HPLC-tandem Mass Spectrometry Research Articles

Quantitative proteomics of dorsolateral prefrontal cortex reveals an early pattern of synaptic dysmaturation in children with idiopathic autism.

Autism spectrum disorder (ASD) is a developmental disorder with a rising prevalence and unknown etiology presenting with deficits in cognition and abnormal behavior. We hypothesized that the investigation of the synaptic component of prefrontal cortex may provide proteomic signatures that may identify the biological underpinnings of cognitive deficits in childhood ASD. Subcellular fractions of synaptosomes from prefrontal cortices of age-, brain area-, and postmortem-interval-matched samples from children and adults with idiopathic ASD vs. controls were subjected to HPLC-tandem mass spectrometry. Analysis of data revealed the enrichment of ASD risk genes that participate in slow maturation of the postsynaptic density (PSD) structure and function during early brain development. Proteomic analysis revealed down regulation of PSD-related proteins including AMPA and NMDA receptors, GRM3, DLG4, olfactomedins, Shank1-3, Homer1, CaMK2α, NRXN1, NLGN2, Drebrin1, ARHGAP32, and Dock9 in children with autism (FDR-adjusted P < 0.05). In contrast, PSD-related alterations were less severe or unchanged in adult individuals with ASD. Network analyses revealed glutamate receptor abnormalities. Overall, the proteomic data support the concept that idiopathic autism is a synaptopathy involving PSD-related ASD risk genes. Interruption in evolutionarily conserved slow maturation of the PSD complex in prefrontal cortex may lead to the development of ASD in a susceptible individual.

Early Chronic Fluoxetine Treatment of Ts65Dn Mice Rescues Synaptic Vesicular Deficits and Prevents Aberrant Proteomic Alterations.

Down syndrome (DS) is the most common form of inherited intellectual disability caused by trisomy of chromosome 21, presenting with intellectual impairment, craniofacial abnormalities, cardiac defects, and gastrointestinal disorders. The Ts65Dn mouse model replicates many abnormalities of DS. We hypothesized that investigation of the cerebral cortex of fluoxetine-treated trisomic mice may provide proteomic signatures that identify therapeutic targets for DS. Subcellular fractionation of synaptosomes from cerebral cortices of age- and brain-area-matched samples from fluoxetine-treated vs. water-treated trisomic and euploid male mice were subjected to HPLC-tandem mass spectrometry. Analysis of the data revealed enrichment of trisomic risk genes that participate in regulation of synaptic vesicular traffic, pre-synaptic and post-synaptic development, and mitochondrial energy pathways during early brain development. Proteomic analysis of trisomic synaptic fractions revealed significant downregulation of proteins involved in synaptic vesicular traffic, including vesicular endocytosis (CLTA, CLTB, CLTC), synaptic assembly and maturation (EXOC1, EXOC3, EXOC8), anterograde axonal transport (EXOC1), neurotransmitter transport to PSD (SACM1L), endosomal-lysosomal acidification (ROGDI, DMXL2), and synaptic signaling (NRXN1, HIP1, ITSN1, YWHAG). Additionally, trisomic proteomes revealed upregulation of several trafficking proteins, involved in vesicular exocytosis (Rab5B), synapse elimination (UBE3A), scission of endocytosis (DBN1), transport of ER in dendritic spines (MYO5A), presynaptic activity-dependent bulk endocytosis (FMR1), and NMDA receptor activity (GRIN2A). Chronic fluoxetine treatment of Ts65Dn mice rescued synaptic vesicular abnormalities and prevented abnormal proteomic changes in adult Ts65Dn mice, pointing to therapeutic targets for potential treatment of DS.

2A2 protein of DHAV-1 induces duck embryo fibroblasts gasdermin E-mediated pyroptosis

