Interplay of Vitamin D and CYP3A4 Polymorphisms in Endocrine Disorders and Cancer.

Vitamin D has received considerable optimistic attention as a potentially important factor in many pathological states over the past few decades. However, the proportion of the active form of vitamin D metabolites responsible for biological activity is highly questionable in disease states due to flexible alterations in the enzymes responsible for their metabolism. For instance, CYP3A4 plays a crucial role in the biotransformation of vitamin D and other drug substances. Food-drug and/or drug-drug interactions, the disease state, genetic polymorphism, age, sex, diet, and environmental factors all influence CYP3A4 activity. Genetic polymorphisms in CYP450-encoding genes have received considerable attention in the past few decades due to their extensive impact on the pharmacokinetic and dynamic properties of drugs and endogenous substances. In this review, we focused on CYP3A4 polymorphisms and their interplay with vitamin D metabolism and summarized the role of vitamin D in calcium homeostasis, bone diseases, diabetes, cancer, other diseases, and drug substances. We also reviewed clinical observations pertaining to CYP3A4 polymorphisms among the aforementioned disease conditions. In addition, we highlighted the future perspectives of studying the pharmacogenetics of CYP3A4, which may have potential clinical significance for developing novel diagnostic genetic markers that will ascertain disease risk and progression.

Sequential lipidomic, metabolomic, and proteomic analyses of serum, liver, and heart tissue specimens from peroxisomal biogenesis factor 11α knockout mice.

Peroxisomes are versatile single membrane-enclosed cytoplasmic organelles, involved in reactive oxygen species (ROS) and lipid metabolism and diverse other metabolic processes. Peroxisomal disorders result from mutations in Pex genes-encoded proteins named peroxins (PEX proteins) and single peroxisomal enzyme deficiencies. The PEX11 protein family (α, ß, and γ isoforms) plays an important role in peroxisomal proliferation and fission. However, their specific functions and the metabolic impact caused by their deficiencies have not been precisely characterized. To understand the systemic molecular alterations caused by peroxisomal defects, here we utilized untreated peroxisomal biogenesis factor 11α knockout (Pex11α KO) mouse model and performed serial relative-quantitative lipidomic, metabolomic, and proteomic analyses of serum, liver, and heart tissue homogenates. We demonstrated significant specific changes in the abundances of multiple lipid species, polar metabolites, and proteins and dysregulated metabolic pathways in distinct biological specimens of the Pex11α KO adult mice in comparison to the wild type (WT) controls. Overall, the present study reports comprehensive semi-quantitative molecular omics information of the Pex11α KO mice, which might serve in the future as a reference for a better understanding of the roles of Pex11α and underlying pathophysiological mechanisms of peroxisomal biogenesis disorders.

Integrating Top-Down and Bottom-Up Mass Spectrometric Strategies for Proteomic Profiling of Iranian Saw-Scaled Viper, Echis carinatus sochureki, Venom.

Saw-scaled or carpet vipers (genus Echis) are considered to cause a higher global snakebite mortality than any other snake. Echis carinatus sochureki (ECS) is a widely distributed snake species, also found across the thirteen provinces of Iran, where it is assumed to be responsible for the most snakebite envenomings. Here, we collected the Iranian specimens of ECS from three different geographically distinct populations, investigated food habits, and performed toxicity assessment and venom proteome profiling to better understand saw-scaled viper life. Our results show that the prey items most commonly found in all populations were arthropods, with scorpions from the family Buthidae particularly well represented. LD50 (median lethal dose) values of the crude venom demonstrate highly comparable venom toxicities in mammals. Consistent with this finding, venom characterization via top-down and bottom-up proteomics, applied to both crude venoms and size-exclusion chromatographic fractions, revealed highly comparable venom compositions among the different populations. By combining all proteomics data, we identified 22 protein families from 102 liquid chromatography and tandem mass spectrometry (LC-MS/MS) raw files, including the most abundant snake venom metalloproteinases (SVMPs, 29-34%); phospholipase A2 (PLA2s, 26-31%); snake venom serine proteinases (SVSPs, 11-12%); l-amino acid oxidases (LAOs, 8-11%), C-type lectins/lectin-like (CTLs, 7-9%) protein families, and many newly detected ones, e.g., renin-like aspartic proteases (RLAPs), fibroblast growth factors (FGFs), peptidyl-prolyl cis-trans isomerases (PPIs), and venom vasodilator peptides (VVPs). Furthermore, we identified and characterized methylated, acetylated, and oxidized proteoforms relating to the PLA2 and disintegrin toxin families and the site of their modifications. It thus seems that post-translational modifications (PTMs) of toxins, particularly target lysine residues, may play an essential role in the structural and functional properties of venom proteins and might be able to influence the therapeutic response of antivenoms, to be investigated in future studies.

