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The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in hundreds of millions of infections and millions of deaths globally. Although vaccination campaigns are mitigating the pandemic, emerging viral variants continue to pose challenges. The spike (S) protein of SARS-CoV-2 plays a critical role in viral entry by binding to the angiotensin-converting enzyme 2 (ACE2) receptor, making both proteins essential targets for therapeutic and vaccine development. The glycosylation of these proteins influences their structure and function. This underscores the need for detailed site-specific glycoproteomic analysis. In this study, we characterized the N- or O-glycosylation profiles of the recombinant receptor-binding domain (RBD) of spike protein and ACE2 proteins expressed from Expi293F cells, as well as the S2 subunit of spike protein expressed in plant (N. benthamiana) cells. Using a high-resolution Orbitrap Eclipse Tribrid mass spectrometer equipped with the Ultimate 3000 RSLCnano and I-GPA (Integrated GlycoProteome Analyzer) developed in a previous study, 148 N- and 28 O-glycopeptides from RBD, 71 N-glycopeptides from the S2 subunit, and 139 N-glycopeptides from ACE2 were characterized. In addition, we report post-translational modifications (PTMs) of glycan, including mannose-6-phosphate (M6P) and GlcNAc-1-phosphate-6-O-mannose in N-glycan of RBD and ACE2, and O-acetylation in O-glycan of RBD, identified for the first time in these recombinant proteins. The relative abundance distribution according to glycosites and glycan types were analyzed by quantified site-specific N- and O (only from RBD)-glycopeptides from RBD, S2, and ACE2 using I-GPA. Asn331 for RBD, Asn1098 for S2, and Asn103 for ACE2 were majorly N-glycosylated, and dominant glycan-type was complex from RBD and ACE2 and high-mannose from S2. These findings will provide valuable insights into the glycosylation patterns that influence protein function and immunogenicity and offer new perspectives for the development of vaccines and antibody-based therapies against COVID-19. [ABSTRACT FROM AUTHOR]

We have previously shown that 2-thiouridine (S2U), either as a single nucleoside or as an element of RNA chain, is effectively desulfurized under applied in vitro oxidative conditions. The chemically induced desulfuration of S2U resulted in two products: 4-pyrimidinone nucleoside (H2U) and uridine (U). Recently, we investigated whether the desulfuration of S2U is a natural process that also occurs in the cells exposed to oxidative stress or whether it only occurs in the test tube during chemical reactions with oxidants at high concentrations. Using different types of eukaryotic cells, such as baker's yeast, human cancer cells, or modified HEK293 cells with an impaired antioxidant system, we confirmed that 5-substituted 2-thiouridines are oxidatively desulfurized in the wobble position of the anticodon of some tRNAs. The quantitative LC-MS/MS-MRMhr analysis of the nucleoside mixtures obtained from the hydrolyzed tRNA revealed the presence of the desulfuration products of mcm5S2U: mcm5H2U and mcm5U modifications. We also observed some amounts of immature cm5S2U, cm5H2U and cm5U products, which may have indicated a disruption of the enzymatic modification pathway at the C5 position of 2-thiouridine. The observed process, which was triggered by oxidative stress in the living cells, could impair the function of 2-thiouridine-containing tRNAs and alter the translation of genetic information. [ABSTRACT FROM AUTHOR]

A QuEChERS (quick, easy, cheap, effective, rugged, and safe)-based multi-mycotoxin method was developed, analyzing 24 (17 free and 7 modified) Alternaria and Fusarium toxins in cereals via ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). A modified QuEChERS approach was optimized for sample preparation. Quantification was conducted using a combination of stable isotope dilution analysis (SIDA) for nine toxins and matrix-matched calibration for ten toxins. Quantification via a structurally similar internal standard was conducted for four analytes. Alternariol-9-sulfate (AOH-9-S) was measured qualitatively. Limits of detection (LODs) were between 0.004 µg/kg for enniatin A1 (ENN A1) and 3.16 µg/kg for nivalenol (NIV), while the limits of quantification were between 0.013 and 11.8 µg/kg, respectively. The method was successfully applied to analyze 136 cereals and cereal-based foods, including 28 cereal-based infant food products. The analyzed samples were frequently contaminated with Alternaria toxins, proving their ubiquitous occurrence. Interestingly, in many of those samples, some modified Alternaria toxins occurred, mainly alternariol-3-sulfate (AOH-3-S) and alternariol monomethyl ether-3-sulfate (AME-3-S), thus highlighting the importance of including modified mycotoxins in the routine analysis as they may significantly add to the total exposure of their parent toxins. Over 95% of the analyzed samples were contaminated with at least one toxin. Despite the general contamination, no maximum or indicative levels were exceeded. [ABSTRACT FROM AUTHOR]