The duck hepatitis A virus type 1 (DHAV-1) causes rapid death in ducklings by triggering a severe cytokine storm. Pyroptosis is an inflammatory form of programmed cell death that is directly related to an increase in pro-inflammatory cytokine levels. Only a few studies have explored the mechanisms underlying pyroptosis in virus-infected avian cells. In this study, we established an avian infection model in vitro by infecting duck embryo fibroblasts (DEFs) with the virulent DHAV-1 LY0801 strain. DHAV-1 infection induced pyroptosis in the DEFs by activating gasdermin E (GSDME) protein via caspase-3-mediated cleavage. The genes encoding the different structural and non-structural DHAV-1 proteins were cloned into eukaryotic expression plasmids, and the 2A2 protein was identified as the key protein involved in pyroptosis. The HPLC-tandem mass spectrometry (HPLC-MS/MS) and co-immunoprecipitation (Co-IP) analysis established that DHAV-1 2A2 directly interacted with the mitochondrial anti-viral signaling protein (MAVS) both intracellularly and in vitro. Furthermore, we got the results that N-terminal 1–130 aa of 2A2 was involved in the interaction with MAVS and the C-terminal TM domain of MAVS is necessary for the interaction with 2A2 by Co-IP analysis. To our knowledge, this is the first study to reveal that DHAV-1 protein interacts with host proteins to induce pyroptosis. Our findings provide new insights into the molecular pathogenesis of DHAV-1 infection, and a scientific basis for the prevention and treatment of duck viral hepatitis.

Investigation of the human metabolism and disposition of the prolyl hydrolase inhibitor daprodustat using IV microtracer with Entero-Test bile string.

Daprodustat is an oral small molecule hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor (PHI) approved in Japan and the United States for the treatment of anemia associated with chronic kidney disease. This phase 1, nonrandomized, 2-period, crossover study in 6 healthy men characterized and quantified the metabolites generated after a microtracer IV infusion of 50 μg (125 nCi) [14 C]-daprodustat administered concomitantly with a nonradiolabeled therapeutic dose of a 6-mg daprodustat tablet, followed by a single oral solution dose of 25 mg (62.5 μCi) [14 C]-daprodustat. High-performance liquid chromatography (HPLC) coupled with radioactivity detection (TopCount or AMS) and HPLC-tandem mass spectrometry (HPLC-MSn ) were used for quantitative measurement and structural identification of radioactive metabolites in plasma, urine, feces, and bile. Following oral administration of [14 C]-daprodustat, unchanged daprodustat was the principal circulating drug-related component, accounting for 40% of plasma radioactivity. Predominant oxidative metabolites M2, M3, M4, and M13 individually represented 6-8% of the plasma radioactivity and together accounted for the majority of radioactivity in urine and feces (53% in both matrices; 12% and 41% of dose, respectively). Unchanged daprodustat was not detected in urine and was only 0.7% of total radioactivity in feces (<0.5% of dose), with the remainder of the dose accounted for by oxidative metabolites. The radio-metabolic profile of duodenal bile following IV infusion of [14 C]-daprodustat was similar to that observed in feces after oral administration. The data suggested that oral daprodustat was extensively absorbed, cleared exclusively by oxidative metabolism, and eliminated via hepatobiliary (primary) and urinary (secondary) excretion.

Procine recombinant NK-lysin inhibits hepatocellular carcinoma metastasis by downregulating FKBP3 and inhibiting oxidative phosphorylation and glycolysis: a proteomic analysis

To investigate the potential mechanisms that mediate the inhibitory effect of porcine recombinant NKlysin (prNK-lysin) against liver cancer cell metastasis. HPLC-tandem mass spectrometry was used to identify the differentially expressed proteins in prNK-lysin-treated hepatocellular carcinoma SMMOL/LC-7721 cells in comparison with the control and PBS-treated cells. GO functional annotation and KEGG pathway analysis of the differentially expressed proteins were performed using GO and KEGG databases. RT-qPCR was used to determine the mRNA expression levels of polypeptide-N-acetylgalactosaminotransferase 13 (GALNT13), transmembrane protein 51 (TMEM51) and FKBP prolyl isomerase 3 (FKBP3) in the cells, and the protein expression of FKBP3 was verified using Western blotting. Proteomic analysis identified 1989 differentially expressed proteins in prNK-lysin-treated cells compared with the control cells, and 2753 compared with PBS-treated cells. Fifteen proteins were differentially expressed between PBS-treated and the control cells, and 1909 were differentially expressed in prNK- lysin group compared with both PBS and control groups. These differentially expressed proteins were involved mainly in the viral process, translational initiation and RNA binding and were enriched mainly in ribosome, protein process in endoplasmic reticulum, and RNA transport pathways. RT-qPCR showed that compared with the control group, prNK-lysin treatment significantly increased the mRNA expressions of GALNT13 (P < 0.05) and TMEM51 (P < 0.01) and lowered FKBP3 mRNA expression (P < 0.05). Western blotting also showed a significantly decreased expression of FKBP3 protein in prNK-lysin-treated cells (P < 0.001). Treatment with prNK-lysin causes significant changes in protein expression profile of SMMOL/LC-7721 cells and inhibits hepatocellular carcinoma metastasis by downregulating FKBP3 protein and affecting the cellular oxidative phosphorylation and glycolysis pathways.

