into human food chain, through rice consumption. To unravel the conundrum of oxidative vs. reductive stress, the differential root-system architecture (RSA) was studied under As (a ROS producer) and thiourea (TU; a ROS scavenger) alone treatments, which indicated 0.80- and 0.74-fold reduction in the number of lateral roots (NLR), respectively compared with those of control. In case of As+TU treatment, NLR was increased by 4.35-fold compared with those of As-stress, which coincided with partial restoration of redox-status and auxin transport towards the root-tip. The expression levels of 16 ROS related genes, including RBOHC, UPB-1 C, SHR1, PUCHI, were quantified which provided the molecular fingerprint, in accordance with endogenous ROS signature. LC-MS based untargeted and targeted metabolomics data revealed that As-induced oxidative stress was metabolically more challenging than TU alone-induced reductive stress. Cis/trans-ferruloyl putrescine and γ-glutamyl leucine were identified as novel As-responsive metabolites whose levels were decreased and increased, respectively under As+TU than As-treated roots. In addition, the overall amino acid accumulation was increased in As+TU than As-treated roots, indicating the improved nutritional availability. Thus, the study revealed dynamic interplay between "ROS-metabolites-RSA", to the broader context of TU-mediated amelioration of As-stress in rice.

Characterizations of SARS-CoV-2 mutational profile, spike protein stability and viral transmission.

The recent pandemic of SARS-CoV-2 infection has affected more than 3.0 million people worldwide with more than 200 thousand reported deaths. The SARS-CoV-2 genome has the capability of gaining rapid mutations as the virus spreads. Whole-genome sequencing data offers a wide range of opportunities to study mutation dynamics. The advantage of an increasing amount of whole-genome sequence data of SARS-CoV-2 intrigued us to explore the mutation profile across the genome, to check the genome diversity, and to investigate the implications of those mutations in protein stability and viral transmission. We have identified frequently mutated residues by aligning ~660 SARS-CoV-2 genomes and validated in 10,000 datasets available in GISAID Nextstrain. We further evaluated the potential of these frequently mutated residues in protein structure stability of spike glycoprotein and their possible functional consequences in other proteins. Among the 11 genes, surface glycoprotein, nucleocapsid, ORF1ab, and ORF8 showed frequent mutations, while envelop, membrane, ORF6, ORF7a and ORF7b showed conservation in terms of amino acid substitutions. Combined analysis with the frequently mutated residues identified 20 viral variants, among which 12 specific combinations comprised more than 97% of the isolates considered for the analysis. Some of the mutations across different proteins showed co-occurrences, suggesting their structural and/or functional interaction among different SARS-COV-2 proteins, and their involvement in adaptability and viral transmission. Analysis of protein structure stability of surface glycoprotein mutants indicated the viability of specific variants and are more prone to be temporally and spatially distributed across the globe. A similar empirical analysis of other proteins indicated the existence of important functional implications of several variants. Identification of frequently mutated variants among COVID-19 patients might be useful for better clinical management, contact tracing, and containment of the disease.

Lipidomic Analysis of Cancer Cell and Tumor Tissues.

Due to their role in cellular structure, energetics, and signaling, characterization of changes in cellular and extracellular lipid composition is of key importance to understand cancer biology. In addition, several mass spectrometry-based profiling as well as imaging studies have indicated that lipid molecules may be useful to augment existing biochemical and histopathological methods for diagnosis, staging, and prognosis of cancer. Therefore, analysis of lipidomic changes associated with cancer cells and tumor tissues can be useful for both fundamental and translational studies. Here, we provide a high-throughput single-extraction-based method that can be used for simultaneous lipidomic and metabolomic analysis of cancer cells or healthy or tumor tissue samples. In this chapter, a modified Bligh-Dyer method is described for extraction of lipids followed by analysis of fatty acid composition by gas chromatography-mass spectrometry (GC-MS) or untargeted lipidomics using electrospray ionization mass spectrometry (ESIMS) coupled with reverse-phase (RP) ultraperformance liquid chromatography (UPLC) followed by multivariate data analysis to identify features of interest.

Mass Spectrometry-Based Profiling of Metabolites in Human Biofluids.

Cancer poses a daunting challenge to researchers and clinicians alike. Early diagnosis, accurate prognosis, and prediction of therapeutic response remain elusive in most types of cancer. In addition, lacunae in our understanding of cancer biology continue to hinder advancement of therapeutic strategies. Metabolic reprogramming has been identified as integral to pathogenesis and progression of the disease. Consequently, analysis of biofluid metabolome has emerged as a promising approach to further our understanding of disease biology as well as to identify cancer biomarkers. However, unbiased identification of robust and meaningful differences in metabolic signatures remains a non-trivial task. This chapter describes a generalized strategy for global metabolic profiling of human biofluids using ultra-performance liquid chromatography (UPLC) and mass spectrometry, which together offer a sensitive, high-throughput, and versatile platform. A step-by-step protocol for performing untargeted metabolic profiling of urine and serum (or plasma), using hydrophilic interaction liquid chromatography (HILIC) or reverse-phase (RP) chromatography coupled with electrospray ionization mass spectrometry (ESI-MS) to multivariate data analysis and identification of metabolites of interest has been detailed.

Assessment of Metabolic Signature for Cancer Diagnosis Using Desorption Electrospray Ionization Mass Spectrometric Imaging.