The composition of the protein corona covering any nanoparticle (NP) when it enters a biological fluid determines the parameters of the NP's interaction with the body. To "control" these parameters, it is important to know the composition of the protein corona, the determination of which is a complex task associated with the two-layer organization of the corona (hard and soft coronas). In a previous publication, we reported obtaining lipid-coated NPs with a full protein corona, isolating them, and proving the presence of the corona on the surface of the NPs. This work reports on the preparation, isolation, and purification of lipid-coated NPs bearing a hard corona. The protein corona composition was determined by using the LC–MS/MS method. Thirty-seven serum proteins were identified with a high degree of reliability. The hard corona contained various apolipoproteins, including apolipoprotein E, which can potentially affect the penetration of NPs into the cell. [ABSTRACT FROM AUTHOR]

Wildlife faces an increasing threat from extreme climatic events, such as heatwaves, which can have a severe impact on various species, including crucial pollinators like bumblebees. Bumblebees are cold‐adapted and heterothermic, possessing the ability to regulate their internal temperature. The impact of heat stress seems species specific in bumblebees. While most species are impacted, some bumblebee species manage to survive, potentially by employing physiological mechanisms, including the modulation of their protein profile (e.g. Heat Shock Proteins). However, there is limited understanding of how their protein profiles are associated with heat exposure. In this study, we examined the global variation in the protein profile of males from two bumblebee species sampled in the wild: the heat‐tolerant Bombus terrestris and the heat‐sensitive Bombus magnus. After subjecting them to heat stupor at 40°C in controlled condition, it was observed that nearly all B. terrestris survived the stress, while over 50% of B. magnus individuals succumbed to the heat exposure. Through off‐gel bottom‐up proteomics and LC–MS/MS analysis of the hemolymph proteome, we identified 164 proteins in both species with a large part of differentially expressed proteins after heat exposure. Additionally, quantitative analysis of fat bodies revealed that the relative mass was stable in B. terrestris, while it was significantly lower in B. magnus exposed to heat stress. Our data suggest that compared with B. magnus, B. terrestris displays a higher adaptability of its hemolymph proteome in response to heat stress. This adaptability could be a key factor contributing to the high physiological resistance of B. terrestris and its ability to adapt to new, stressful environments expected due to climate change. Understanding these mechanisms of protein regulation in bumblebees could provide valuable insights into their resilience and vulnerability facing environmental stresses. [ABSTRACT FROM AUTHOR]

Arisarum vulgare O. Targ. Tozz. (Araceae), locally known as "Elbgouga," holds significant traditional importance in Algeria for the treatment of various human ailments, including pain, infections, inflammation, digestive disorders, cancer, skin problems, eczema, wounds, and burns. The aim of this study was to explore for the first time the phytochemical profile, antioxidant, antibacterial, and inhibitory effects on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes, DNA protection, and capacity to promote wound healing. Preliminary phytochemical experiments were conducted to evaluate the major classes of bioactive compounds, in addition to the total phenol and flavonoid amounts in AVEE. An LC‐MS/MS analysis was conducted to clarify the phytochemical composition of this particular botanical species. The antioxidant capacity was assessed using DPPH· and ABTS·+ radical scavenging tests. The agar diffusion approach was used to ascertain the antibacterial efficacy against four bacteria (Staphylococcus aureus, Enterococcus faecium, Escherichia coli, and Salmonella typhimurium). Cholinesterase inhibition was evaluated using a colorimetric method that relies on Ellman's reaction. The protective effects of AVEE on pBR322 plasmid DNA damaged by H2O2 and UV treatment were assessed by their DNA‐breaking forms. The in vivo acute dermal toxicity and wound healing potential of the AVEE ointment (1–5% AVEO) were also investigated, and histological analyses were carried out on biological samples. Five protein targets were the subject of an in silico molecular docking investigation (TNFα, TGFBR1, IL‐1ß, GSK‐3ß, and NOS) and ADMET studies of the main components of the extracts were performed. The screening of phytochemicals revealed a significant concentration of phenolic compounds (176.00 ± 1.08 mg of gallic acid equivalents/g of dry extract), mainly flavonoids (81.21 ± 1.24 mg of quercetin equivalents/g of dry extract). Three major compounds were identified by LC‐MS/MS analysis, belonging mainly to the class of flavonoids, with rutin as the most abundant compound (11.85 mg/g extract), followed by hesperidin (4.125 mg/g extract). A conspicuous presence of isoquercitrin (1.327 mg/g extract) was also found. Small amounts of flavonoids and phenolic acids were also detected (catechin, chlorogenic acid, syringic acid, salicylic acid, kaempferol, and luteolin). DPPH assay showed higher antioxidant capacity compared to the ABTS·+ assay (142.60 ± 5.52 µg/mL and 236.10 ± 0.22 µg/mL, respectively). AVEE was effective against all selected bacterial strains; however, the highest zone of inhibition (36.00 ± 0.1 mm) was noted against E. faecium. The extract of the plant significantly inhibited both AChE and BChE (89.98 ± 18.76 µg/mL inhibitory activity against AChE and 98.28 ± 44.68 µg/mL inhibitory activity against BChE, respectively). The ethanolic extract of A. vulgare and quercetin exhibited more significant DNA protection action in form I, with percentages of 90.41 and 94.23%, respectively, compared to form II, where the percentages were 27.91 and 51.92%, respectively. The AVEO formulation may be safely administered by topical application. A statistically significant wound contraction was discovered in the group treated with 5% AVEO compared to the groups that were not treated or treated with petroleum jelly. Furthermore, there was no notable disparity detected between the group treated with 5% AVEO and the group treated with the reference drug. The 5% AVEO‐treated group showed, after the research was complete, the most significant percentage of wound contraction (96.90 ± 0.42%). Moreover, the topical application of the formulation improved histological parameters. In silico study showed that rutin, hesperidin, and isoquercitrin had a high affinity to the five main targets that might contribute to the wound healing potential of A. vulgare ethanolic extract. The impressive biological capabilities of A. vulgare indicate that the plant has the ability to be a valuable source of bioactive chemicals with various medical applications. [ABSTRACT FROM AUTHOR]

