Liquid Chromatography-tandem Mass Spectrometry Technology Research Articles

Sulfur-containing amino acids and risk of schizophrenia

BackgroundSchizophrenia is a chronic and complex severe psychiatric disorder. Male and female are different in their risks for schizophrenia for the biologic and sociocultural reasons. Homocysteine (Hcy), Cysteine (Cys), and methionine (Met) play important roles in metabolism, and the three amino acids may also be involved in pathogenesis of schizophrenia. ObjectiveThis study aimed to test the associations between sulfur-containing amino acid blood levels and risk of schizophrenia, evaluating the different risk in male and female. MethodsWe organized a case-control study on 876 individuals with schizophrenia and 913 age- and sex-matched healthy subjects as control group. The concentrations of Hcy, Cys and Met were measured by liquid chromatography-tandem mass spectrometry technology. Subsequently, restricted cubic spline was applied to explore full-range associations of these amino acids with schizophrenia. Interactions between levels of the three amino acids and sex on additive scale were also tested. ResultsHcy levels at ≤29 μmol/L were associated with sharply increased risk of schizophrenia, inversely, Met was associated with sharply decreased risk of schizophrenia at levels ≤22 μmol/L. Increased Cys levels were associated with decreased risk of schizophrenia. Almost inverse associations were observed between Cys/Hcy and Met/Hcy ratios and schizophrenia. Significant synergistic interactions between levels of all the three amino acids and sex were discovered on an additive scale. ConclusionsOur study suggests a close association between sulfur-containing amino acids and schizophrenia with different risk in male and female. Future studies are demanded to clarify the pathogenic role of Hcy, Cys and Met in schizophrenia.

Widely targeted metabolomics unveils baicalin-induced hippocampal metabolic alternations in a rat model of chronic unpredictable mild stress

Baicalin has various neuroprotective effects in models of nervous system disease. Our study has shown baicalin could alleviate depressive-like behaviors in a neuroendocrine mouse model. But the systematic metabolic characteristic and particular targets of baicalin in regulating depressive behaviors have never been investigated. Therefore, this study aims to reveal the hippocampal metabolic profiling of chronic unpredictable mild stress (CUMS) induced depressive rats and the potential metabolic variations after baicalin treatment. We first used the sucrose preference test and open field test to access the antidepressant effects of baicalin. Then, metabolites of the hippocampus after baicalin therapy were monitored by widely-targeted metabolomics based on ultra-performance liquid chromatography-tandem mass spectrometry technology. Finally, the potential mechanism associated with neurogenesis obtained from metabolomics was verified by immunohistochemistry. The results showed that baicalin(40,80 mg/kg) could significantly alleviate depressive behaviors induced by CUMS as demonstrated by an increase in sucrose preference and movement distance and stand-up times in open field test. In the metabolomic analysis, a total of 733 metabolites were identified after baicalin treatment including 15 differential metabolites such as organic acid and its derivatives, heterocyclic compounds, fatty acid, bile acids, amino acid and its metabolites, and so on. Enrichment for differential metabolites showed that the differential metabolites might be involved in the process of folate and cofactor biosynthesis, cholesterol metabolism, primary bile acid biosynthesis, tyrosine metabolism and dopaminergic synapse. Moreover, immunohistochemical analysis confirmed baicalin could facilitate hippocampal neurogenesis of depressive rats in CUMS model. These results suggested baicalin might exert antidepressant effects through regulating the differential metabolites which might play a crucial role in inhibiting oxidative stress and improving neurogenesis. Our findings wish to discover the potential mechanism of baicalin on depression from the metabolomics perspective and promote its clinical application.

DNA methyltransferase inhibitor exposure-response: Challenges and opportunities.