The behavior of cathepsin D during milk processing and its contribution to bitterness in a model fresh cheese

The bovine endopeptidase cathepsin D was investigated regarding its temperature-dependent inactivation and ability to form bitter peptides within a spiked model fresh cheese. Cathepsin D was found to be more susceptible than other milk endogenous peptidases to temperature treatments in skim milk. Inactivation kinetics revealed decimal reduction times of 5.6 min to 10 s in a temperature range from 60 to 80°C. High temperature and ultra-high temperature (UHT) treatments from 90 to 140°C completely inactivated cathepsin D within 5 s. A residual cathepsin D activity of around 20% was detected under pasteurization conditions (72°C for 20 s). Therefore, investigations were done to estimate the effect of residual cathepsin D activity on taste in a model fresh cheese. The UHT-treated skim milk was spiked with cathepsin D and acidified with glucono-δ-lactone to produce a model fresh cheese. A trained bitter-sensitive panel was not able to distinguish cathepsin D-spiked model fresh cheeses from the control model fresh cheeses in a triangle test. Model fresh cheese samples were also analyzed for known bitter peptides derived from casein fractions using a HPLC-tandem mass spectrometry (MS) approach. In accordance with the sensory evaluation, the MS analyses revealed that the bitter peptides investigated within the cathepsin D-spiked model fresh cheese were not found or were below the limit of detection. Even though cathepsin D may be present during the fermentation of pasteurized milk, it does not seem to be responsible for bitter peptide formation from milk proteins on its own.

Plasma arginine as a candidate predictive biomarker for response to immune checkpoint inhibition (ICI) in metastatic colorectal cancer (mCRC): Analysis of the CCTG CO.26 trial.

3545 Background: Nutritional stress is one of the mechanisms used by tumour cells to evade the immune system. Arginine (ARG), an amino acid involved in several cellular functions including immunomodulation, is important in regulating T-lymphocyte cell activity and the anti-tumour response. ARG deficiency in the tumour microenvironment has been shown to impair T-cell response while ARG supplementation may promote anti-tumour immune activity. In this exploratory post-hoc analysis of the Phase II CO.26 trial (NCT02870920), we investigated the role of plasma ARG in predicting response to ICI in patients (pts) with refractory mCRC. Methods: CO.26 was a phase II trial which randomized pts with refractory mCRC to durvalumab plus tremelimumab (D+T) versus best supportive care (BSC). Plasma ARG concentrations were determined from blood samples pre-treatment using HPLC-tandem mass spectrometry. The median plasma ARG value was used as a cut-off stratifying pts into ARG-high (≥10650 ng/ml) versus ARG-low ( &lt; 10650 ng/ml) groups. Progression-free (PFS) and overall survival (OS) were estimated using the Kaplan-Meier method and compared between groups using the log-rank test. Cox proportional hazard models were used to analyze prognostic and predictive impacts of ARG on PFS and OS. Results: Of 180 pts enrolled in CO.26, 162 pts (N = 115 treated with D+T and 47 BSC) had pre-treatment blood samples for baseline ARG analysis. There were no significant differences in baseline characteristics between pts included in this analysis and the total study pts, or between ARG-high and ARG-low pts. In pts treated with D+T, ARG-high was associated with more favourable prognosis (ARG-high median OS 7.62 months vs. ARG-low 5.49 months, multivariable hazard ratio [HR] 0.60, 95% confidence interval [CI] 0.40-0.91, p = 0.016). In ARG-high pts, D+T significantly improved OS (median OS 7.62 months with D+T vs 3.61 months BSC; HR 0.61, 95% CI 0.37-0.99, p = 0.04). In ARG-low pts there was no OS benefit with D+T (median OS 5.49 months D+T vs 4.27 months BSC; HR 0.84, 95% CI 0.50-1.41, p = 0.51. Interaction p = 0.037). Baseline ARG values had no association with PFS or disease control rate. Conclusions: Baseline plasma ARG was prognostic in pts with mCRC treated with D+T, and high ARG was predictive of improved OS with ICI. Prospective studies should be done to validate ARG as a biomarker identifying mCRC pts likely to derive benefit from ICI. Therapeutic approaches targeting the ARG pathway should be investigated in future studies.