Salt is a potent abiotic stress that arrests plant growth by impairing their physio-biochemical and molecular processes. However, it is unknown how the ABA signaling system and vacuolar-type Na+/H+ antiporter proteins induce stress tolerance in wheat (Triticum aestivum L.) seedlings. The present study aimed to identify salt-responsive proteins and signaling pathways involved in the resistance of wheat to salt stress. We explored the proteome profile, 20 amino acids, 14 carbohydrates, 8 major phytohormones, ion content, and salt tolerance genes in wheat (Triticum aestivum L., cv.) under 200 mM NaCl with control plants for six days. The results showed that amino acids such as alanine, serine, proline, glutamine, and aspartic acid were highly expressed under salt stress compared with control plants, suggesting that amino acids are the main players in salinity tolerance. The ABA signaling system was activated in response to salinity stress through the modulation of protein phosphatase 2C (PP2C) and ABA-responsive element binding factor (ABF), resulting in an ABA-mediated downstream response. Additionally, the vacuolar-type Na+/H+ antiporter was identified as a key protein in salt stress tolerance via compartmentalizing Na+ in the vacuole. Furthermore, a significant increase in the abundance of the 14-3-3 protein was noticed in salt-fed plants, suggesting that this protein plays an important role in Na+ compartmentalization. Moreover, up-regulation of ascorbate peroxidase (APX), glutathione-S-transferase (GST), and thioredoxin-scavenged reactive oxygen species resulted in improved plant growth under salt stress. These data will help to identify salt-responsive proteins that can be used in future breeding programs to develop salt-tolerant varieties. [ABSTRACT FROM AUTHOR]

Limited or absent activity of the enzyme α-galactosidase A (α-Gal A), due to mutation in the related gene on the X chromosome, leads to the development of a rare hereditary and genetic disease known as Fabry disease (FD). This pathology involves a progressive accumulation in various organs of the substrates of the enzyme e.g., globotriaosylceramide (Gb3) and its deacylated form, globotriaosylsphingosine (Lyso-Gb3), suggesting these molecules as biomarkers of Fabry disease. The present paper describes the development of an analytical strategy for the identification and quantification of Gb3 and Lyso-Gb3, in serum and blood samples by using liquid chromatography (LC) coupled to mass spectrometry in multiple reaction monitoring (MRM/MS) ion mode. The best experimental conditions were obtained by extracting the glycolipids with chloroform/methanol/H2O (2/1/0.3) and by separating them on a C4 column with a linear gradient (A: H2O with 2 mM ammonium formate. B: methanol with 1 mM ammonium formate, both acidified with 0.2% formic acid). The best transitions (a combination of precursor and fragment ions—m/z) were 786.8 m/z > 268.3 m/z for Lyso-GB3, 1137.3 m/z > 264.3 m/z for Gb3, 1039.3 m/z > 264.4 m/z for N-heptadecanoyl-ceramide trihexoside, and 843.5 m/z > 264.3 m/z for N-glycinated lyso-ceramide trihexoside, the latter being used as an internal standard. The developed method provided a reliable, fast, and effective procedure for direct measurements of GB3 and Lyso-GB3 in serum and blood for diagnosis of Fabry disease, suggesting this method as a complementary assay to the current enzymatic test. Therefore, this approach could open new insights into the clinical diagnostics of lysosomal storage disorders. [ABSTRACT FROM AUTHOR]

Sulfoxaflor is a systemic sulfoximine insecticide, used to control sucking pests in different agricultural crops including wheat. However, the information regarding its residual fate in wheat agroecosystem under tropical environment and the associated dietary risk are still obscure. Therefore, a supervised field experiment was conducted in wheat comprising the application of sulfoxaflor 12% (w/v) SC at 30 and 60 g a.i. ha−1 in two consecutive seasons. An optimised method for estimation of sulfoxaflor residues using modified QuEChERS technique was developed and validated. The extracting solvent ethyl acetate + cyclohexane (9 + 1 v/v) along with the clean-up combinations viz. 75 mg PSA+25 mg GCB+150 mg magnesium sulphate for wheat plant and straw and 75 mg PSA+75 mg C18 + 150 mg magnesium sulphate for grain produced satisfactory recovery, minimum matrix interference and optimum peak shape. The average recovery, repeatability and within-laboratory reproducibility of sulfoxaflor in wheat substrates ranged between 90.66% and 103.55%, 2.67–5.17% and 3.05–8.15%, respectively, and the limit of quantification was estimated 0.01 mg kg−1. Sulfoxaflor was stable up to 60 days in wheat matrices under storage (−20°C). The dissipation of sulfoxaflor in wheat plants followed first-order kinetics and the overall shorter persistence in Season I and II ranged between 1.05–1.14 and 2.44–2.47 days (T1 – T2), respectively. The deviation in the persistence of the compound between two seasons due to variation in climatic parameters was observed to be statistically significant. The estimated pre-harvest interval of sulfoxaflor ranged between 4.95–5.34 (Season I) and 11.37–11.55 days (Season II). Besides, the terminal residues of sulfoxaflor were below quantification limit in the harvested wheat products, and the estimated long-term dietary risk of sulfoxaflor was observed to be negligible. Therefore, it was expected that the application of this insecticide in wheat cultivation should pose no residual toxicity and the harvested wheat products can be considered safe for human consumption. [ABSTRACT FROM AUTHOR]