Although DNA methyltransferase inhibitors (DNMTis), such as azacitidine and decitabine, are used extensively in the treatment of myelodysplastic syndromes and acute myeloid leukemia, there remain unanswered questions about DNMTi's mechanism of action and predictors of clinical response. Because patients often remain on single-agent DNMTis or DNMTi-containing regimens for several months before knowing whether clinical benefit can be achieved, the development and clinical validation of response-predictive biomarkers represents an important unmet need in oncology. In this review, we will summarize the clinical studies that led to the approval of azacitidine and decitabine, as well as the real-world experience with these drugs. We will then focus on biomarker development for DNMTis-specifically, efforts at determining exposure-response relationships and challenges that remain impacting the broader clinical translation of these methods. We will highlight recent progress in liquid-chromatography tandem mass spectrometry technology that has allowed for the simultaneous measurement of decitabine genomic incorporation and global DNA methylation, which has significant potential as a mechanism-of-action based biomarker in patients on DNMTis. Last, we will cover important research questions that need to be addressed in order to optimize this potential biomarker for clinical use.

The Pharmacokinetic and Absolute Bioavailability of Cyclosporine (Atopica for Cats®) in Cats.

This study aimed to evaluate the absolute bioavailability of cyclosporine in cats by investigating the pharmacokinetic profile after intravenous and oral administration, respectively. Twenty-four clinically healthy cats were enrolled in this study and randomly divided into four groups, namely the intravenous group (3 mg/kg), low oral group (3.5 mg/kg), medium oral group (7 mg/kg), and high oral group (14 mg/kg). Whole blood was obtained at the scheduled time points after a single dose administration and cyclosporine was determined using ultra-performance liquid chromatography-tandem mass spectrometry technology (UPLC-MS/MS). Pharmacokinetic parameters were calculated using the WinNonlin 8.3.4 software via compartmental and non-compartmental models. As a result, the bioavailability values for the low, medium, and high oral groups were 14.64%, 36.98%, and 13.53%, respectively. The nonlinear pharmacokinetic profile was observed in the range from 3.5 mg/kg to 14 mg/kg in cats following oral administration. Whole blood concentrations taken 4 h after oral administration were better correlated with the area under the blood concentration-time curve AUC0-24 with a high regression coefficient (R2 = 0.896). This concentration would be a greater predictor in the following therapeutic drug monitoring. No adverse effect was observed in the whole study process.

Peptidomics analysis of human breast milk from women with postpartum depression reveals its potential pathogenesis.

Postpartum depression (PPD) is a prevalent psychiatric disorder during the postnatal period, and it exerts adverse impacts not only on mothers but also on infants, impairing the well-being of the whole family. However, the role of peptides in the breast milk of mothers with PPD has not been studied. The purpose of this study was to unveil the peptidomic profile of PPD from breast milk samples. We performed comparative peptidomic profiling of human breast milk from PPD and control mothers using liquid chromatography-tandem mass spectrometry technology with iTRAQ-8 labelling. GO and KEGG pathway analyses of precursor proteins were used to predict the underlying biological functions of differentially expressed peptides (DEPs). Ingenuity Pathway Analysis (IPA) was further performed to explore the interactions and involved pathways of DEPs. A total of 294 peptides from 62 precursor proteins were identified to be differentially expressed in the breast milk of PPD mothers compared with the control group. Bioinformatics analysis predicted that these DEPs were associated with ECM-receptor interaction, neuroactive ligand-receptor interaction, cell adhesion molecule binding and oxidative stress in macrophages. These results indicate that DEPs from human breast milk may play a part in PPD and become promising noninvasive biomarkers.

Evaluating the Properties of Ginger Protease-Degraded Collagen Hydrolysate and Identifying the Cleavage Site of Ginger Protease by Using an Integrated Strategy and LC-MS Technology.

