Several secondary metabolite gene clusters in the genomes of ten Penicillium spp. raise the risk of multiple mycotoxin occurrence in chestnuts.

Penicillium spp. produce a great variety of secondary metabolites, including several mycotoxins, on food substrates. Chestnuts represent a favorable substrate for Penicillium spp. development. In this study, the genomes of ten Penicillium species, virulent on chestnuts, were sequenced and annotated: P. bialowiezense. P. pancosmium, P. manginii, P. discolor, P. crustosum, P. palitans, P. viridicatum, P. glandicola, P. taurinense and P. terrarumae. Assembly size ranges from 27.5 to 36.8 Mb and the number of encoded genes ranges from 9,867 to 12,520. The total number of predicted biosynthetic gene clusters (BGCs) in the ten species is 551. The most represented families of BGCs are non ribosomal peptide synthase (191) and polyketide synthase (175), followed by terpene synthases (87). Genome-wide collections of gene phylogenies (phylomes) were reconstructed for each of the newly sequenced Penicillium species allowing for the prediction of orthologous relationships among our species, as well as other 20 annotated Penicillium species available in the public domain. We investigated in silico the presence of BGCs for 10 secondary metabolites, including 5 mycotoxins, whose production was validated in vivo through chemical analyses. Among the clusters present in this set of species we found andrastin A and its related cluster atlantinone A, mycophenolic acid, patulin, penitrem A and the cluster responsible for the synthesis of roquefortine C/glandicoline A/glandicoline B/meleagrin. We confirmed the presence of these clusters in several of the Penicillium species conforming our dataset and verified their capacity to synthesize them in a chestnut-based medium with chemical analysis. Interestingly, we identified mycotoxin clusters in some species for the first time, such as the andrastin A cluster in P. flavigenum and P. taurinense, and the roquefortine C cluster in P. nalgiovense and P. taurinense. Chestnuts proved to be an optimal substrate for species of Penicillium with different mycotoxigenic potential, opening the door to risks related to the occurrence of multiple mycotoxins in the same food matrix.

Metabolic regulation mechanism of melatonin for reducing cadmium accumulation and improving quality in rice.

Melatonin acts as a potential regulator of cadmium (Cd) tolerance in rice. However, its practical value in rice production remains unclear. To validate the hypothesis that melatonin affects Cd accumulation and rice quality, a series of experiments were conducted. The results showed that exogenous melatonin application was associated with reduced Cd accumulation (23-43%) in brown rice. Fourier transform infrared spectroscopy (FTIR) analysis showed that exogenous melatonin affected the rice protein secondary structure and starch short-range structure. Metabolomics based on LC-MS/MS revealed that exogenous melatonin altered the brown rice metabolic profile, decreased fatty acid metabolite content, but increased amino acid metabolite, citric acid, melatonin biosynthetic metabolite, and plant hormone contents. These findings indicate that exogenous melatonin can effectively reduced Cd accumulation and improve rice quality through metabolic network regulation, serving as an effective treatment for rice cultivated in Cd-contaminated soil.

Evaluation of transport mechanisms of methotrexate in human choriocarcinoma cell lines by LC-MS/MS.

Methotrexate (MTX) is commonly prescribed as the initial treatment for gestational trophoblastic neoplasia (GTN), but MTX monotherapy may not be effective for high-risk GTN and choriocarcinoma. The cellular uptake of MTX is essential for its pharmacological activity. Thus, our study aimed to investigate the cellular pharmacokinetics and transport mechanisms of MTX in choriocarcinoma cells. For the quantification of MTX concentrations in cellular matrix, a liquid chromatography-tandem mass spectrometry method was created and confirmed initially. MTX accumulation in BeWo, JEG-3, and JAR cells was minimal. Additionally, the mRNA levels of folate receptor α (FRα) and breast cancer resistance protein (BCRP) were relatively high in the three choriocarcinoma cell lines, whereas proton-coupled folate transporter (PCFT), reduced folate carrier (RFC), and organic anion transporter (OAT) 4 were low. Furthermore, the expression of other transporters was either very low or undetectable. Notably, the application of inhibitors and small interfering RNAs (siRNAs) targeting FRα, RFC, and PCFT led to a notable decrease in the accumulation of MTX in BeWo cells. Conversely, the co-administration of multidrug resistance protein 1 (MDR1) and BCRP inhibitors increased MTX accumulation. In addition, inhibitors of OATs and organic-anion transporting polypeptides (OATPs) reduced MTX accumulation, while peptide transporter inhibitors had no effect. Results from siRNA knockdown experiments and transporter overexpression cell models indicated that MTX was not a substrate of nucleoside transporters. In conclusion, the results indicate that FRα and multiple transporters such as PCFT, RFC, OAT4, and OATPs are likely involved in the uptake of MTX, whereas MDR1 and BCRP are implicated in the efflux of MTX from choriocarcinoma cells. These results have implications for predicting transporter-mediated drug interactions and offer potential directions for further research on enhancing MTX sensitivity.