Straw from no-till cropping systems, in addition to increasing the soil organic matter content, may also impede the movement of applied herbicides into the soil and, thus, alter the behavior and fate of these compounds in the environment. Rain or irrigation before or after an herbicide treatment can either help or hinder its movement through the straw, influencing weed control. Our objective was to develop a system for herbicide application and rain simulation, enabling the evaluation of the movement of various herbicides either in dry or wet straw under different rainfall volumes (25, 50, 75, and 100 mm). The amount of the applied herbicides that moved through the straw were collected and measured using a liquid chromatograph with a tandem mass spectrometry system (LC-MS/MS). Measurements obtained with the developed system showed a high herbicide treatment uniformity across all replications. The movement of the active ingredients through the straw showed variability that was a function of the applied herbicide, ranging from 17% to 99%. In wet straw, the collected herbicide remained constant from 50 to 100 mm of simulated rainfall. For the wet straw, the decreasing percentages of the herbicide movement through straw to the soil were sulfentrazone (99%), atrazine and diuron (91% each), hexazinone (84%), fomesafen (80.4%), indaziflam (79%), glyphosate (63%), haloxyfop-p-methyl (45%), and S-metolachlor (27%). On the dry straw, the decreasing percentages of the herbicide movement were fomesafen (88%), sulfentrazone (74%), atrazine (69.4%), hexazinone (69%), diuron (68.4%), glyphosate (48%), indaziflam (34.4%), S-metolachlor (22%), and haloxyfop-p-methyl (18%). Overall, herbicide movement was higher in wet straw (with a previous 25 mm simulated rainfall layer) than in dry straw. Some herbicides, like haloxyfop-p-methyl and indaziflam, exhibited over 50% higher movement in wet straw than dry straw after 100 mm of simulated rain. The developed system can be adapted for various uses, serving as a valuable tool to evaluate the behavior of hazardous substances in different agricultural and environmental scenarios. [ABSTRACT FROM AUTHOR]

A method was developed for the determination of 26 drugs of abuse from different classes, including illicit drugs in quantitative dried blood spots (qDBSs), with the aim to provide a convenient method for drug testing by using only 10 μL of capillary blood. A satisfactory limit of quantification (LOQ) of 2.5 ng/mL for 9 of the compounds and 5 ng/mL for 17 of the compounds and a limit of detection (LOD) of 0.75 ng/mL for 9 of the compounds and 1.5 ng/mL for 17 of the compounds were achieved for all analytes. Reversed-phase liquid chromatography was applied on a C18 column coupled to MS, providing selective detections with both +ESI and -ESI modes. Extraction from the qDBS was performed using AcN-MeOH, 1:1 (v/v), with recovery ranging from 84.6% to 106%, while no significant effect of the hematocrit was observed. The studied drugs of abuse were found to be stable over five days under three different storage conditions (at ambient temperature 21 °C, at −20 °C, and at 35 °C), thus offering a highly attractive approach for drug screening by minimally invasive sampling for individuals that could find application in forensic toxicology analysis. [ABSTRACT FROM AUTHOR]

Since antiquity, Centaurea species have been used in folk medicine to treat several diseases owing to their potential biological activities that distinguish this genus such as antioxidant, anticancer, and anti-inflammatory effect. The current study aimed to investigate the possible neuroprotective effects of the n-butanol extract of Centaurea maroccana (BECM) against cisplatin (CP) induced neurotoxicity in mice. BECM's potential neuroprotective properties were studied in vitro and in vivo models. Male Swiss albino mice were orally received BECM (200 mg/kg) for 10 days before a single intraperitoneal injection of cisplatin (8 mg/kg). Vitamin E (100 mg/kg) was given daily by gavage as a positive control. In vitro results revealed that BECM inhibited lipid peroxidation (LPO) levels and acetylcholinesterase (AChE) activity. In vivo findings showed that BECM pretreatment was able to regulate lactate dehydrogenase (LDH) levels and to improve CP-induced cholinergic dysfunction by inhibiting AChE activity in mice brains. Moreover, BECM attenuated CP-provoked oxidative stress by suppressing LPO levels, increasing total antioxidant capacity (TAC) and enhancing the activities of antioxidant enzymes (catalase (CAT), superoxide dismutase (SOD), reduced glutathione (GSH), glutathione peroxidase (GPx) and glutathione S-transferase (GST)) in both brain cytosolic and mitochondrial fractions. The histological analysis exhibited neurotoprotective effect of BECM by protecting the cerebral cortex and reducing the histomorphological alterations resulted by cisplatin. Interestingly, our extract achieved neuroprotection comparable to vitamin E in most evaluated parameters. It appears that protective potency of BECM against CP-induced neurotoxicity could be related to its richness in polyphenols confirmed by liquid-chromatography tandem mass spectrometry analysis (LC-MS/MS). [ABSTRACT FROM AUTHOR]