(1) Methods: An integrated strategy, including in vitro study (degree of hydrolysis (DH) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity) and in vivo study (absorption after oral administration in rats), was developed to evaluate the properties of the fish skin gelatin hydrolysates prepared using different proteases (pepsin, alkaline protease, bromelain, and ginger protease). Meanwhile, in order to identify the hydrolysis site of ginger protease, the peptides in the ginger protease-degraded collagen hydrolysate (GDCH) were comprehensively characterized by liquid chromatography/tandem mass spectrometry (LC-MS) method. (2) Results: The GDCH exhibited the highest DH (20.37%) and DPPH radical scavenging activity (77.73%), and in vivo experiments showed that the GDCH was more efficiently absorbed by the gastrointestinal tract. Further oral administration experiments revealed that GDCH was not entirely degraded to free amino acids and can be partially absorbed as dipeptides and tripeptides in intact forms, including Pro-Hyp, Gly-Pro-Hyp, and X-Hyp-Gly tripeptides. LC-MS results determined the unique substrate specificity of ginger protease recognizing Pro and Hyp at the P2 position based on the amino acids at the P2 position from the three types of tripeptides (Gly-Pro-Y, X-Hyp-Gly, and Z-Pro-Gly) and 136 identified peptides (>4 amino acids). Interestingly, it suggested that ginger protease can also recognize Ala in the P2 position. (3) Conclusions: This study comprehensively evaluated the properties of GDCH by combining in vitro and in vivo strategies, and is the first to identify the cleavage site of ginger protease by LC-MS technique. It provides support for the follow-up study on the commercial applications of ginger protease and bioactivities of the hydrolysate produced by ginger protease.

Metabolomics and microbiome reveal potential root microbiota affecting the alkaloidal metabolome in Aconitum vilmorinianum Kom.

BackgroundThe plant microbiome is vital for plant health, fitness, and productivity. Interestingly, plant metabolites and the plant microbiome can influence each other. The combination of metabolomics and microbiome may reveal the critical links between the plant and its microbiome. It is of great significance to agricultural production and human health, especially for Chinese medicine research. Aconitum vilmorinianum Kom. is a herb with alkaloid activities, and its roots are the raw material for some Chinese medicines. Former studies have investigated alkaloidal metabolites and antibacterial activities of endophytes in A. vilmorinianum roots. However, there are limited reports on the root microbiota that can influence the alkaloidal metabolome of A. vilmorinianum.ResultsThis research used ultra performance liquid chromatography-tandem mass spectrometry technology and high-throughput sequencing to examine the alkaloidal metabolome, bacterial microbiota, and fungal microbiota in A. vilmorinianum roots at two different sites in China. The results revealed that the samples from the two sites were rich in distinct alkaloidal metabolites and recruited significantly different root microbiota. Based on bioinformatics analysis, we found the potential bacterial and fungal microbiota impacting the alkaloidal metabolome in A. vilmorinianum.ConclusionOur findings reveal the composition of the alkaloidal metabolome, bacterial root microbiota, and fungal root microbiota in A. vilmorinianum roots at two different sites. Potential root microbiota that can influence the alkaloidal metabolome of A. vilmorinianum are indicated. This study provides a strategy for the cultivation and research of A. vilmorinianum and other Chinese herbs.

The active ingredients analysis of Herba Lysimachiae treating osteoarthritis based on the LC-MS/MS technology and public bioinformatics platforms.

Herba Lysimachiae inhibits synovial damage in osteoarthritis via regulating two bio labels (integrin alpha 2b/beta 3). However, the relevant active ingredients are still unknown. Here, the active ingredients of herbal medicines were analyzed based on the liquid chromatography-tandem mass spectrometry technology and public bioinformatics platforms. The liquid chromatography-tandem mass spectrometry technology was used for compound analysis, and public databases (PubChem BioAssay and STRING) were applied to establish the links between herbal compounds and both bio labels, and identify which herbal compounds may regulate these bio labels. Subsequently, the osteoarthritis model was used to confirm the results. Totally, ninety compounds in Herba Lysimachiae were identified based on the liquid chromatography-tandem mass spectrometry technology. Bioinformatics analysis showed that five compounds (myricetin, fisetin, esculetin, 7-hydroxycoumarin-4-acetic acid, and caffeic acid) may synergistically regulate bio labels through 11 targets, which may be the active ingredients of Herba Lysimachiae for osteoarthritis treatment. In the verification experiments, five compounds markedly suppressed the overexpression of bio labels in the synovium of the osteoarthritis model. In conclusion, the present study effectively and rapidly analyzed the active ingredients of Herba Lysimachiae for osteoarthritis treatment.