Untargeted lipidomics of bronchopulmonary dysplasia induced by hyperoxia exposure in rats.

Background: Bronchopulmonary dysplasia (BPD), characterized by impaired lung development, remains a leading cause of morbidity and mortality in premature infants. The synthesis and metabolism of lipids play a critical role in normal lung development, such as dipalmitoylphosphatidylcholine, a key component of pulmonary surfactant (PS). Therefore, we conducted a lipidomics study of rat lung tissue to explore the changes of pulmonary lipid composition in the progression of BPD disease. Methods: In this study, we exposed neonatal Sprague-Dawley (SD) rats to hyperoxia for 14 days. After hyperoxia exposure, the lung tissues of rats were analyzed pathologically, and untargeted lipidomics was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results: Hematoxylin-eosin (H&E) staining showed that the alveoli enlarged, the number of alveoli decreased and the pulmonary surfactant-associated protein D (SFTPD) decreased in hyperoxia-exposed rats. A total of 620 pulmonary lipids were detected by LC-MS/MS, covering 27 lipid categories. The most common lipids were triacylglycerol (TAG), followed by phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Conclusions: Compared with those rats exposed to normoxic conditions, the lipid levels in the lungs of rats exposed to hyperoxia for 14 days generally decreased, with the levels of TAG and PC decreasing most significantly. In short, our results provide a clue for finding therapeutic targets and biomarkers of a BPD rat model lung liposome.

Pterin Profiling in Serum, Dried Blood Spot, and Urine Samples Using LC-MS/MS in Patients with Inherited Hyperphenylalaninemia.

Background: Hyperphenylalaninemia (HPA) is defined as blood phenylalanine (Phe) levels exceeding the normal values (>120 µmol/L or >2 mg/dL) and is caused by a deficiency in the enzyme phenylalanine hydroxylase (PAH). The widespread screening of Phe levels in newborn screening programs has led to a very high number of patients with HPA. Methods: The samples were collected at various ages, not at the point of diagnosis. Nine pterin derivatives, including isoxanthopterin, sepiapterin, xanthopterin, primapterin, biopterin, neopterin, 7,8-dihydrobiopterin, 7,8-dihydroneopterin, and tetrahydrobiopterin (BH4), were analyzed in different HPA classes in serum, dried blood spots (DBS), and urine samples. A total of 18 patients, including six classical phenylketonuria (PKU), eight BH4-responsive PKU, and four mild HPA patients, were included in the study. Results: Among the nine pterin derivatives measured, a significant increase was observed in the levels of isoxanthopterin, biopterin, and 7,8-dihydrobiopterin in serum, dried blood spots (DBS), and urine samples of patients with HPA compared to the control group. However, elevations in isoxanthopterin, biopterin, and 7,8-dihydrobiopterin were observed in all HPA groups, although the extent of elevation varied among the different disease groups. There were also significant differences between HPA subgroups among these high values. Conclusion: In this study, it might be suggested that pterin profiling shows promising potential for its effective utilization in the differential diagnosis of HPA. Pterin profiling demonstrated its efficacy in accurately categorizing patients into distinct subtypes. This approach offers several notable advantages, including the ability to simultaneously screen multiple HPA subtypes through a single test, establish disease decision limits for pterins, shorten the time required for HPA differential diagnosis, reduce the risk of misdiagnosis, and increase overall diagnostic accuracy. This study is the most comprehensive study examining the association between HPA pterin in the literature. In our study, samples obtained from BH4-responsive PKU patients were on treatment. This may have affected the results. Preliminary findings on pterin profiles may need to be replicated in a prospective cohort of samples collected at the time of diagnosis to confirm the results.