Acne vulgaris is a chronic inflammatory disease with high incidence, diverse clinical manifestations, poor clinical efficacy, and easy recurrence. Recent studies have found that the occurrence of acne is related to metabolic factors such as insulin resistance; however, the specific mechanism of action remains unclear. This study aimed to identify significantly different metabolites and related metabolic pathways in the serum of acne vulgaris patients with or without insulin resistance. LC–MS/MS was used to analyze serum samples from patients about acne with insulin resistance (n = 51) and acne without insulin resistance (n = 69) to identify significant metabolites and metabolic pathways. In this study, 18 significant differential metabolites were screened for the first time. In the positive-ion mode, the upregulated substances were creatine, sarcosine, D-proline, uracil, Phe–Phe, L-pipecolic acid, and DL-phenylalanine; the downregulated substances were tridecanoic acid (tridecylic acid), L-lysine, cyclohexylamine, sphingomyelin (d18:1/18:0), gamma-L-Glu-epsilon-L-Lys, and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine. In the negative-ion mode, the upregulated substance was cholesterol sulfate, and the downregulated substances were D(-)-beta-hydroxybutyric acid, myristic acid, D-galacturonic acid, and dihydrothymine. Cholesterol sulfate showed the most significant expression among all differential metabolites (VIP = 7.3411). Based on the KEGG database, necroptosis and ABC transporters were the most significantly enriched metabolic pathways in this experiment. The differential metabolites and pathways identified in this study may provide new possibilities for the clinical diagnosis and development of targeted drugs for acne patients with insulin resistance. [ABSTRACT FROM AUTHOR]

Chenopodium murale (Syn. Chenopodiastrum murale) (amaranthaceae) is used in the rural Egypt to treat oral ulcers in newborn children. The current study aimed to discover new natural products suitable for treating candidiasis disease with minimal side effects. Characterization of bioactive compounds by LC-QTOF-HR-MS/MS from Chenopodium murale fresh leaves' juice (CMJ) was carried out in order to elucidate their potential anti-fungal and immunomodulatory effects in oral candidiasis in immunosuppressed rats. An oral ulcer candidiasis model was created in three stages: (i) immunosuppression by drinking dexamethasone (0.5 mg/L) for two weeks; (ii) Candida albicans infection (3.00 × 106 viable cell/mL) for one week; and (iii) treatment with CMJ (0.5 and 1.0 g/kg orally) or nystatin (1,000,000 U/L orally) for one week. Two doses of CMJ exhibited antifungal effects, for example, through a significant reduction in CFU/Petri (236.67 ± 37.86 and 4.33 ± 0.58 CFU/Petri), compared to the Candida control (5.86 × 104 ± 1.21 CFU/Petri), p ≤ 0.001. In addition, CMJ significantly induced neutrophil production (32.92% ± 1.29 and 35.68% ± 1.77) compared to the Candida control level of 26.50% ± 2.44. An immunomodulatory effect of CMJ at two doses appeared, with a considerable elevation in INF-γ (103.88 and 115.91%), IL-2 (143.50, 182.33%), and IL-17 (83.97 and 141.95% Pg/mL) compared with the Candida group. LC-MS/MS analysis operated in negative mode was used for tentative identification of secondary (SM) metabolites based on their retention times and fragment ions. A total of 42 phytoconstituents were tentatively identified. Finally, CMJ exhibited a potent antifungal effect. CMJ fought Candida through four strategies: (i) promotion of classical phagocytosis of neutrophils; (ii) activation of T cells that activate IFN-γ, IL-2, and IL-17; (iii) increasing the production of cytotoxic NO and H2O2 that can kill Candida; and (iv) activation of SOD, which converts superoxide to antimicrobial materials. These activities could be due to its active constituents, which are documented as anti-fungal, or due to its richness in flavonoids, especially the active compounds of kaempferol glycosides and aglycone, which have been documented as antifungal. After repetition on another type of small experimental animal, their offspring, and an experimental large animal, this study may lead to clinical trials. [ABSTRACT FROM AUTHOR]