Repeated Low-Level Blast Exposure Alters Urinary and Serum Metabolites

Repeated exposure to low-level blast overpressures can produce biological changes and clinical sequelae that resemble mild traumatic brain injury (TBI). While recent efforts have revealed several protein biomarkers for axonal injury during repetitive blast exposure, this study aims to explore potential small molecule biomarkers of brain injury during repeated blast exposure. This study evaluated a panel of ten small molecule metabolites involved in neurotransmission, oxidative stress, and energy metabolism in the urine and serum of military personnel (n = 27) conducting breacher training with repeated exposure to low-level blasts. The metabolites were analyzed using HPLC—tandem mass spectrometry, and the Wilcoxon signed-rank test was used for statistical analysis to compare the levels of pre-blast and post-blast exposures. Urinary levels of homovanillic acid (p &lt; 0.0001), linoleic acid (p = 0.0030), glutamate (p = 0.0027), and serum N-acetylaspartic acid (p = 0.0006) were found to be significantly altered following repeated blast exposure. Homovanillic acid concentration decreased continuously with subsequent repeat exposure. These results suggest that repeated low-level blast exposures can produce measurable changes in urine and serum metabolites that may aid in identifying individuals at increased risk of sustaining a TBI. Larger clinical studies are needed to extend the generalizability of these findings.

Arylnaphthalene Lignans with Anti-SARS-CoV-2 and Antiproliferative Activities from the Underground Organs of Linum austriacum and Linum perenne.

Diphyllin (1) and justicidin B (2) are arylnaphthalene lignans with antiviral and antiproliferative effects. Compound 1 is also known as an effective inhibitor of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). To evaluate the in vitro antiviral and cytotoxic potency of both lignans in SARS-CoV-2 -infected cells and various cancer cell lines, respectively, 1 and 2 were isolated from the underground organs of Linum austriacum and Linum perenne. Two previously undescribed arylnaphthalene lignans, denominated linadiacin A and B (3 and 4), were also isolated and identified. In acidic media, 3 was converted by a two-step reaction into 2 via the intermediate 4. Optimum acid treatment conditions were determined to isolate lignans by one-step preparative high-performance liquid chromatography (HPLC). The results of the conversion, HPLC-tandem mass spectrometry, nuclear magnetic resonance spectroscopy, and molecular modeling studies allowed complete structure analysis. Compounds 1 and 2 were the most effective against SARS-CoV-2 with a 3-log reduction in the viral copy number at a 12.5 μM concentration. Ten human cancer cell lines showed sensitivity to at least one of the isolated lignans.

Effect of Club cell secretory proteins on the association of tobacco smoke and PAH co-exposure with lung function decline: A longitudinal observation of Chinese coke oven workers.

Exposure to polycyclic aromatic hydrocarbons (PAH) and tobacco smoke is associated with epithelial damage and reduced lung function. Club cell secretory protein (CC16) is a known biomarker for lung epithelial cells. However, the potential relationships between PAH and tobacco smoke exposure, CC16 levels, and reduced lung function remain unclear. This longitudinal study aimed to explore the potential role of CC16 in the association of tobacco smoke and PAH co-exposure with lung function. We enrolled 313 workers from a coking plant in China in 2014 and followed them up in 2019. The concentrations of PAH and nicotine metabolites in urine were determined using high-performance liquid chromatography (HPLC) with a fluorescence detector and HPLC-tandem mass spectrometry, respectively. The plasma CC16 concentration was determined using an enzyme-linked immunosorbent assay. An analysis of the generalized estimating equation showed that each 1-unit increase in log-transformation of the last tertile of trans-3'-hydroxycotinine (3HC) was associated with a 3.30ng/ml decrease in CC16. Restricted cubic spline analysis revealed a significant nonlinear dose-effect association between cotinine (COT) and CC16 (Pnonlinear=0.018). In the low- CC16 subgroup, we found a significant association between total nicotine metabolites and forced vital capacity (FVC%) (β: 1.45, 95% CI: 2.87, -0.03), and the associations of nicotine (NIC), COT, and 3HC with FVC% were all of marginal significance. High levels of total hydroxyl polycyclic aromatic hydrocarbons (ΣOH-PAH) and NIC in the urine had an interactive effect on the decline of CC16 (P<0.05). Cross-lagged panel analysis indicated that the decrease in CC16 preceded the decrease in FVC%. CC16 mediated the association between elevated nicotine metabolites and decreased FVC% in the low- CC16 subgroup. CC16 plays an essential role in the association of PAH and tobacco smoke exposure with reduced lung function. Coke oven workers with low plasma CC16 levels are more likely to experience decreased lung function after tobacco smoke exposure.