High-resolution atmospheric-pressure MALDI mass spectrometry imaging workflow for lipidomic analysis of late fetal mouse lungs.

Mass spectrometry imaging (MSI) provides label-free, non-targeted molecular and spatial information of the biomolecules within tissue. Lipids play important roles in lung biology, e.g. as surfactant, preventing alveolar collapse during normal and forced respiration. Lipidomic characterization of late fetal mouse lungs at day 19 of gestation (E19) has not been performed yet. In this study we employed high-resolution atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionization MSI for the lipidomic analysis of E19 mouse lungs. Molecular species of different lipid classes were imaged in E19 lung sections at high spatial and mass resolution in positive- and negative-ion mode. Lipid species were characterized based on accurate mass and on-tissue tandem mass spectrometry. In addition, a dedicated sample preparation protocol, homogenous deposition of matrices on tissue surfaces and data processing parameters were optimized for the comparison of signal intensities of lipids between different tissue sections of E19 lungs of wild type and Pex11ß-knockout mice. Our study provides lipid information of E19 mouse lungs, optimized experimental and data processing strategies for the direct comparison of signal intensities of metabolites (lipids) among the tissue sections from MSI experiments. To best of our knowledge, this is the first MSI and lipidomic study of E19 mouse lungs.

A perspective view of top-down proteomics in snake venom research.

The venom produced by snakes contains complex mixtures of pharmacologically active proteins and peptides which play a crucial role in the pathophysiology of snakebite diseases. The deep understanding of venom proteomes can help to improve the treatment of this "neglected tropical disease" (as expressed by the World Health Organization [WHO]) and to develop new drugs. The most widely used technique for venom analysis is liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based bottom-up (BU) proteomics. Considering the fact that multiple multi-locus gene families encode snake venom proteins, the major challenge for the BU proteomics is the limited sequence coverage and also the "protein inference problem" which result in a loss of information for the identification and characterization of toxin proteoforms (genetic variation, alternative mRNA splicing, single nucleotide polymorphism [SNP] and post-translational modifications [PTMs]). In contrast, intact protein measurements with top-down (TD) MS strategies cover almost complete protein sequences, and prove the ability to identify venom proteoforms and to localize their modifications and sequence variations.

Quantitative lipidomic analysis of mouse lung during postnatal development by electrospray ionization tandem mass spectrometry.

Lipids play very important roles in lung biology, mainly reducing the alveolar surface tension at the air-liquid interface thereby preventing end-expiratory collapse of the alveoli. In the present study we performed an extensive quantitative lipidomic analysis of mouse lung to provide the i) total lipid quantity, ii) distribution pattern of the major lipid classes, iii) composition of individual lipid species and iv) glycerophospholipid distribution pattern according to carbon chain length (total number of carbon atoms) and degree of unsaturation (total number of double bonds). We analysed and quantified 160 glycerophospholipid species, 24 sphingolipid species, 18 cholesteryl esters and cholesterol from lungs of a) newborn (P1), b) 15-day-old (P15) and c) 12-week-old adult mice (P84) to understand the changes occurring during postnatal pulmonary development. Our results revealed an increase in total lipid quantity, correlation of lipid class distribution in lung tissue and significant changes in the individual lipid species composition during postnatal lung development. Interestingly, we observed significant stage-specific alterations during this process. Especially, P1 lungs showed high content of monounsaturated lipid species; P15 lungs exhibited myristic and palmitic acid containing lipid species, whereas adult lungs were enriched with polyunsaturated lipid species. Taken together, our study provides an extensive quantitative lipidome of the postnatal mouse lung development, which may serve as a reference for a better understanding of lipid alterations and their functions in lung development and respiratory diseases associated with lipids.