Application of newly developed and validated LC-MS/MS method for pharmacokinetic study of adagrasib and pembrolizumab simultaneously in rat plasma.

Non-small cell lung cancer (NSCLC) is a significant subtype of lung cancer, and poses a dangerous global threat. One of the current approaches of NSCLC treatment is a combination therapy of adagrasib and pembrolizumab. Accurate monitoring of these drug concentrations in biological fluids is critical for treatment efficacy. Since no method was reported for simultaneous estimation of these drugs, this study focuses on the development of a validated LC-MS/MS bioanalytical method for simultaneous quantification of Adagrasib and Pembrolizumab in rat plasma. The analytes were extracted from the biological matrix through liquid-liquid extraction techniques using acetonitrile as extraction solvent. The analytes were separated on a Waters X-bridge phenyl C18 column, with a mixture of acetonitrile: 0.1 % TFA in water (50: 50 v/v) as mobile phase at an isocratic flow rate of 1.0 mL/min with a runtime of about 5 min. Adagrasib (m/z 605.12 → 201.62), Pembrolizumab (m/z 146.32 → 85.15), and Sotorasib (m/z 561.59 → 218.92) were determined by recording the mass spectra through multiple reaction monitoring in positive mode. The method was validated according to USFDA guidelines. The results demonstrate satisfactory linearity with an r2 value of 0.9998 in the ranges of 40-800 and 10-200 ng/mL, accuracy with mean percentage recovery of 95.22-98.59 % and 96.98-98.57 %, precision indicated with %RSD ranged between 0.39-1.91 % and 0.85-9.03 % for Adagrasib and Pembrolizumab respectively, and other key parameters. The developed method can determine the pharmacokinetic parameters to indicate the efficacy and safety of the drugs, and also can quantify selected drugs simultaneously in biological samples.

An insight into the antioxidant activities and the exploration of record high quantities of quercetin and luteolin in the plant parts of Myxopyrum smilacifolium (wall.) Blume.

This study attempts to reveal antioxidants in the plant parts of Myxopyrum smilacifolium (Wall.) Blume using antioxidant assays and LC-MS/MS analysis. Methanol is the most effective solvent for collecting antioxidants. The roots-derived methanol extract demonstrates the greatest antioxidant activity, corresponding to the extremely low IC50 values of 16.39 µg/mL and 19.80 µg/mL for DPPH and ABTS radicals, respectively. The high phenolic and flavonoid contents are the primary reason for outstanding total antioxidant capacity (TAC; i.e. 247.73 ± 1.62 mg GA/g or 163.93 ± 0.83 mg AS/g) of the root extract. LC-MS/MS quantification of five phenolic compounds reveals exceptionally high amounts of quercetin and luteolin in the root extract, ranging from 238.86 ± 5.74 to 310.99 ± 1.44 µg/g and from 201.49 ± 7.84 to 234.10 ± 2.54 µg/g, respectively, in the root-derived methanol extract. The achievement highlights M. smilacifolium as a promising source of natural antioxidants for large-scale medical applications.

Discovery of anti-melanogenic components in persimmon (Diospyros kaki) leaf using LC-MS/MS-MN, AlphaFold2-enabled virtual screening and biological validation.

Persimmon (Diospyros kaki) leaf is widely used as a tea substitute in East Asia, offering potential health benefits. Although studies have highlighted their effects on hyperpigmentation disorders, the active components remain unidentified. This study introduces a novel approach combining LC-MS/MS-based molecular networking with AlphaFold2-enabled virtual screening to expedite the identification of bioactive components in persimmon leaf. A total of 105 compounds were identified by MS/MS analysis. Further, virtual screening identified five flavonoids with potential anti-melanogenic properties. Bioassays confirmed myricetin, quercetin, and kaempferol inhibited melanogenesis in human melanocytes in a dose-dependent manner. Biolayer interferometry assays revealed strong binding affinity between these flavonols and hsTYR, with KD values of 23.26 ± 11.77 for myricetin, 12.43 ± 0.37 for quercetin, and 14.99 ± 3.80 µM for kaempferol. Molecular dynamics simulations provided insights into the binding interactions of these flavonols with hsTYR, particularly highlighting the essential role of the 3-OH group on the C-ring. This study elucidates the bioactive components responsible for the anti-melanogenic effects of persimmon leaf, supporting their use in product development.