Responses to β-alanine and carnosine supplementation of mussel Mytilus coruscus as revealed by UPLC–MS/MS based untargeted metabolomics

Histidine-containing dipeptides (HCDs) have been demonstrated with important physiological functions in vertebrates. This study was executed to investigate the contents and metabolic profiles of HCDs in an invertebrate, the mussel Mytilus coruscus was selected. The carnosine/anserine analysis indicated that M. coruscus contain anserine with a higher concentration than that of carnosine, and the increases in HCDs concentrations due to β-alanine injection and carnosine injection were observed, confirming that Mytilus belongs to a species with the ability of HCDs synthesis. Further metabolomics analysis was performed based on ultra-performance liquid chromatography-tandem mass spectrometry technology to explore the effects of β-alanine injection and carnosine injection, respectively, on the whole mussel tissue. A total of 1,868 significantly differential metabolites (SDMs) were detected between the β-alanine injected, carnosine injected, and the control M. coruscus groups. The SDMs induced by β-alanine injection were enriched mainly in energy metabolism, lipid metabolism, and amino acid metabolism of mussel, while carnosine injection mainly disrupted arachidonic acid metabolism, steroid hormone biosynthesis, and retinol metabolism in the mussel. The activities of antioxidant enzymes from the whole mussel tissue extracts before and after β-alanine/carnosine injection were measured, and both treatments showed significant elevation of enzymatic activities of superoxide dismutase and catalase. Considering the positive effects of β-alanine injection on mussel metabolism, this study provided a clue for the potential application of β-alanine supplementation in mussel farming.

Proteomic mapping of Drosophila transgenic elav.L-GAL4/+ brain as a tool to illuminate neuropathology mechanisms

Drosophila brain has emerged as a powerful model system for the investigation of genes being related to neurological pathologies. To map the proteomic landscape of fly brain, in a high-resolution scale, we herein employed a nano liquid chromatography-tandem mass spectrometry technology, and high-content catalogues of 7,663 unique peptides and 2,335 single proteins were generated. Protein-data processing, through UniProt, DAVID, KEGG and PANTHER bioinformatics subroutines, led to fly brain-protein classification, according to sub-cellular topology, molecular function, implication in signaling and contribution to neuronal diseases. Given the importance of Ubiquitin Proteasome System (UPS) in neuropathologies and by using the almost completely reassembled UPS, we genetically targeted genes encoding components of the ubiquitination-dependent protein-degradation machinery. This analysis showed that driving RNAi toward proteasome components and regulators, using the GAL4-elav.L driver, resulted in changes to longevity and climbing-activity patterns during aging. Our proteomic map is expected to advance the existing knowledge regarding brain biology in animal species of major translational-research value and economical interest.

ITRAQ-Based Proteomics Analysis of Plasma of Myasthenia Gravis Patients Treated with Jia Wei Bu Zhong Yi Qi Decoction.

Myasthenia gravis (MG) is an autoimmune disease. A proportion of MG patients did not get satisfactory results after treatment with pyridostigmine and prednisone. Jia Wei Bu Zhong Yi Qi (Jia Wei BZYQ) decoction, a water extract from multiple herbs, has been demonstrated to be effective in the treatment of multiple “Qi deficiency type” diseases including MG in China. In this text, we investigated protein alterations in the plasma from healthy volunteers (C), MG patients without any treatment (T1), MG patients with routine western medical treatment (T2), and MG patients with combined treatments of Jia Wei BZYQ decoction and routine western medicines (T3) and identified some potential proteins involved in the pathogenesis and treatment of MG. iTRAQ (isobaric tags for relative and absolute quantitation) and 2D-LC-MS/MS (two-dimensional liquid chromatography-tandem mass spectrometry technologies) were employed to screen differentially expressed proteins. The identification, quantification, functional annotation, and interaction of proteins were analyzed by matching software and databases. In our project, 618 proteins were identified, among which 447 proteins had quantitative data. The number of differentially expressed proteins was 110, 117, 143, 115, 86, and 158 in T1 vs. C, T2 vs. C, T2 vs. T1, T3 vs. C, T3 vs. T1, and T3 vs. T2 groups, respectively. Functional annotation results showed that many differentially expressed proteins were closely associated with immune responses. For instance, some key proteins such as C-reactive protein, apolipoprotein C-III, apolipoprotein A-II, alpha-actinin-1, and thrombospondin-1 have been found to be abnormally expressed in T3 group compared to T1 group or T2 group. Interaction network analyses also provided some potential biomarkers or targets for MG management.