A Nested Case-Control Study of Allopregnanolone and Preterm Birth in the Healthy Start Cohort.

Chronic stress is a risk factor for preterm birth; however, objective measures of stress in pregnancy are limited. Maternal stress biomarkers may fill this gap. Steroid hormones and neurosteroids such as allopregnanolone (ALLO) play important roles in stress physiology and pregnancy maintenance and therefore may be promising for preterm birth prediction. We evaluated maternal serum ALLO, progesterone, cortisol, cortisone, pregnanolone, and epipregnanolone twice in gestation to evaluate associations with preterm birth. We performed a nested case-control study using biobanked fasting serum samples from the Healthy Start prebirth cohort. We included healthy women with a singleton pregnancy and matched preterm cases with term controls (1:1; N = 27 per group). We used a new HPLC-tandem mass spectrometry assay to quantify ALLO and five related steroids. We used ANOVA, Fisher exact, χ2, t test, and linear and logistic regression as statistical tests. Maternal serum ALLO did not associate with preterm birth nor differ between groups. Mean cortisol levels were significantly higher in the preterm group early in pregnancy (13w0d-18w0d; P < 0.05) and higher early pregnancy cortisol associated with increased odds of preterm birth (at 13w0d; odds ratio, 1.007; 95% CI, 1.0002-1.014). Progesterone, cortisone, pregnanolone, and epipregnanolone did not associate with preterm birth. The findings from our pilot study suggest potential utility of cortisol as a maternal serum biomarker for preterm birth risk assessment in early pregnancy. Further evaluation using larger cohorts and additional gestational timepoints for ALLO and the other analytes may be informative.

Atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging of Neospora caninum-infected cell monolayers.

Neospora caninum is an obligate intracellular protozoan parasite of the phylum Alveolata (subphylum Apicomplexa) which has not been studied extensively in a biochemical context. N. caninum is a primary cause of reproductive disorders causing mummification and abortion not only in cattle but also in other small ruminant species resulting in a substantial economic impact on the livestock industry. In canids, which are the final hosts of N. caninum, clinical disease includes neuromuscular symptoms, ataxia, and ascending paralysis. Fatal outcomes of neosporosis have also been reported depending on the host species, age and immune status, however, its zoonotic potential is still uncertain. Therefore, N. caninum should be thoroughly investigated. Matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry (MS) and MS imaging (MSI) were used, combined with high-performance liquid chromatography (HPLC) to investigate these intracellular parasites. The aim of this study was to identify molecular biomarkers for N. caninum tachyzoite-infected host cells and to further clarify their functions. By atmospheric-pressure scanning microprobe MALDI MS(I), sections of N. caninum-infected and non-infected host cell pellets were examined in order to determine potential markers. In vivo, N. caninum infects different types of nucleated cells, such as endothelial cells which represent a highly immunoreactive cell type. Therefore, primary bovine umbilical vein endothelial cells were here used as a suitable infection system. For comparison, the permanent MARC-145 cell line was used as an additional, simplified in vitro cell culture model. HPLC-tandem MS (HPLC-MS/MS) experiments combined with database search were employed for structural verification of markers. The statistically relevant biomarkers found by MS and identified by HPLC-MS/MS measurements were partly also found in infected monolayers. Marker signals were imaged in cell layers of N. caninum-infected and non-infected host cells at 5µm lateral resolution.

Serum concentrations of oncometabolite, 2-hydroxyglutarate (2HG), as biomarkers for isocitrate dehydrogenase (IDH1/2) mutations in cholangiocarcinoma (ICCA).