Milk protein concentrate (MPC) is considered an ideal substitute of cow milk because of its similar protein and nutrition. In this study, MPC was hydrolyzed to peptides by alcalase and neutrase, and the properties of hydrolysate were evaluated. When MPC was hydrolyzed at the ratio of alcalase and neutrase of 1:1 and enzyme to substrate ratio of 6000 U/g MPC at 50°C, pH 8.5 for 3 h, the proportion of peptides with molecular weights <1 kDa was 85.31%, and the antigenicity reduction rates of casein and β‐lactoglobulin were 33.76% and 22.38%, respectively. Moreover, LC–MS/MS peptide identification revealed that the alcalase and neutrase disrupted a total of 65 epitopes of casein and 21 epitopes of whey protein, which further elucidated the mechanism of complex enzyme hydrolysis to reduce milk protein allergenicity. [ABSTRACT FROM AUTHOR]

This study describes the analysis of five quinolizidine alkaloids (QA), i.e. 13-OH-lupanine, lupanine, lupinine, angustifoline and sparteine in 30 samples of lupine flours, lupine seeds and derived products collected 2019–2021 on the German retail market. QA occur as secondary metabolites in plants of lupine species. Certain QA are of toxicological relevance. The analytical determination performed by LC-MS/MS revealed some samples with high QA concentrations (up to 21,000 mg/kg), particularly in bitter lupine seeds. As those concentrations would result in substantial exceedances of the maximum tolerable intake values proposed by health authorities, they must be considered as a health concern. [ABSTRACT FROM AUTHOR]

Zivzik pomegranate (Punica granatum) has recently sparked considerable interest due to its nutritional and antioxidant properties. To evaluate the antioxidant capacities of P. granatum juice, ethanol (EEZP), and water (WEZP) extracts from peel and seed, the antioxidant methods of 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid radical (ABTS•+) scavenging, 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH•) scavenging, Fe3+-2,4,6-tris(2-pyridyl)-S-triazine (TPTZ) reducing, Fe3+ reducing, and Cu2+ reducing methods were used. The antioxidant capacities of samples were compared with the most commonly used synthetic antioxidants, i.e., BHA, BHT, α-tocopherol, and Trolox. In terms of setting an example, the IC50 values of EEZP for ABTS•+ and DPPH• scavenging activities were found to be lower than standards, at 5.9 and 16.1 μg/mL, respectively. The phenolic and flavonoid contents in EEZP peel were 59.7 mg GAE/g and 88.0 mg QE/g, respectively. Inhibition of α-glycosidase, α-amylase, acetylcholinesterase, and human carbonic anhydrase II (hCA II) enzymes was also investigated. EEZP demonstrated IC50 values of 7.3 μg/mL against α-glycosidase, 317.7 μg/mL against α-amylase, 19.7 μg/mL against acetylcholinesterase (AChE), and 106.3 μg/mL against CA II enzymes. A total of 53 phenolic compounds were scanned, and 30 compounds were determined using LC-MS/MS. E. coli and S. aureus bacteria were resistant to all four antibiotics used as standards in hospitals. [ABSTRACT FROM AUTHOR]

The determination of the selected antihypertensive drugs in human plasma samples with the novel solvent front position extraction (SFPE) technique is presented. The SFPE procedure combined with LC-MS/MS analysis was used for the first time to prepare a clinical sample containing the drugs mentioned above from different therapeutic groups. The effectiveness of our approach was compared with the precipitation method. The latter technique is usually used to prepare biological samples in routine laboratories. During the experiments, the substances of interest and the internal standard were separated from other matrix components using a prototype horizontal chamber for thin-layer chromatography/high-performance thin-layer chromatography (TLC/HPTLC) with a moving pipette powered by a 3D mechanism, which distributed the solvent on the adsorbent layer. Detection of the six antihypertensive drugs was performed by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) in multiple reaction monitoring (MRM) mode. Results obtained by SFPE were very satisfactory (linearity R2 ≥ 0.981; %RSD ≤ 6%; LOD and LOQ were in the range of 0.06–9.78 ng/mL and 0.17–29.64 ng/mL, respectively). The recovery was in the range of 79.88–120.36%. Intra-day and inter-day precision had a percentage coefficient of variation (CV) in the range of 1.10–9.74%. The procedure is simple and highly effective. It includes the automation of TLC chromatogram development, which significantly reduced the number of manual operations performed, the time of sample preparation and solvent consumption. [ABSTRACT FROM AUTHOR]