Analysis of Metabolic Changes in Endogenous Metabolites and Diagnostic Biomarkers for Various Diseases Using Liquid Chromatography and Mass Spectrometry.

Analysis of endogenous metabolites in various diseases is useful for searching diagnostic biomarkers and elucidating the molecular mechanisms of pathophysiology. The author and collaborators have developed some LC/tandem mass spectrometry (LC/MS/MS) methods for metabolites and applied them to disease-related samples. First, we identified urinary conjugated cholesterol metabolites and serum N-palmitoyl-O-phosphocholine serine as useful biomarkers for Niemann-Pick disease type C (NPC). For the purpose of intraoperative diagnosis of glioma patients, we developed the LC/MS/MS analysis methods for 2-hydroxyglutaric acid or cystine and found that they could be good differential biomarkers. For renal cell carcinoma, we searched for various biomarkers for early diagnosis, malignancy evaluation and recurrence prediction by global metabolome analysis and targeted LC/MS/MS analysis. In pathological analysis, we developed a simultaneous LC/MS/MS analysis method for 13 steroid hormones and applied it to NPC cells, we found 6 types of reductions in NPC model cells. For non-alcoholic steatohepatitis (NASH), model mice were prepared with special diet and plasma bile acids were measured, and as a result, hydrophilic bile acids were significantly increased. In addition, we developed an LC/MS/MS method for 17 sterols and analyzed liver cholesterol metabolites and found a decrease in phytosterols and cholesterol synthetic markers and an increase in non-enzymatic oxidative sterols in the pre-onset stage of NASH. We will continue to challenge themselves to add value to clinical practice based on cutting-edge analytical chemistry methodology.

Dereplication of 4-Quinolone Alkaloids from Waltheria indica (Malvaceae) Tissues Using Molecular Network Tools.

Waltheria indica (Malvaceae) is a plant popularly used in folk medicine by traditional African and indigenous communities, and in various countries worldwide, to treat general inflammation. Several biological activities of this plant have been reported, including acetylcholinesterase inhibition and potential anti-human immunodeficiency virus (HIV), antinociceptive, analgesic, antifungal, anticancer, anti-inflammatory, leishmanicidal, trypanocidal, antioxidant, and antibacterial activities. The chemical profile of Waltheria indica was assessed by dereplication analysis using UPLC-MS/MS, and data acquisition was performed using chemoinformatics tools, such as Mass Spectrometry-Data Independent AnaLysis (MS-DIAL) and MS-FINDER softwares. The preprocessed data were sent to the GNPS to build a feature-based molecular network (FBMN). Thirty-three 4-quinolone alkaloids were annotated in the extracts and fractions of stems and roots, whereas 12 were annotated in the extracts and fractions of flowers and leaves. This represents an inaugural chemical investigation study employing UPLC-Q-TOF-MS/MS analysis, along with a molecular network approach, within this species and genus.

A new method based on dispersive solid phase microextraction with commercial MOFs coupled to LC-MS/MS for the determination of isoflavones in soy drinks.

For the first time, a method based on dispersive solid phase microextraction (D-µSPE) using commercial metal-organic frameworks coupled to liquid chromatography-triple quadrupole tandem mass spectrometry (LC-MS/MS) has been proposed for the determination of isoflavones in soy drinks. The use of commercial sorbents simplifies the sample treatment procedure and allows their application to routine analysis. Optimization of the parameters involved in the microextraction process was carried out using a Box-Behnken experimental design. Under the optimized conditions, the limits of detection ranged between 2 and 7 µg L-1; the intra-day and inter-day precision were <10 and 20%, respectively, and the recoveries were in the range of 61-120%. No significant matrix effect was found, which allowed the use of external standard calibration method. The method was successfully applied to the determination of isoflavones in commercial soy milk samples.

Ziziphus spina-christi L. extract attenuates bleomycin-induced lung fibrosis in mice via regulating TGF-ß1/SMAD pathway: LC-MS/MS Metabolic profiling, chemical composition, and histology studies.