Analysis of ketone-based neurosteroids by reactive low-temperature plasma mass spectrometry.

RATIONALE: Neurosteroids are important signalling molecules that modulate neuronal activity. Their low concentrations and low volatility make neurosteroid detection and quantification by ambient mass spectrometry challenging. Here we develop a reactive low-temperature plasma mass spectrometry (LTP-MS) method and demonstrate its potential for fast screening and quantification of neurosteroids in mouse brain. METHODS: Ketone-based neurosteroids were analysed with the LTP-MS method. The plasma of the LTP was heated in order to improve the desorption efficiency of low-volatility neurosteroids. Methylamine with a concentration of 500 ppbv was employed as the reactive reagent. Neurosteroids in mouse brain tissue extracts were detected in 70 s with mass errors less than ±3 ppm due to coupling of the ion source with a high-performance mass spectrometer. RESULTS: Reaction between neurosteroids and methylamine, seeded into the LTP gas stream, resulted in the formation of protonated methylamine-neurosteroid adducts with 5- to 100-fold abundances, compared to [M + H]+ ions detected in non-reactive LTP-MS. The lowest detectable concentrations of neurosteroid standards were in the range of ng/mL. Concentrations of neurosteroids in male and female mouse brain extracts as determined with reactive LTP-MS were on the level of ng/g, comparable to results obtained with high-performance liquid chromatography-tandem mass spectrometry. CONCLUSIONS: The developed reactive LTP-MS is capable of providing sensitive identification and quantification of ketone-based neurosteroids in mouse brain extracts with minimal sample treatment, and showcases the potential of reactive LTP-MS as a tool for fast screening of neurosteroid levels in brain.

A New Immunomodulatory Role for Peroxisomes in Macrophages Activated by the TLR4 Ligand Lipopolysaccharide.

Peroxisomes are proposed to play an important role in the regulation of systemic inflammation; however, the functional role of these organelles in inflammatory responses of myeloid immune cells is largely unknown. In this article, we demonstrate that the nonclassical peroxisome proliferator 4-phenyl butyric acid is an efficient inducer of peroxisomes in various models of murine macrophages, such as primary alveolar and peritoneal macrophages and the macrophage cell line RAW264.7, but not in primary bone marrow-derived macrophages. Further, proliferation of peroxisomes blocked the TLR4 ligand LPS-induced proinflammatory response, as detected by the reduced induction of the proinflammatory protein cyclooxygenase (COX)-2 and the proinflammatory cytokines TNF-α, IL-6, and IL-12. In contrast, disturbing peroxisome function by knockdown of peroxisomal gene Pex14 or Mfp2 markedly increased the LPS-dependent upregulation of the proinflammatory proteins COX-2 and TNF-α. Specifically, induction of peroxisomes did not affect the upregulation of COX-2 at the mRNA level, but it reduced the half-life of COX-2 protein, which was restored by COX-2 enzyme inhibitors but not by proteasomal and lysosomal inhibitors. Liquid chromatography-tandem mass spectrometry analysis revealed that various anti-inflammatory lipid mediators (e.g., docosahexaenoic acid) were increased in the conditioned medium from peroxisome-induced macrophages, which blocked LPS-induced COX-2 upregulation in naive RAW264.7 cells and human primary peripheral blood-derived macrophages. Importantly, LPS itself induced peroxisomes that correlated with the regulation of COX-2 during the late phase of LPS activation in macrophages. In conclusion, our findings identify a previously unidentified role for peroxisomes in macrophage inflammatory responses and suggest that peroxisomes are involved in the physiological cessation of macrophage activation.