Metabolic reprogramming is a hallmark of tumor development. A technique that can map this complex biochemical shift by taking a snapshot of various metabolites in a tissue specimen (biopsy) is of high utility in the context of cancer diagnosis. Desorption electrospray ionization mass spectrometric imaging (DESI-MSI) is such a powerful and emerging analytical technique to simultaneously visualize the distributions of hundreds of metabolites, lipids, and other small molecules in the biological tissue. In DESI-MSI, a fine spray of high-velocity charged microdroplets rapidly extracts molecular species from the tissue surface and subsequently transfers them to the mass spectrometer, while the sample is continuously moved in two dimensions under the impinging spray of microdroplets. This allows a detailed multiplex molecular mapping of the tissue. DESI-MSI enables simultaneous examination of hundreds of putative metabolic biomarkers, an approach that lends much more predictive power than simply evaluating one or a few candidate biomarkers. The speed, versatility, lack of complicated sample preparation, and operation at ambient conditions make DESI-MSI extremely promising as a rapid diagnostic and prognostic tool.

Inverse association of vitamin D3 levels with lung cancer mediated by genetic variation.

Vitamin D is an essential micronutrient required for normal physiological function and recognized for its role regulating calcium metabolism. Recent work is beginning to emerge demonstrating a role for vitamin D in chronic illnesses, such as cancer. Circulating serum levels of 25(OH)D2/3 were quantitatively measured using sensitive ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) in 406 lung cancer cases and 437 population controls, while 1,25(OH)2 D2/3 levels were measured in a subset of 90 cases and 104 controls using the same method, from the NCI-MD case-control cohort. 25(OH)D3 levels were inversely associated with lung cancer status across quartiles (Q2 vs. Q1: ORadjusted  = 0.5, 95% CI = 0.3-0.8; Q3 vs. Q1: ORadjusted  = 0.5, 95% CI = 0.3-0.8; Q4 vs. Q1: ORadjusted  = 0.5, 95% CI = 0.2-0.9; Ptrend  = 0.004). Levels of 1,25(OH)2 D3 were also inversely associated with lung cancer status (Q2 vs. Q1: ORadjusted  = 0.2, 95% CI = 0.03-0.7; Q3 vs. Q1: ORadjusted  = 0.1, 95% CI = 0.01-0.4; Q4 vs. Q1: ORadjusted  = 0.04, 95% CI = 0.01-0.3; Ptrend <0.0001). Although the observed trends were similar for the 25(OH)D2 (Ptrend  = 0.08), no significant associations were seen between vitamin D2 and lung cancer status. Additionally, genotyping of 296 SNPs in the same subjects resulted in findings that 27 SNPs, predominantly in CYP24A1 and VDR genes, were significantly associated with lung cancer status, affected mRNA expression, and modulated vitamin D levels. These findings suggest a protective role for vitamin D3 in lung cancer, with similar trends but insignificant findings for D2 . Vitamin D3 levels appeared to be modulated by genetic variation in CYP24A1 and VDR genes. Additional research to illuminate the mechanism(s) through which vitamin D exacerbates effects against lung carcinogenesis is warranted.

Tuning the substrate specificity by engineering the active site of cytochrome P450cam: a rational approach.

Rational design of the active site of cytochrome P450cam has been carried out to catalyse oxygenation of various potentially important chemical reactions. The modeling studies showed that the distal pocket of the heme consisting of the Y96, T101, F87 and L244 residues could be suitably mutated to change the substrate specificity of the enzyme. We found that the mutant enzymes could catalyse oxygenation of indole to produce indigo. While Y96F was found to be several times better as a catalyst for conversion of indole to indigo, the double mutant Y96F/L244A showed the highest NADH oxidation rate as well as yield of indigo. The oxidative catalysis using H(2)O(2) as the oxygen source was found to produce a higher purity of indigo, and lesser or no formation of indirubin was detected. The enzymatic oxygenation of aromatic hydrocarbons such as coumarin and analogues was also found to be enhanced on mutation of Y96 and L244 residues in the enzyme. The studies also showed that mutation of suitable residues can alter the regio-selectivity of hydroxylation of the aromatic hydrocarbons.

Role of threonine 101 on the stability of the heme active site of cytochrome P450cam: multiwavelength circular dichroism studies.

The role of the threonine 101 residue that resides close to the heme propionic acid side chain of cytochrome P450cam on the conformational properties of the active site of the enzyme has been investigated by circular dichroism (CD) spectroscopy. Site-specific mutation of the threonine by valine has been carried out that does not affect the size of the residue but significantly alters the hydropathy index. The T101V mutant of cytochrome P450cam showed distinct differences in the CD spectra near the heme region, indicating a subtle effect of the mutation on the properties of the heme active site. Thermal stabilities of the mutant and wild-type enzyme have been studied by temperature dependence of the ellipticity (intensity of the CD band) in the far-UV region for the secondary structure and at different wavelengths in the visible region that arise from the heme moiety for the tertiary structure around the prosthetic group. The thermal unfolding data from variations of the CD intensity at different wavelengths were analyzed using a generalized multistep unfolding model, and two distinct equilibrium intermediate conformational states of the enzyme were identified. The mutation of the T101 residue by valine was found to decrease the thermal stability of both the intermediates in the presence of the substrate. On the other hand, this mutation had no apparent effect on the thermal stability of the enzyme in the absence of the substrate. These results suggested that the threonine residue stabilizes the protein cavity around the heme center in the case of the substrate-bound species, possibly by hydrogen bonding with one of the propionate side chains of the heme moiety. Such hydrogen bonding of the heme propionate with threonine is absent in the substrate-free form of the enzyme.