Ancient Egyptians (including Bedouins and Nubians) have long utilized Ziziphus spina-christi (L.), a traditional Arabian medicinal herb, to alleviate swellings and inflammatory disorders. It is also mentioned in Christian and Muslim traditions. Ziziphus spina-christi L. (Family: Rhamnaceae) is a plentiful source of polyphenols, revealing free radical scavenging, antioxidant, metal chelating, cytotoxic, and anti-inflammatory activities. Herein, different classes of the existing bioactive metabolites in Z. spina-christi L. were detected using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the first time. The study also aimed to assess the anti-inflammatory and antifibrotic properties of Z. spina-christi L. extract against bleomycin-induced lung fibrosis in an experimental mouse model. 32 male Swiss Albino mice were assigned into 4 groups; the first and second were the normal control group and the bleomycin positive control (single 2.5 U/kg bleomycin intratracheal dose). The third and fourth groups received 100 and 200 mg/kg/day Z. spina-christi L. extract orally for 3 weeks, 2 weeks before bleomycin, and 1 week after. The bioactive metabolites in Z. spina-christi L. extract were identified as phenolic acids, catechins, flavonoids, chalcones, stilbenes, triterpenoid acids, saponins, and sterols. The contents of total phenolic compounds and flavonoids were found to be 196.62 mg GAE/gm and 33.29 mg QE/gm, respectively. In the experimental study, histopathological examination revealed that lung fibrosis was attenuated in both Z. spina-christi L.- treated groups. Z. spina-christi L. extract downregulated the expression of nuclear factor kappa B (NF-κB) p65 and decreased levels of the inflammatory markers tumor necrosis factor-alpha (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and c-Jun N-terminal kinase (JNK) in lung tissue. Z. spina-christi L. also downregulated the expression of the fibrotic parameters collagen-1, alpha-smooth muscle actin (α-SMA), transforming growth factor-beta 1 (TGF-ß1), matrix metalloproteinase-9 (MMP-9) and SMAD3, with upregulation of the antifibrotic SMAD7 in lung tissue. Overall, the present study suggests a potential protective effect of Z. spina-christi L. extract against bleomycin-induced lung fibrosis through regulation of the TGF-ß1/SMAD pathway.

LC-MS/MS quantification of Δ8-THC, Δ9-THC, THCV ISOMERS and their main metabolites in human plasma.

BACKGROUND: In recent years, potential therapeutic applications of several different cannabinoids, such as Δ9-tetrahydrocannabinol (Δ9-THC), its isomer Δ8-THC and Δ9-tetrahydrocannabivarin (Δ9-THCV), have been investigated. Nevertheless, to establish dose-effect relationship and to gain knowledge of their pharmacokinetics and metabolism, sensitive and specific analytical assays are needed to measure these compounds in patients. For this reason, we developed and validated an online extraction high-performance liquid chromatography- tandem mass spectrometry (LC/LC-MS/MS) method for the simultaneous quantification of 13 cannabinoids and metabolites including the Δ8 and Δ9 isomers of THC, THCV and those of their major metabolites in human plasma. METHODS: Plasma was fortified with cannabinoids at varying concentrations within the working range of the respective compound and 200 µL were extracted using a simple one-step protein precipitation procedure. The extracts were analyzed using online trapping LC/LC-atmospheric pressure chemical ionization (APCI)-MS/MS running in the positive multiple reaction monitoring (MRM) mode. RESULTS: The lower limit of quantification ranged from 0.5 to 2.5 ng/mL and the upper limit of quantification was 400 ng/mL for all analytes. Inter-day analytical accuracy and imprecision ranged from 82.9 to 109% and 4.3 to 20.3% (coefficient of variance), respectively. Of 534 plasma samples following controlled oral administration of Δ8-THCV, 236 were positive for Δ8-THCV (median; interquartile ranges: 3.5 ng/mL; 1.8 - 11.9 ng/mL), 383 for the major metabolite (-)-11-nor-9-carboxy-Δ8-tetrahydrocannabivarin (Δ8-THCV-COOH) (95.4 ng/mL; 20.7 - 328 ng/mL), 260 for (-)-11-nor-9-carboxy-Δ9-tetrahydrocannabivarin (Δ9-THCV-COOH) (5.8 ng/mL; 2.5 - 16.1 ng/mL), 157 for (-)-11-hydroxy-Δ8-tetrahydrocannabivarin (11-OH-Δ8-THCV) (1.7 ng/mL; 1.0 - 3.7 ng/mL), 49 for Δ8-THC-COOH (1.7 ng/mL; 1.4 - 2.3 ng/mL) and 42 for Δ9-THCV (1.3 ng/mL; 0.8 - 1.6 ng/mL). CONCLUSIONS: We developed and validated the first LC/LC-MS/MS assay for the specific quantification of Δ8-THC, Δ9-THC and THCV isomers and their respective metabolites in human plasma. Δ8-THCV-COOH, 11-hydroxy-Δ8-THCV and Δ9-THCV-COOH were the major Δ8-THCV metabolites in human plasma after oral administration of 98.6% pure Δ8-THCV.