3049 Background: Mutations in IDH1/2 genes are common in low-grade glioma (GM) and occur in approximately 20% of intrahepatic cholangiocarcinoma (ICCA). Mutant IDHs leads to preferential accumulation of the R-relative (R2HG) to the S-enantiomer (S2HG) of 2HG. We investigated the utility of circulating R2HG, total R2HG+S2HG (tRS) and ratio of R2HG/S2HG (rRS) as biomarkers in GM and ICCA patients (pts). Methods: Blood and tumor tissues samples were obtained from pts with IDH1/2-mutant GM and ICCA. IDH mutation was confirmed by either immunohistochemistry (GM pts) or next generation sequencing (ICCA pts). Samples were analyzed for S2HG and R2HG using a validated HPLC tandem mass spectrometry method. Tissue and serum R2HG, tRS and rRS were compared with paired t-tests for GM pts. Serum S2HG, R2HG, tRS and rRS were compared with unpaired t-tests between GM and ICCA pts. A p&lt;0.05 was considered statistically significant. Results: Blood and tumor samples were collected from 21 pts (GM = 11, ICCA=10). Tumor tissues were insufficient for analysis in 1 GM and 8 ICCA pts. Tissue S2HG, R2HG, tRS, and rRS were 0.72±0.68 ng/g, 48.92±58.92 ng/g, 66.87±80.35 ng/g and 55.77±36.23 for GM pts. There were no differences in R2HG and tRS between tissue and serum samples, while S2HG was significantly higher in serum samples ( p = 0.001) and rRS was significantly higher in tissue samples for GM pts ( p = 0.001). rRS were 144.8 and 244.9 respectively in 2 ICCA pts with sufficient tissues. There was no difference in serum S2HG between GM and ICCA pts. However, serum R2HG, tRS and rRS were significantly higher in ICCA pts. Conclusions: In IDH1/2 mutant GM and ICCA pts, R2HG was increased relative to S2HG in tumor tissues. In GM pts, the accumulation of R2HG in tumor tissues was not reflected in blood, likely due to the inability of 2HG to diffuse across the blood-brain barrier. Serum R2HG, tRS and rRS were significantly elevated in ICCA pts. These biomarkers have no clinical utilities in GM pts. However, they can potentially be used to identify IDH mutations in ICCA pts, especially given the inability to obtain tumor tissue in some ICCA pts. Clinical trial information: NCT03991832. [Table: see text]

A comparative in vitro study on monomer release from bisphenol A-free and conventional temporary crown and bridge materials.

This study aimed to investigate the release of common monomers from two conventional and two bisphenol A (BPA)-free temporary crown and bridge materials. Cylindrical samples of all materials were prepared (N=90; five samples for each material and cycle of analysis). All samples were immersed in high-performance liquid chromatography (HPLC)-grade water and incubated for 1 h, 12 h, 24 h, and 7 days in an incubation shaker at 37°C and 112rpm. Extraction was performed in accordance with ISO 10993-12. Eluted monomers were detected and quantified by HPLC coupled with ultraviolet-visible spectroscopy and mass spectrometry (HPLC-UV/Vis-MS). Analysis of BPA was performed by HPLC coupled with ultraviolet-visible spectroscopy (HPLC-UV/Vis) and positive results were verified by HPLC-tandem mass spectrometry (HPLC-MS/MS). Neither bisphenol A-glycidyl methacrylate (Bis-GMA) nor BPA was quantifiable in any of the crown and bridge samples investigated in the present study. However, all samples contained triethylene glycol dimethacrylate (TEGDMA) and/or urethane dimethacrylate (UDMA) after 24 h of incubation. Statistical analysis showed that significantly more UDMA was released from the BPA-free materials than from the conventional materials. All concentrations of UDMA measured were below the effective cytotoxic concentrations previously reported. However, for a few materials, especially BPA-free temporary crown and bridge materials, the levels of UDMA were above previously reported potentially harmful concentrations for local cells. As BPA-free materials were introduced as being more biocompatible than materials containing BPA, substitution of Bis-GMA with UDMA should be further investigated.

HPLC Method for Separation of Cannabidiol Hemp Seed Oil with Skin Lipids and Tandem HRMS Technology for Characterization of a Chemical Marker

