Unraveling the Mechanisms of Ch-SeNP Cytotoxicity against Cancer Cells: Insights from Targeted and Untargeted Metabolomics.

Although chitosan-stabilized selenium nanoparticles (Ch-SeNPs) have emerged as a promising chemical form of selenium for anticancer purposes, gathering more profound knowledge related to molecular dysfunctions contributes significantly to the promotion of their evolution as a chemotherapeutic drug. In this sense, metabolites are the end products in the flow of gene expression and, thus, the most sensitive to changes in the physiological state of a biological system. Therefore, metabolomics provides a functional readout of the biochemical activity and cell state. In the present study, we evaluated alterations in the metabolomes of HepG2 cells after the exposure to Ch-SeNPs to elucidate the biomolecular mechanisms involved in their therapeutic effect. A targeted metabolomic approach was conducted to evaluate the levels of four of the main energy-related metabolites (adenosine triphosphate (ATP); adenosine diphosphate (ADP); nicotinamide adenine dinucleotide (NAD+); and 1,4-dihydronicotinamide adenine dinucleotide (NADH)), revealing alterations as a result of exposure to Ch-SeNPs related to a shortage in the energy supply system in the cell. In addition, an untargeted metabolomic experiment was performed, which allowed for the study of alterations in the global metabolic profile as a consequence of Ch-SeNP exposure. The results indicate that the TCA cycle and glycolytic pathways were impaired, while alternative pathways such as glutaminolysis and cysteine metabolism were upregulated. Additionally, increased fructose levels suggested the induction of hypoxia-like conditions. These findings highlight the potential of Ch-SeNPs to disrupt cancer cell metabolism and provide insights into the mechanisms underlying their antitumor effects.

Super-SILAC Quantitative Proteome Profiling of Zebrafish Larvae.

The super-SILAC approach enables the quantitative proteome profiling of highly complex samples such as biological tissues or whole organisms. In this approach, a super-SILAC mix consisting of heavy isotope-labeled cells representative of the tissue or organism to be analyzed is mixed with the unlabeled samples of interest, such that the labeled proteins act as a spike-in standard, thus allowing the relative quantification of proteins between the samples of interest. In this chapter, we thoroughly describe the protocol to carry out the super-SILAC approach using a common in vivo model such as zebrafish larvae.

Short-chain fatty acids during pregnancy in multiple sclerosis: A prospective cohort study.

BACKGROUND AND PURPOSE: Short-chain fatty acids (SCFAs) can have pro- or anti-inflammatory properties, but their relationship with multiple sclerosis (MS) relapses during pregnancy remains unknown. This study aimed to explore SCFA profiles in MS patients during pregnancy and to assess their association with the appearance of relapses during pregnancy and postpartum. METHODS: We prospectively included 53 pregnant MS patients and 21 healthy control women. Patients were evaluated during pregnancy and puerperium. SCFAs were measured by liquid chromatography-mass spectrometry. RESULTS: Sixteen patients (32%) had relapses during pregnancy or puerperium, and 37 (68%) did not. All MS patients showed significant increases in acetate levels during pregnancy and the postpartum period compared to non-MS women. However, propionate and butyrate values were associated with disease activity. Their values were higher in nonrelapsing patients and remained similar to the control group in relapsing patients. The variable that best identified active patients was the propionate/acetate ratio. Ratios of <0.36 during the first trimester were associated with higher inflammatory activity (odds ratio = 165, 95% confidence interval = 10.2-239.4, p < 0.01). Most nonrelapsing patients showed values of >0.36, which were similar to those in healthy pregnant women. CONCLUSIONS: Low propionate/acetate ratio values during the first trimester of gestation identified MS patients at risk of relapses during pregnancy and the postpartum period.

Integration of Transcriptomics and Metabolomics to Reveal the Molecular Mechanisms Underlying Rhodium Nanoparticles-Based Photodynamic Cancer Therapy.