Serum Organophosphorus compound levels as a surrogate marker for severity assessment and management in Acute Organophosphorus Poisoning - A novel approach.

Organophosphorus (OP) compounds are the most extensively used pesticides' class worldwide; cause most self­poisoning deaths especially in India. Thus, it is utmost important for early identification and aggressive management of OP poisoning from the clinical perspective to prevent serious complications by using sophisticated LC-MS/MS approach. This was a prospective study involving 103 patients of OP cases admitted to Karnataka Institute of Medical Sciences from June 2022 to May 2023, based on the inclusion and exclusion criteria patients were subjected to study. On admission, venous blood was collected from patient with Malathion and Profenofos OP poisoning history and subjected to serum biomarker and to LC-MS/MS analysis. Out of the 103 patients, 68 patients consumed Profenofos (66%) and 35 patients consumed Malathion (34%). Pseudocholinesterase levels among the of OP cases revealed that the 33 patients had mild toxicity, 40 patients had moderate toxicity and 30 patients had severe toxicity of OP poisoning. Subsequently LC-MS/MS analysis showed that the results obtained are not in correlation with indirect serum marker pseudocholinesterase levels. On the other side, LC-MS/MS results are in correlation with the clinical outcome of the patients with respect to morbidity and mortality. Thus, LC-MS/MS approach to assess the OP levels in patients could be used as potential diagnostic and prognostic marker for the absolute quantification of OP compounds compared to indirect OP levels estimation.

Okadaic acid as a major problem for the seafood safety (Mytilus galloprovincialis) and the dynamics of toxic phytoplankton in the Slovenian coastal sea (Gulf of Trieste, Adriatic Sea).

This article presents the first results on shellfish toxicity in the Slovenian sea (Gulf of Trieste, Adriatic Sea) since the analytical methods for the detection of biotoxins (PSP, ASP, DSP and other lipophilic toxins) in bivalve molluscs were included in the national monitoring program in 2013. In addition to toxins, the composition and abundance of toxic phytoplankton and general environmental characteristics of the seawater (surface temperature and salinity) were also monitored. During the 2014-2019 study period, only lipophilic toxins were detected (78 positive tests out of 446 runs), of which okadaic acid (OA) predominated in 97 % of cases, while dinophysistoxin-2 and yessotoxins only gave a positive result in one sampling event each. The number of samples that did not comply with the EC Regulation for the OA group was 17 or 3.8 % of all tests performed, all of which took place from September to November, while a few positive OA tests were also recorded in December, April, and May. This toxicity pattern was consistent with the occurrence pattern of the five most common DSP-producing dinoflagellates, which was supported by the development of warm and thermohaline stratified waters: Dinophysis caudata, D. fortii, D. sacculus, D. tripos and Phalacroma rotundatum. The strong correlation (r = 0.611, p < 0.001) between D. fortii, reaching abundances of up to 950 cells L-1, and OA suggests that D. fortii is the main cause of OA production in Slovenian waters. Strong interannual variations in OA and phytoplankton dynamics, exacerbated by the effects of anthropogenic impacts in this coastal ecosystem, reduce the predictability of toxicity events and require continuous and efficient monitoring. Our results also show that the introduction of the LC-MS/MS method for lipophilic toxins has improved the management of aquaculture activities, which was not as accurate based on mouse bioassays.