Simple Summary: In our work, we described and proposed a novel LC-MS/MS based approach for early lung cancer screening based on the VOC marker. Through this approach, two key volatile organic compounds (VOCs) used in the early screening and diagnosis of lung cancer were determined as 3-hydroxy-2-butanone and 2-pentanone. 3-hydroxy-2-butanone in the human body was found for the first time to be derived from the sugar metabolism of oral bacteria and we found a correlation between the bacteria that metabolize 3-hydroxy-2-butanone and lung cancer. (1) Background: lung cancer is the world's deadliest cancer, but early diagnosis helps to improve the cure rate and thus reduce the mortality rate. Annual low-dose computed tomography (LD-CT) screening is an efficient lung cancer-screening program for a high-risk population. However, LD-CT has often been characterized by a higher degree of false-positive results. To meet these challenges, a volatolomic approach, in particular, the breath volatile organic compounds (VOCs) fingerprint analysis, has recently received increased attention for its application in early lung cancer screening thanks to its convenience, non-invasiveness, and being well tolerated by patients. (2) Methods: a LC-MS/MS-based volatolomics analysis was carried out according to P/N 5046800 standard based breath analysis of VOC as novel cancer biomarkers for distinguishing early-stage lung cancer from the healthy control group. The discriminatory accuracy of identified VOCs was assessed using subject work characterization and a random forest risk prediction model. (3) Results: the proposed technique has good performance compared with existing approaches, the differences between the exhaled VOCs of the early lung cancer patients before operation, three to seven days after the operation, as well as four to six weeks after operation under fasting and 1 h after the meal were compared with the healthy controls. The results showed that only 1 h after a meal, the concentration of seven VOCs, including 3-hydroxy-2-butanone (TG-4), glycolaldehyde (TG-7), 2-pentanone (TG-8), acrolein (TG-11), nonaldehyde (TG-19), decanal (TG-20), and crotonaldehyde (TG-22), differ significantly between lung cancer patients and control, with the invasive adenocarcinoma of the lung (IAC) having the most significant difference. (4) Conclusions: this novel, non-invasive approach can improve the detection rate of early lung cancer, and LC-MS/MS-based breath analysis could be a promising method for clinical application. [ABSTRACT FROM AUTHOR]

Veterinary drugs are widely used to treat livestock diseases, but the emergence of problems related to drug resistance prompted the government of the Republic of Korea to enact laws regulating the use of veterinary drugs in livestock. Monitoring the contents of residual drugs in livestock products at regular intervals is necessary to assess whether the regulations have resulted in the effective management of food product safety and prevent and manage unexpected problems related to this issue. A 2019–2023 livestock product post-marketing monitoring program launched by the Ministry of Food and Drug Safety (MFDS) of the Republic of Korea inspected 1135 livestock products. Sixty-two different veterinary drugs were detected among 167 monitored, with a detection rate of 28.2% (320/1135 samples; chicken [125], pork [114], milk [35], beef [30], and egg [16]). Diclazuril was detected most frequently, followed by flumequine, clopidol, and enrofloxacin. Over the past five years, two samples that did not conform to the regulations were identified to contain 20 and 215 ng/g, diclazuril and toltrazuril sulfone, respectively. The estimation of exposure and risk assessment revealed that the hazard index of diclazuril was below 75%. However, in case of toltrazuril sulfone, it showed a hazard index exceeding 138% in the scenario of the 95th percentile high consumption of chicken. The results of this study may provide a useful reference in initiating more targeted work in the future, such as the further fine-tuning of food safety monitoring programs and developing food safety standards. In future studies, the MFDS shall continue to monitor the statuses of residual veterinary drugs in commercial livestock products. • We investigated 151 veterinary drug residues in 1135 livestock samples from the Republic of Korea. • Among the analyzed samples, 28.2% (n = 320) contained veterinary drug residues. • Two violations of the maximum residual limits set by the Republic of Korea were observed. • Various residues of veterinary drugs were often detected in combination. • The current levels of exposure to diclazuril and toltrazuril sulfone did not present a significant risk. [ABSTRACT FROM AUTHOR]

Previous reports revealed that peel extracts of Ficus carica (fig) have a wide range of pharmacological and biological activities. The current study aimed to determine the phytochemical components of the ethanol extracts of Peggy Red fig (PRF) and Green fig (GF) peels by liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis, along with its antioxidant properties and neuroprotective effect in Caenorhabditis elegans. LC–MS/MS analysis confirmed 50 compounds in the extract, which revealed the presence of phenols, flavonoids, and anthocyanins, and exhibited in vitro antioxidant activity. PRF and GF peel had 163.25 (mg gallic acid equivalent [mg GAE]) g−1, 125.32 (mg GAE) g−1 of total phenolic content, 62.52 (mg rutin equivalent [mg RE]) g−1, and 43.36 (mg RE) g−1 flavonoids content, respectively. In all antioxidant assays, the extract of PRF peel showed higher antioxidant activity than the GF peel, and the extract of PRF peel could effectively reduce the aggregation of amyloid-beta (Aβ), decrease the paralysis of the body, and increase the antioxidant enzyme activities to reduce the toxicity of Aβ1–42 in Alzheimer's disease (AD) transgenic C. elegans CL4176. Therefore, PRF peel extract may have potential applications as a new source for drug development against AD. [ABSTRACT FROM AUTHOR]

The main objective of the present study is to estimate, through differential analysis, various biological activities of total phenolics content in alcoholic extracts of three date palm varieties sensitive or resistant to Fusarium oxysporum. sp Albidinis. Here, stilbene products with antioxidant and bioactive capacities were evidenced in resistant variety Taabdount (TAAR). Furthermore, the methanolic fraction of the TAAR-resistant date palm variety contains a significant product, determined by LC-MS/MS and 1H, 13C NMR, belonging to the family of hydroxystilbenes, which exhibits antioxidant capacities, inhibits the mushroom tyrosinase activity, and activates and exerts a protective effect on hypochlorite-induced damage in 20S proteasome of human dermal fibroblast aged cells. Altogether, the present results indicate that hydroxystilbene present in resistant Phoenix dactylifera L. should be studied to understand the way that the stilbene could exert anti-aging ability. [ABSTRACT FROM AUTHOR]