Rhodium nanoparticles have recently been described as promising photosensitizers due to their low toxicity in the absence of near-infrared irradiation, but their high cytotoxicity when irradiated. Irradiation is usually carried out with a laser source, which allows the treatment to be localized in a specific area, thus avoiding undesirable side effects on healthy tissues. In this study, a multi-omics approach based on the combination of microarray-based transcriptomics and mass spectrometry-based untargeted and targeted metabolomics has provided a global picture of the molecular mechanisms underlying the anti-tumoral effect of rhodium nanoparticle-based photodynamic therapy. The results have shown the ability of these nanoparticles to promote apoptosis by suppressing or promoting anti- and pro-apoptotic factors, respectively, and by affecting the energy machinery of tumor cells, mainly blocking the ß-oxidation, which is reflected in the accumulation of free fatty acids and in the decrease in ATP, ADP and NAD+ levels.

Transcriptome Analysis Identifies Novel Mechanisms Associated with the Antitumor Effect of Chitosan-Stabilized Selenium Nanoparticles.

Selenium nanoparticles (SeNPs) have been receiving special attention in recent years due to their antioxidant capacity and antitumor properties. However, the mechanisms associated with these properties remain to be elucidated. For this reason, a global transcriptome analysis has been designed in this work and it was carried out using human hepatocarcinoma cells and chitosan-stabilized SeNPs (Ch-SeNPs) to identify new targets and pathways related to the antitumor mechanisms associated with Ch-SeNPs. The results obtained confirm the alteration of the cell cycle and the effect of Ch-SeNPs on different tumor suppressors and other molecules involved in key mechanisms related to cancer progression. Furthermore, we demonstrated the antioxidant properties of these nanoparticles and their capacity to induce senescence, which was further confirmed through the measurement of ß-galactosidase activity.

Herpesvirus Antibodies, Vitamin D and Short-Chain Fatty Acids: Their Correlation with Cell Subsets in Multiple Sclerosis Patients and Healthy Controls.

Although the etiology of multiple sclerosis (MS) is still unknown, it is commonly accepted that environmental factors could contribute to the disease. The objective of this study was to analyze the humoral response to Epstein-Barr virus, human herpesvirus 6A/B and cytomegalovirus, and the levels of 25-hydroxyvitamin D (25(OH)D) and the three main short-chain fatty acids (SCFA), propionate (PA), butyrate (BA) and acetate (AA), in MS patients and healthy controls (HC) to understand how they could contribute to the pathogenesis of the disease. With this purpose, we analyzed the correlations among them and with different clinical variables and a wide panel of cell subsets. We found statistically significant differences for most of the environmental factors analyzed when we compared MS patients and HC, supporting their possible involvement in the disease. The strongest correlations with the clinical variables and the cell subsets analyzed were found for 25(OH)D and SCFAs levels. A correlation was also found between 25(OH)D and PA/AA ratio, and the interaction between these factors negatively correlated with interleukin 17 (IL-17)-producing CD4+ and CD8+ T cells in untreated MS patients. Therapies that simultaneously increase vitamin D levels and modify the proportion of SCFA could be evaluated in the future.

Acetate correlates with disability and immune response in multiple sclerosis.

BACKGROUND: Gut microbiota has been related to multiple sclerosis (MS) etiopathogenesis. Short-chain fatty acids (SCFA) are compounds derived from microbial metabolism that have a role in gut-brain axis. OBJECTIVES: To analyse SCFA levels in plasma of MS patients and healthy donors (HD), and the possible link between these levels and both clinical data and immune cell populations. METHODS: Ninety-five MS patients and 54 HD were recruited. Patients were selected according to their score in the Expanded Disability Status Scale (EDSS) (49 EDSS ≤ 1.5, 46 EDSS ≥ 5.0). SCFA were studied in plasma samples by liquid chromatography-mass spectrometry. Peripheral blood mononuclear cells were studied by flow cytometry. Gender, age, treatments, EDSS and Multiple Sclerosis Severity Score (MSSS) were evaluated at the recruitment. RESULTS: Plasma acetate levels were higher in patients than in HD (p = 0.003). Patients with EDSS ≥ 5.0 had higher acetate levels than those with EDSS≤ 1.5 (p = 0.029), and HD (p = 2.97e-4). Acetate levels correlated with EDSS (r = 0.387; p = 1.08e-4) and MSSS (r = 0.265; p = 0.011). In untreated MS patients, acetate levels correlated inversely with CD4+ naïve T cells (r =  - 0.550, p = 0.001) and directly with CD8+ IL-17+ cells (r = 0.557; p = 0.001). CONCLUSIONS: Plasma acetate levels are higher in MS patients than in HD. In MS there exists a correlation between plasma acetate levels, EDSS and increased IL-17+ T cells. Future studies will elucidate the role of SCFA in the disease.