Nrf2 pathway activation promotes the expression of genes related to glutathione metabolism in alcohol-exposed astrocytes.

Introduction: Oxidative and antioxidant pathways play essential roles in the development of alcohol-induced brain injury. The Nrf2 pathway is an endogenous antioxidant response pathway, but there has been little research on the role of Nrf2 in alcohol-related diseases. Thus, we examined the effects of alcohol and an Nrf2 agonist (TBHQ) on astrocyte function, mRNA expression, and metabolite content to further explore the protective mechanisms of Nrf2 agonists in astrocytes following alcohol exposure. Methods: CTX TNA2 astrocytes were cultured with alcohol and TBHQ and then subjected to transcriptome sequencing, LC-MS/MS analysis, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and malondialdehyde (MDA) and superoxide dismutase (SOD) activity assays. Results: Alcohol exposure significantly increased malondialdehyde (MDA) levels while decreasing superoxide dismutase (SOD) levels in astrocytes. Treatment with TBHQ effectively reversed these effects, demonstrating its protective role against oxidative stress induced by alcohol. Transcriptome sequencing and qRT-PCR analysis revealed that TBHQ specifically upregulates genes involved in glutathione metabolism, including a notable increase in the expression of the glutathione S-transferase A5 (GSTA5) gene, which was suppressed by alcohol exposure. Additionally, metabolomic analysis showed that TBHQ regulates key components of ether lipid metabolism in alcohol-exposed astrocytes, with significant reductions in the levels of lysophosphatidylcholine (18:0) (LysoPC (18:0)) and 2-acetyl-1-alkyl-sn-glycero-3-phosphocholine, both of which are critical markers in the ether lipid metabolic pathway. Discussion: The findings underscore the role of TBHQ as an Nrf2 agonist in mitigating alcohol-induced oxidative damage in astrocytes by modulating glutathione metabolism and ether lipid metabolism. The regulation of GSTA5 gene expression emerges as a key mechanism through which Nrf2 agonists confer neuroprotection against oxidative stress and lipid oxidation. These insights pave the way for potential therapeutic strategies targeting the Nrf2 pathway to protect astrocytes from alcohol-induced damage.

Triethanolamine-based esterquat in sediments: New analytical method, environmental occurrence, and homologue composition.

The cationic surfactant triethanolamine-based esterquat (TEAQ) is a main ingredient in commercial fabric softeners and is produced and used in large quantities. However, little information is available for its occurrence in the environment, particularly in sediments. Here, we developed an analytical method for quantifying TEAQ in sediment and investigated TEAQ contamination in Japanese river and lake sediments. In our analytical method, TEAQ concentrations were measured by liquid chromatography-tandem mass spectrometry using a polymer-based size-exclusion column, which resulted in excellent peak shapes. TEAQ was detected at significant levels in procedural blanks, resulting in a method limit of detection in the sediment of 8.9-97 µg/kg-dry for TEAQ monoesters and 0.6-24 µg/kg-dry for TEAQ diesters. TEAQ was detected in 22 out of 26 sediment samples, with the sum of all homologue concentrations being up to 1340 µg/kg-dry. The concentration of TEAQ in sediments was high at locations where the concentrations of benzalkoniums and the organic carbon content were also high. TEAQ was detected in 8 out of 14 commercial fabric softeners at concentrations of 1.7-7.4 wt%. TEAQ homologues containing only saturated fatty acids accounted for 83 ± 5% of the total TEAQ in the sediments, whereas those with unsaturated fatty acids accounted for 71 ± 14% of the total TEAQ in a commercial technical mixture and the softener products. The results of this study will be useful for the environmental risk assessment of esterquats.

LC-MS/MS analysis of didehydrostemofoline from Stemona collinsiae roots extracts in rats plasma and pharmacokinetics profile after oral administration.