Cannabidiol (CBD) hemp seed oil is a commercial raw material with antioxidant and anti-inflammatory benefits that has been formulated into body wash and skin care products. The biggest analytical challenge is how to simultaneously quantify CBD and hemp seed oil as they deposited on the skin surface. CBD is easily separated and quantified from skin surface extracts via a HPLC-mass spectrometry methodology. However, the structural skeleton of triacylglycerides (TAGs) in hemp seed oil is same as those from the skin surface sebum. The strong hydrophobicity with subtle structural difference challenges their separation. In this project, a new reverse phase HPLC-high resolution mass spectrometry methodology was developed with a strong mobile phase normal propanol. The separated hemp seed oil TAGs in the chromatogram were identified and characterized using data-dependent acquisition (DDA) technology. Based on the daughter ion characterization, the separated peak with an ammonium adduct at 890.7226 [M + NH4]+ was confirmed as the parent ion of glycerol with three omega-3 fatty acid chains. This is the first time TAG structure with direct HPLC-tandem mass spectrometry technology has been elucidated without a hydrolysis reaction. The confirmed TAG structure with an ammonium adduct at 890.7226 ± 0.0020 can be used as a representative chemical marker for the hemp seed oil quantification.

Monolith/aminated graphene oxide composite-based electric field-assisted solid phase microextraction for efficient capture of phenoxycarboxylic acids herbicides in environmental waters

Efficient capture of strongly polar, ionizable and trace phenoxycarboxylic acids herbicides (PCAHs) from aqueous samples is essential and challenging for environmental monitoring. In the present work, electric field-assisted solid-phase microextraction (EFA-SPME) based on monolith/aminated graphene oxide composite was developed for the first time to efficiently extract trace PCAHs prior to HPLC-tandem mass spectrometry (HPLC-MS/MS) quantification. First, poly (1-allyl–3-methylimidazole difluoromethanesulfonylamide salt-co-divinylbenzene/ethylene dimethacrylate) monolith/aminated graphene oxide composite (MAC) was prepared on the surface of stainless steel wire and employed as the extraction phase of SPME. After that, the MAC-based fiber and a stainless steel wire were connected to a DC power supply that allowed the implement of variable electric fields during adsorption and desorption processes. Various key factors influencing the extraction performance were inspected in detailed. Results well evidenced that the exertion of electric fields improved the enrichment performance, accelerated the trap and release procedures. The proposed MAC/EFA-SPME-HPLC-MS/MS method achieved wide linear ranges (0.005–50.0 μg/L), low limits of detection (0.54–1.3 ng/L) and good precision (2.7–7.0%) for the quantification of PCAHs. The related extraction mechanism was deduced. Additional, the current approach was successfully applied to monitor studied PCAHs at trace contents in environment waters, and the accuracy was confirmed by confirmatory experiments.

Novel bacteriocins produced by Lactobacillus fermentum strains with bacteriostatic effects in milk against selected indicator microorganisms

The aim of this work was to isolate and characterize bacteriocins produced by 2 Lactobacillus fermentum strains isolated from artisanal Mexican Cocido cheese. Fractions (F ≤3 kDa) obtained from cell-free supernatants of Lb. fermentum strains J23 and J32 were further fractionated by reversed-phase HPLC on a C18 column. Antimicrobial activities of F ≤3 kDa and bacteriocin-containing fractions (BCF), obtained from fractionation of F ≤3 kDa against 4 indicator microorganisms, were determined by the disk diffusion method and growth inhibition in milk. Subsequently, isolated BCF were analyzed by reversed-phase HPLC tandem mass spectrometry. Results showed that BCF presented antimicrobial activity against the 4 indicator microorganisms tested. For J23, one of the fractions (F3) presented the highest activity against Escherichia coli and was also inhibitory against Staphylococcus aureus, Listeria innocua, Salmonella Typhimurium, and Salmonella Choleraesuis. Similarly, fractions F3 and F4 produced by J32 presented antimicrobial activity against all indicator microorganisms. Furthermore, generation time and growth rate showed that F3 from J23 presented significantly higher antimicrobial activity against the 4 indicator microorganisms (2 gram-positive and 2 gram-negative) when inoculated in milk compared with F3 from J32. Interestingly, this fraction presented a broader antimicrobial spectrum in milk than nisin (control). Reversed-phase HPLC tandem mass spectrometry analysis revealed the presence of several peptides in BCF; however, F3 from J23 that was the most active fraction of all presented only 1 bacteriocin. The chemical characterization of this bacteriocin suggested that it was a novel peptide with 10 hydrophobic AA residues in its sequence and a molecular weight of 2,056 Da. This bacteriocin and its producing strain, J23, may find application as a biopreservative against these indicator microorganisms in dairy products.

Comparison of Proteomics Profiles Between Xenografts Derived from Cell Lines and Primary Tumors of Thyroid Carcinoma.