Development of a Nitric Oxide-Responsive Labeling Reagent for Proteome Analysis of Live Cells.

Nitric oxide (NO) is a pleiotropic signaling molecule involved in the regulation of diverse physiological and pathophysiological mechanisms in cardiovascular, nervous, and immunological systems. To understand the biological functions of NO in detail, comprehensive characterization of proteins found in high-NO concentration environments is crucial. Herein, we describe the design of NO-responsive protein labeling reagents based on N-alkoxyacyl- o-phenylenediamine as an optimal reactive scaffold. The designed molecules can label proteins in murine macrophage cells in response to endogenously produced NO. The combination of NO-responsive protein labeling and liquid chromatography-tandem mass spectrometry technology allowed the characterization of the proteome under NO-generated conditions. Moreover, we demonstrated that our reagent was able to selectively mark and be used to fluorescently visualize NO-producing cells in a mixed cell culture system.

Multicenter study of environmental contamination with cyclophosphamide, ifosfamide, and methotrexate in 66 Canadian hospitals: A 2016 follow-up study

ABSTRACTPurpose: Oncology workers are occupationally exposed to antineoplastic drugs. This exposure can induce adverse health effects. To reduce their exposure, contamination on surfaces should be kept as low as possible. The main objective of this study was to monitor environmental contamination with cyclophosphamide, ifosfamide, and methotrexate in oncology pharmacy and patient care areas in Canadian centers. The secondary objective was to describe the impact of some factors that may limit contamination.Methods: This is a descriptive study. Twelve standardized sites were sampled in each participating center (six in the pharmacy and six in patient care areas). Samples were analyzed for the presence of cyclophosphamide, ifosfamide, and methotrexate by ultra-performance liquid chromatography-tandem mass spectrometry technology. Descriptive statistical analyses were done and results were compared with a Kolmogorov–Smirnov test for independent samples.Results: In 2016, 66 centers participated in this study (66/202, 32.7%). Overall, 43.4% (326/752) of the samples were positive for cyclophosphamide, 13.2% (99/752) for ifosfamide and 6.9% (52/752) for methotrexate. The 75th percentile value of cyclophosphamide surface concentration was 6.8 pg/cm2 and lower than the limit of detection for ifosfamide and methotrexate. Centers who prepared more antineoplastic drugs per year (p < 0.0001), who used more cyclophosphamide per year (p < 0.0001) and who primed antineoplastic IV tubing in patient care unit by nurses (p = 0.004) showed significantly higher surface contamination to cyclophosphamide.Conclusion: Environmental surveillance is one part of a comprehensive approach for minimizing hazardous exposures in healthcare. This study highlights a low level of contamination of three hazardous drugs amongst 66 Canadian centers. Regular environmental monitoring is a good practice to maintain contamination as low as reasonably achievable.

Identification of human serum protein targets of Qianggu Decoction () in primary type I osteoporosis based on tandem mass tag labeling and liquid chromatography-tandem mass spectrometry technology.