Stemona collinsiae Craib., Stemonaceae, has been traditionally used as medicinal plants for insecticides, treatment of parasitic worms and various diseases in Southeast Asian countries. Its ethanolic root extract has been postulated for anthelminthic activities which has a potential for development for human gnathostomiasis drug. To investigate the pharmacokinetic profile, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method for the quantification of didehydrostemofoline in rats' plasma was developed and validated. The chromatographic separation was performed on a C18 column using 1 mM ammonium acetate in water and methanol (50:50, v/v). Tetrahydropalmatine was used as an internal standard. The multiple reaction monitoring mode was used for quantitative analysis. The validated method showed good sensitivity, linearity, precision, and accuracy. The results of stability showed that didehydrostemofoline was stable in the extracted samples in auto-sampler for 24 h and in the plasma samples under room temperature for 24 h, -20 °C for 1 month, and after three freeze-thaw processes. The developed method was applied to the pharmacokinetic study of didehydrostemofoline after oral administration of S. collinsiae root extract. Didehydrostemofoline was rapidly absorbed from the gastrointestinal tract. The time to peak drug concentration was 1.75 ± 0.62 h with maximum drug concentration of 1152.58 ± 271.18 ng/mL. Didehydrostemofoline was rapidly eliminated from the body with terminal half-life of 1.86 ± 0.50 h. Calculated drug clearance of didehydrostemofoline was 96.82 ± 23.51 L/h and volume of distribution was 260.40 ± 96.81 L. The present study provided useful data for understanding drug disposition in the body with dynamic time-course which could be beneficial for further clinical trials.

Urine Proteomic Signatures of Mild Hypothermia Treatment in Cerebral Ischemia-Reperfusion Injury in Rats.

Mild hypothermia (MH) is an effective measure to alleviate cerebral ischemia-reperfusion (I/R) injury. However, the underlying biological mechanisms remain unclear. This study set out to investigate dynamic changes in urinary proteome due to MH in rats with cerebral I/R injury and explore the neuroprotective mechanisms of MH. A Pulsinelli's four-vessel occlusion (4-VO) rat model was used to mimic global cerebral I/R injury. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to profile the urinary proteome of rats with/without MH (32 °C) treatment after I/R injury. Representative differentially expressed proteins (DEPs) associated with MH were validated by western blotting in hippocampus. A total of 597 urinary proteins were identified, among which 119 demonstrated significant changes associated with MH. Gene Ontology (GO) annotation of the DEPs revealed that MH significantly enriched in endopeptidase activity, inflammatory response, aging, response to oxidative stress and reactive oxygen species, blood coagulation, and cell adhesion. Notably, changes in 12 DEPs were significantly reversed by MH treatment. Among them, 8 differential urinary proteins were previously reported to be closely associated with brain disease, including NP, FZD1, B2M, EPCR, ATRN, MB, CA1and VPS4A. Two representative proteins (FZD1, B2M) were further validated by western blotting in the hippocampus and the results were shown to be consistent with urinary proteomic analysis. Overall, this study strengthens the idea that urinary proteome can sensitively reflect pathophysiological changes in the brain, and appears to be the first study to explore the neuroprotective effects of MH by urinary proteomic analysis. FZD1 and B2M may be involved in the most fundamental molecular biological mechanisms of MH neuroprotection.

Detection of 13 emerging soil pollutant compounds using a dual extraction method (QuEChERS and solid phase extraction) and a liquid chromatography/mass spectrometry LC-MS/MS method.

Emerging pollutants derived from human and animal sources, are present in soils and pose significant environmental and health impacts, even at low concentrations. Their detection in soil is analytically complex due to soil interference and the rapid degradation of compounds in the matrix. In this study, a protocol was optimized for quantifying hormonal steroids (n = 7), human drugs (n = 3), and antibiotics (n = 3) by a dual-phase extraction using QuEChERS and Solid Phase Extraction (SPE), followed by analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The double extraction phase allows an accurate and effective purification of the target compounds while eliminating the interference in the soil matrix. The method is optimized to detect environmental concentrations of these pollutants, to suit large-scale sampling campaigns and to maintain the efficiency of extraction while reducing analysis time. The limits of detection (LODs) of these compounds ranged between 0.0043 and 0.13 ng/g and recovery rates between 75.9 % and 105.39 %.•Enhanced Analyte Purification: Implements QuEChERS and SPE for robust removal of matrix interferences, optimizing target compound isolation.•Precision at Trace Levels: Secures LODs as minimal as 0.0043 ng/g, enabling accurate detection of low-concentration contaminants.•Adapted for Broad-scale sampling: Modifies extraction and analysis durations to accommodate large-scale environmental assessments.