Patient-consistent xenograft model is a challenge for all cancers but particularly for thyroid cancer, which shows some of the greatest genetic divergence between human tumors and cell lines. In this study, proteomic profiles of tumor tissues from patients, included anaplastic thyroid carcinoma (ATC) and papillary thyroid carcinoma, and xenografts (8305C, 8505C, FRO, BAPAP and IHH4) were obtained using HPLC-tandem mass spectrometry and compared based on all proteins detected (3,961), cancer-related proteins and druggable proteins using pairwise Pearson's correlation analysis. The human tissue showed low proteomic similarity to the ATC cell lines (8305C, r = 0.344-0.416; 8505C, 0.47-0.579; FRO, 0.267-0.307) and to PTC cell lines (BCPAP, 0.303-0.468; IHH4, 0.262-0.509). Human tissue showed the following similarity to cell lines at the level of 135 cancer-related pathways. The ATC cell lines contained 47.4% of the cancer-related pathways (19.26%-33.33%), while the PTC cell lines contained 40% (BCPAP, 25.93%; IHH4, 28.89%). In patient tumor tissues, 44-60 of 76 and 52-53 of 93 druggable proteins were identified in ATC and PTC tumors, respectively. Ten and 29 druggable proteins were not identified in any of the ATC and PTC xenografts, respectively. We provide a reference for CDX selecting in in vivo studies of thyroid cancer.

Identifying and characterizing the components related to the brown color of Chinese sugar-smoked chicken during processing

The desired color is a key indicator for consumer acceptability of Chinese sugar-smoked chicken. To investigate the formation of color attributes of Chinese sugar-smoked chicken during processing, color values, structural characteristics, and components of brown pigment were evaluated in 2 groups, which were defined as brown skin (BS) and normal skin (NS) of Chinese sugar-smoked chicken based on their color values. Compared with the NS samples, the BS samples showed significantly lower values of lightness, redness, and yellowness and higher content of malondialdehyde and 5-hydroxymethylfurfural. UV–visible and Fourier-transform infrared spectra suggested that the structure of brown pigment was similar to melanin. The brown pigment consisted of multiple chemical components including the polymer of fructose and glucose, and derivatives produced by lipid oxidation, which were identified by HPLC–tandem mass spectrometry. The polymer content of glucose and fructose, which was demonstrated as sucrose by HPLC analysis, was higher in the BS group than in the NS group. Our results indicated that the higher content of the polymer of glucose and fructose was mainly responsible for the brown color of Chinese sugar-smoked chicken.

Serum Levels of Vitamin A and Vitamin D and Their Association With Symptoms in Children With Attention Deficit Hyperactivity Disorder

Objective: To measure levels of vitamin A (VA) and vitamin D (VD) and the symptomatic association of their co-deficiencies on attention deficit hyperactivity disorder (ADHD) in Chinese children (6–9 years).Methods: Eighty-two children (69 boys and 13 girls; mean age = 7.1 ± 0.9 years at the time of the diagnosis) with ADHD were recruited as ADHD group. A total of 106 healthy children were recruited as the healthy control (HC) group. Serum levels of retinol and 25-hydroxyvitamin D (25(OH)D) of all children were evaluated using high-performance liquid chromatography (HPLC) and HPLC-tandem mass spectrometry. The Swanson, Nolan, and Pelham IV Rating Scale (SNAP-IV) was employed to assess the clinical symptoms of ADHD.Results: Children suffering from ADHD had significantly reduced serum levels of retinol and 25(OH)D compared with those of HCs, and the prevalence of VA deficiency and VD deficiency were higher in children suffering from ADHD. Serum concentrations of 25(OH)D and retinol were linked closely with the presence or absence of ADHD after adjustment for age, body mass index, season of blood sampling, and sun exposure. Serum concentrations of 25(OH)D and retinol showed a negative correlation with the total scores of SNAP-IV. Children with ADHD as well as VA and VD co-deficiency had increased SNAP-IV total scores and ADHD inattention subscale scores.Conclusion: VA deficiency and VD deficiency in children with ADHD were increased in comparison with that in HCs. VA and VD co-deficiency associated with ADHD symptom severity. Attention should be paid to regular testing of VA levels and VD levels. However, the mechanism of VA and VD in ADHD needs to be further studied. Interventional studies on VA and VD supplementation are recommended to further verify the relationship between VA and VD co-deficiency and ADHD.