To investigate the serum protein targets of Qianggu Decoction (, QGD) on treating osteoporosis by the proteomics analysis using tandem mass tag (TMT) and liquid chromatographytandem mass spectrometry (LC-MS/MS). Twenty serum protein samples were recruited (10 patients with primary type I osteoporosis before and after QGD treatment) and the high abundance ratios protein was removed, two serum samples were extracted and labeled with TMT reagent. Then, mass spectrometric detection, identification of differentially expressed proteins and bioinformatics analysis of differentially expressed proteins were carried out. A total of 60 proteins were identified, within a 99% confidence interval, to be differentially regulated of which, 34 proteins were up-regulated and 26 proteins were down-regulated. Differentially expressed proteins analyzed by Gene Ontology (GO) annotation mainly get involved in 12 different biological processes, 7 types of cellular components, and 6 kinds of molecular functions. Angiotensinogen (AGT), stromelysin-1 (MMP3), heparanase (HPSE) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were screened as candidate protein targets of QGD treatment, which were related to metabolic mechanism of bone remodeling and/or bone collagen of osteoporosis. By the utilization of the protein-protein interaction network analysis tool named STRING10.0, it showed that AGT, MMP3, HPSE and GAPDH were located in the key node of the protein-protein interactions network. Furthermore, AGT, MMP3, HPSE and GAPDH were found to be directly related to BMP, MAPK, Wnt, SMAD and tumor necrosis factor ligand superfamily member 11 (TNFSF11) families. The proteomics analysis by using TMT combined with LC-MS/MS was a feasible method for screening the potential therapeutic targets associated with QGD treatment. It suggests that AGT, MMP3, HPSE and GAPDH may be candidate protein targets of QGD treatment which can be used as therapeutic effect monitor and early diagnosis of primary type I osteoporosis.

Analyte stability during the total testing process: studies of vitamins A, D and E by LC-MS/MS.

There are limited evidence based studies demonstrating the stability of fat-soluble vitamins (FSV) measured in blood. This study aimed to examine the effects of light, temperature and time on vitamins A, D and E throughout the total testing process. Four experiments were conducted. Three investigated the sample matrix, of whole blood, serum and the extracted sample, against the variables of temperature and light; and the fourth experiment investigated the sample during the extraction process against the variable of light. All samples were analysed via our simultaneous FSV method using liquid chromatography-tandem mass spectrometry technology. The allowable clinical percentage change was calculated based on biological variation and desirable method imprecision for each analyte. The total change limit was ±7.3% for 25-OH-vitamin D3, ±11.8% for retinol and ±10.8% for α-tocopherol. Vitamins D and E were stable in the investigated conditions (concentration changes <4%) in the pre-analytical and analytical stages. Vitamin A showed photosensitivity in times >48 h with concentration changes of -6.8% (blood) and -6.5% (serum), both are within the allowable clinical percentage change. By contrast, the extracted retinol sample demonstrated a concentration change of -18.4% after 48 h of light exposure. However, vitamin A in the serum and extracted solution was stable for one month when stored at -20°C. Blood samples for vitamins D and E analyses can be processed in normal laboratory conditions of lighting and temperature. The required conditions for vitamin A analysis are similar when performed within 48 h. For longer-term storage, serum and vitamin A extracts should be stored at -20°C.

A High-Calcium and Phosphate Rescue Diet and VDR-Expressing Transgenes Normalize Serum Vitamin D Metabolite Profiles and Renal Cyp27b1 and Cyp24a1 Expression in VDR Null Mice.

Vitamin D receptor (VDR)-mediated 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-dependent gene expression is compromised in the VDR null mouse. The biological consequences include: hypocalcemia, hypophosphatemia, elevated parathyroid hormone (PTH) and 1,25(OH)2D3, and consequential skeletal abnormalities. CYP24A1 is a cytochrome P450 enzyme that is involved in the side chain oxidation and destruction of both 1,25(OH)2D3 and 25-hydroxyvitamin D3 (25-OH-D3). In the current studies, we used liquid chromatography-tandem mass spectrometry technology to compare the metabolic profiles of VDR null mice fed either a normal or a calcium and phosphate-enriched rescue diet and to assess the consequence of transgenic expression of either mouse or human VDR genes in the same background. Serum 1,25(OH)2D3 levels in VDR null mice on normal chow were highly elevated (>3000 pg/mL) coincident with undetectable levels of catabolites such as 24,25-(OH)2D3 and 25-OH-D3-26,23-lactone normally observed in wild-type mice. The rescue diet corrected serum Ca(++), PTH, and 1,25(OH)2D3 values and restored basal expression of Cyp24a1 as evidenced by both renal expression of Cyp24a1 and detection of 24,25-(OH)2D3 and the 25-OH-D3-26,23-lactone. Unexpectedly, this diet also resulted in supranormal levels of 3-epi-24,25-(OH)2D3 and 3-epi-25-OH-D3-26,23-lactone. The reappearance of serum 24,25-(OH)2D3 and renal Cyp24a1 expression after rescue suggests that basal levels of Cyp24a1 may be repressed by high PTH. Introduction of transgenes for either mouse or human VDR also normalized vitamin D metabolism in VDR null mice, whereas this metabolic pattern was unaffected by a transgene encoding a ligand binding-deficient mutant (L233S) human VDR. We conclude that liquid chromatography-tandem mass spectrometry-based metabolic profiling is an ideal analytical method to study mouse models with alterations in calcium/phosphate homeostasis.