Integration of untargeted and targeted mass spectrometry-based metabolomics provides novel insights into the potential toxicity associated to surfynol.

The intake of toxic compounds through the diet as a result of migration processes from food packaging is of increasing concern. It has been shown that the surfactant commercially known as surfynol, which is commonly used in food-contact materials, is capable of migrating from multilayer containers into the food, reaching potentially harmful concentration levels. In the present study, the integration of an untargeted and a targeted metabolomics approach has been carried out using NTERA-2 germinal cells as in-vitro model, to make further progress in elucidating the molecular mechanisms associated with the toxicity of surfynol. This study has allowed the identification of different altered metabolites mainly related with energy-acquiring, cell development and cellular defense mechanisms. While glutamine, L-threonine, propanoate, octadecanoate and carbamate were found at higher concentrations in cells exposed tu surfynol, L-valine, oxalate, phosphate, phenylalanine and myoinositol were found inhibited. Additionally, concentrations of ATP, ADP and NAD+ were found significantly inhibited, supporting the idea that surfynol induces glycolysis inactivation. The results obtained strengthen the evidence of the toxicity associated to surfynol; therefore, reinforcing the need for a more comprehensive study on the viability of its use in food packaging.

Rhodium Nanoparticles as a Novel Photosensitizing Agent in Photodynamic Therapy against Cancer.

Photodynamic therapy (PDT) is a promising alternative treatment for different types of cancer due to its high selectivity, which prevents healthy tissues from being damaged. The use of nanomaterials in PDT has several advantages over classical photosensitizing agents, due to their unique properties and their capacity for functionalization. Especially interesting is the use of metallic nanoparticles, which are capable of absorbing electromagnetic radiation and either transferring this energy to oxygen molecules for the generation of reactive oxygen species (ROS) or dissipating it as heat. Although previous reports have demonstrated the capacity of Rh derivatives to serve as anti-tumor drugs, to the best of our knowledge there have been no studies on the potential use of small-sized Rh nanoparticles as photosensitizers in PDT. In this study, 5 nm Rh nanoparticles have been synthesized and their potential in PDT has been evaluated. The results show that treatment with Rh nanoparticles followed by NIR irradiation induces apoptosis in cancer cells through a p53-independent mechanism.

Strategies for Membrane Protein Analysis by Mass Spectrometry.

Membrane proteins are of utmost importance in different cellular processes including: cell signaling, substrate transport, homeostasis control, immune surveillance, etc. In addition, they represent between 60% and 70% of the therapeutic targets currently used. Therefore, the identification and characterization of these proteins is crucial in many fields of research. Although proteomics has undergone an extraordinary advance in recent years thanks to the development of mass spectrometry, the methods used for the identification and quantification of soluble proteins generally fail to be used for membrane proteins, mainly due to their hydrophobic character.In this chapter, we revised the different alternatives, modifications and improvements that have been developed over the years with the aim of adapting the methods used in proteomics to the particular study of membrane proteins, thus allowing to increase the number of membrane proteins identified, as well as their coverage.

Pharmacokinetics and disposition of miltefosine in healthy mice and hamsters experimentally infected with Leishmania infantum.

Miltefosine is the only currently available oral drug for treatment of leishmaniasis. However, information on the pharmacokinetics (PK) of miltefosine is relatively scarce in animals. PK parameters and disposition of the molecule was determined in healthy NMRI mice and Syrian hamsters infected and treated with different miltefosine doses and regimens. Long half-life of the molecule was confirmed and differential pattern of accumulation of the drug was observed in analyzed organs in mice and hamster. Long treatment schedules produced miltefosine levels over IC50 value against L. infantum intracellular amastigotes for at least 24 days in spleen and liver of infected hamsters. The observed differential pattern of organ accumulation of the drug in mice and hamster supports the relevance of both species for translational research on chemotherapy of leishmaniasis.

A common surfactant used in food packaging found to be toxic for reproduction in mammals.