Proteomic Analysis Reveals Resistance Mechanism Against Chlorpyrifos inFrankliniella occidentalis(Thysanoptera: Thripidae)

The western flower thrips is an economically important worldwide pest of many crops, and chlorpyrifos has been used to control western flower thrips for many years. To develop a better resistance-management strategy, a chlorpyrifos-resistant strain of western flower thrips (WFT-chl) was selected in the laboratory. More than 39-fold resistance was achieved after selected by chlorpyrifos for 19 generations in comparison with the susceptible strain (WFT-S). Proteome of western flower thrips (WFT-S and WFT-chl) was investigated using a quantitative proteomics approach with isobaric tag for relative and absolute quantification technique and liquid chromatography-tandem mass spectrometry technologies. According to the functional analysis, 773 proteins identified were grouped into 10 categories of molecular functions and 706 proteins were presented in 213 kinds of pathways. Comparing the proteome of WFT-chl with that of WFT-S, a total of eight proteins were found up-regulated and three down-regulated. The results from functional annotation and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses indicated that the differentially expressed protein functions in binding, catalyzing, transporting, and enzyme regulation were most important in resistance development. A list of proteins functioning in biological processes of metabolism, biological regulation, and response to stimulus was found in WFT-chl, suggesting that they are possibly the major components of the resistance mechanism to chlorpyrifos in western flower thrips. Notably, several novel potential resistance-related proteins were identified such as ribosomal protein, Vg (vitellogenin), and MACT (muscle actin), which can be used to improve our understanding of the resistance mechanisms in western flower thrips. This study provided the first comprehensive view of the complicated resistance mechanism employed by WFT-S and WFT-chl through the isobaric tag for relative and absolute quantification coupled with liquid chromatography-tandem mass spectrometry technologies.

AMG 580: a novel small molecule phosphodiesterase 10A (PDE10A) positron emission tomography tracer.

Phosphodiesterase 10A (PDE10A) inhibitors have therapeutic potential for the treatment of psychiatric and neurologic disorders, such as schizophrenia and Huntington's disease. One of the key requirements for successful central nervous system drug development is to demonstrate target coverage of therapeutic candidates in brain for lead optimization in the drug discovery phase and for assisting dose selection in clinical development. Therefore, we identified AMG 580 [1-(4-(3-(4-(1H-benzo[d]imidazole-2-carbonyl)phenoxy)pyrazin-2-yl)piperidin-1-yl)-2-fluoropropan-1-one], a novel, selective small-molecule antagonist with subnanomolar affinity for rat, primate, and human PDE10A. We showed that AMG 580 is suitable as a tracer for lead optimization to determine target coverage by novel PDE10A inhibitors using triple-stage quadrupole liquid chromatography-tandem mass spectrometry technology. [(3)H]AMG 580 bound with high affinity in a specific and saturable manner to both striatal homogenates and brain slices from rats, baboons, and human in vitro. Moreover, [(18)F]AMG 580 demonstrated prominent uptake by positron emission tomography in rats, suggesting that radiolabeled AMG 580 may be suitable for further development as a noninvasive radiotracer for target coverage measurements in clinical studies. These results indicate that AMG 580 is a potential imaging biomarker for mapping PDE10A distribution and ensuring target coverage by therapeutic PDE10A inhibitors in clinical studies.