Migration from a multilayer plastic material intended for food contact showed that 2,4,7,9-tetramethyl-5-decyne-4,7-diol mixture (surfynol), used as a surfactant in the adhesive employed to build the multilayer, was transferred to water and other food simulants in contact with the plastic. When these multilayer plastics were used for containing seminal doses for artificial insemination, it was found that fertility was seriously damaged in terms of motility, acrosome integrity, mitochondrial activity and penetration capacity in the cells, thus affecting male fertility. Quantitative proteomic analysis of exposed germinal cells demonstrated the inhibition of key proteins involved in the fertilization capacity by affecting the cytoskeleton, sperm motility, the energy machinery and sperm defense mechanisms against oxidation, therefore confirming the surfactant-induced male infertility. These results open up new and interesting perspectives for the study of reprotoxicity caused by different chemicals common in our daily lives. SIGNIFICANCE: This paper demonstrates the toxicity for reproduction of a common surfactant used in food packaging and the scientific reasons why the sperm loses reproductive capacity in presence of this chemical. So, the surfactant affects the male fertility. The surfactant is present in many adhesives used either for building multilayer materials or to glue paper and plastic in food packaging. This is the first time that reprotoxicity is demonstrated for this compound. According to the theoretical approach Threshold of Toxicological Concern (TTC) the compound is highly toxic but experimental data did not exist so far. The study described in this paper and the results obtained open a door to further research in which male infertility caused by chemicals could be demonstrated.

Proteomic evaluation of mouse adipose tissue and liver following hydroxytyrosol supplementation.

BACKGROUND AND AIMS: Hydroxytyrosol (HT) is the primary phenolic compound of olives, virgin olive oil, and their byproducts. Proteomic analysis of metabolically active tissues helps elucidating novel mechanisms of action and potential targets in cardiometabolic disease. Thus, we aimed at determining the impact of long-term HT supplementation on the proteome of adipose and liver tissue, in mice. METHODS: C57BL/6J mice received either a control diet or a diet supplemented with nutritionally relevant doses of HT for eight weeks. RESULTS: HT supplementation differentially affects the adipose and liver tissues proteome, as evaluated by super-SILAC. Some oxidative stress-related proteins were modulated in both tissues, such as the multifunctional protein peroxiredoxin 1, which was consistently repressed by HT supplementation. In some cases tissue-dependent modulation was observed, as in the case of FASN. CONCLUSIONS: This study provides interesting information on the connection between changes seen at tissue proteome level and the metabolic effects of HT. The use of this pertinent proteomics quantification approach may prove quite useful for uncovering novel potential pharmaco-nutritional targets of HT supplementation.

A quantitative proteomic approach for unveiling novel mechanisms associated with MeHg-induced toxicity: effects on the methylation cycle.

Methylmercury (MeHg) is still a major threat for human health and the environment due to its extremely high toxicity that mainly affects the nervous system. Despite the great efforts made during the last few decades, the specific molecular mechanisms involved in MeHg-induced toxicity are still not completely unveiled. In this work we explored such mechanisms using neuroblastoma cells (Neuro-2a) and SILAC as a quantitative proteomic approach. We found that exposure of Neuro-2a cells to 2 mg L-1 MeHg for 8 h decreased the cell viability to 70% and caused significant changes in the morphology of the cells, specially regarding neurite development. Our proteomic results showed different proteins altered upon MeHg exposure that helped to identify pathways related to the toxicity exerted by MeHg. Specifically, we have found that MeHg affects the methylation cycle by inhibiting the expression of key enzymes including MTHFD1 and MTR. Moreover, we demonstrate that inhibition of MTHFD1 is not observed when exposing the cells to inorganic Hg and other heavy metals such as Pb or Cu. Thus, this work sets the stage for dissecting a specific molecular mechanism for MeHg-induced toxicity.

Sample preparation strategies for improving the identification of membrane proteins by mass spectrometry.

Despite enormous advances in the mass spectrometry and proteomics fields during the last two decades, the analysis of membrane proteins still remains a challenge for the proteomic community. Membrane proteins play a wide number of key roles in several cellular events, making them relevant target molecules to study in a significant variety of investigations (e.g., cellular signaling, immune surveillance, drug targets, vaccine candidates, etc.). Here, we critically review the several attempts that have been carried out on the different steps of the sample preparation procedure to improve and modify existing conventional proteomic strategies in order to make them suitable for the study of membrane proteins. We also revise novel techniques that have been designed to tackle the difficult but relevant task of identifying and characterizing membrane proteins.