Analysis of SiO2 nanoparticles binding proteins in rat blood and brain homogenate.

A multitude of nanoparticles, such as titanium oxide (TiO2), zinc oxide, aluminum oxide, gold oxide, silver oxide, iron oxide, and silica oxide, are found in many chemical, cosmetic, pharmaceutical, and electronic products. Recently, SiO2 nanoparticles were shown to have an inert toxicity profile and no association with an irreversible toxicological change in animal models. Hence, exposure to SiO2 nanoparticles is on the increase. SiO2 nanoparticles are routinely used in numerous materials, from strengthening filler for concrete and other construction composites, to nontoxic platforms for biomedical application, such as drug delivery and theragnostics. On the other hand, recent in vitro experiments indicated that SiO2 nanoparticles were cytotoxic. Therefore, we investigated these nanoparticles to identify potentially toxic pathways by analyzing the adsorbed protein corona on the surface of SiO2 nanoparticles in the blood and brain of the rat. Four types of SiO2 nanoparticles were chosen for investigation, and the protein corona of each type was analyzed using liquid chromatography-tandem mass spectrometry technology. In total, 115 and 48 plasma proteins from the rat were identified as being bound to negatively charged 20 nm and 100 nm SiO2 nanoparticles, respectively, and 50 and 36 proteins were found for 20 nm and 100 nm arginine-coated SiO2 nanoparticles, respectively. Higher numbers of proteins were adsorbed onto the 20 nm sized SiO2 nanoparticles than onto the 100 nm sized nanoparticles regardless of charge. When proteins were compared between the two charges, higher numbers of proteins were found for arginine-coated positively charged SiO2 nanoparticles than for the negatively charged nanoparticles. The proteins identified as bound in the corona from SiO2 nanoparticles were further analyzed with ClueGO, a Cytoscape plugin used in protein ontology and for identifying biological interaction pathways. Proteins bound on the surface of nanoparticles may affect functional and conformational properties and distributions in complicated biological processes.

Metabolic Switching of BILR 355 in the Presence of Ritonavir. I. Identifying an Unexpected Disproportionate Human Metabolite

11-Ethyl-5,11-dihydro-5-methyl-8-[2-[(1-oxido-4-quinolinyl)oxy] ethyl]-6H-dipyrido[3,2-b:2',3'-e][1,4]diazepin-6-one (BILR 355) is an inhibitor of the human immunodeficiency virus-1. BILR 355 exhibited a nonlinear pharmacokinetic profile and low exposure after oral administration to humans. This article describes the in vitro metabolism of BILR 355, which is correlated with the in vivo nonlinearity findings. Our in vitro studies had demonstrated that BILR 355 was extensively metabolized by cytochrome P450 3A. Thus, BILR 355 was concomitantly administered with ritonavir (RTV) in an attempt to boost systemic exposure, which did occur in humans. In addition, the expectation was that the overall metabolism of BILR 355 would be decreased with concomitant administration of RTV. Subsequent metabolite profiling was performed using human plasma samples obtained from clinical phase Ib studies with concomitant administration of BILR 355 and RTV. A total of 18 metabolites was observed. Their structures were proposed on the basis of high-performance liquid chromatography-tandem mass spectrometry technologies, and 10 metabolites were confirmed by comparison with synthetic standards. We were surprised to find that a disproportionate human metabolite, BILR 516, was uncovered during this metabolite profiling study and pharmacokinetic analysis of BILR 516 showed that it had a longer half-life and higher exposure than the parent compound at steady state. Of interest, BILR 516 was not detected in human plasma when BILR 355 was administered alone. Therefore, whereas RTV boosted the exposure of BILR 355, it resulted in a significant metabolic switching of BILR 355. Overall, this article demonstrates an unusual example of metabolic switching and raises concern about the consequence of metabolic switching during drug development.