Background: Poplar fungal infections are difficult to control and result in severe economic loss. As a viable alternative to chemical pesticides, biocontrol is an effective safe method for disease control. Results: Inhibitory activity of Bacillus velezensis 33RB and Aspergillus niger 46SF was evaluated against numerous phytopathogens. The bacterial strain displayed the highest inhibitory activity toward Colletotrichum gloeosporioides BJ02 and Fusarium oxysporum 20RF (61.2 and 49.4%, respectively). Also, the maximum inhibitory activity of A. niger 46SF was exhibited (75.51 and 70.83%) against C. gloeosporioides BJ02 and F. oxysporum 20RF, respectively. The minimum volume (6.25 ml) of sterilized cultural filtrate of bacterial and fungal strains significantly inhibited the growth of C. gloeosporioides BJ02 by 73.3 and 83.3%, respectively, and F. oxysporum 20RF reached 40.4 and 78.8%, respectively. B. velezensis 33RB and A. niger 46SF displayed the highest inhibition toward C. gloeosporioides BJ02 and F. oxysporum 20RF at neutral pH and pH 5, respectively. Moreover, the highest inhibitory activity of B. velezensis 33RB and A. niger 46SF was achieved at 37 °C and 28 °C, respectively. Pathogenicity tests on sterilized detached leaves indicated that these isolates could potentially affect anthracnose and fusarium wilt diseases. Several secondary bioactive metabolites that assured the biocontrol efficacy of tested microbes were detected by liquid chromatography–mass spectrometry (LC–MS). The most detectable compounds included organic acids such as fumaric, DL-malic, citric, isobutyric, and glutamic acids. Also, numerous fatty acids such as lauric, linoleic, oleic, stearic, and myristic acids with diverse biological functions, including antimicrobial properties, were determined. Conclusions: Bacillus velezensis 33RB and A. niger 46SF were potential alternatives to chemical pesticides as biological control agents for the phytopathogens C. gloeosporioides BJ02 and F. oxysporum with environmentally friendly and sustainable properties. [ABSTRACT FROM AUTHOR]

Diabetes mellitus is a major endocrine dysfunction that severely threatens the global population. Litsea cubeba (Lour.) Pers. (Lauraceae), commonly known as "mountain pepper", has been utilized in many chronic disorders in different countries. However, the antidiabetic activity of this plant has not been explored to date. The present study aimed to explore the antidiabetic and antihyperlipidemic activities of the methanolic extract of fruits of L. cubeba (LCMF) from the Darjeeling Himalaya, India, through in vivo experimentations and network pharmacological analysis. The HFD-STZ induced diabetic model rats were treated with LCMF for 28 days and were examined for blood glucose, lipid profile, liver and kidney function parameters, hepatic antioxidant status and histopathology of liver, kidney and pancreas. The LCMF treatment effectively lowered (about 25%) high blood glucose levels and improved serum lipid profiles, liver glycogen content, and normalized antioxidant status. A significant increment in endogenous insulin (6.83 mU/L) and C-peptide levels (0.86 ng/mL), along with the regeneration of beta islets, were observed. LC-MS/MS and in silico network-pharmacological analysis showed the presence of some important metabolites, viz. thymol, scopoline, isosafrole, safrole, arecoline, etc. that interact with the target proteins (viz. IL6, ADRB1 etc.), transcription factors (viz. NFKB1, CYP1A1 etc.) and enzymes (viz. α-glucosidase, α-amylase etc.) associated with diabetes and hyperlipidemia. The study for the first time proved that the traditional edible fruits of L. cubeba have potent antidiabetic and antihyperlipidemic properties. The subsequent detailed characterization and mode of action studies would lead to discovering new antidiabetic and antihyperlipidemic drugs for human use. • Litsea cubeba fruits exerted significant hypoglycemic activities in vivo. • Promising antihyperlipidemic activities were also observed. • Regeneration of beta-islet cells of the pancreas was observed. • Endogenous insulin and C-peptide levels were found to be increased. • Network pharmacology revealed interactions of metabolites with target proteins. [Display omitted] [ABSTRACT FROM AUTHOR]

Mesobuthus tamulus (Indian Red Scorpion) sting is a severe but neglected health issue in India. The accomplishment of in-patient scorpion sting management is highly dependent on the safety, efficacy, and homogeneity of scorpion antivenom preparation. Therefore, in this study, the above qualities of commercial anti-scorpion antivenoms manufactured in India were assessed by in vitro laboratory analyses. Biophysical characterization of venom by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, size exclusion chromatography, and proteomics analysis demonstrated that anti-scorpion antivenoms (ASAs) mostly contain F(ab') 2 molecules with a trace amount of undigested immunoglobulin (Ig) G. The physicochemical characterization, electron microscopy, and dynamic light scattering studies revealed that ASAs were prepared according to the guidelines of World Health Organization (WHO), and were devoid of aggregate content and virus particles. ASAs did not show IgE contamination and bacterial endotoxin but demonstrated moderate complement activation properties, which may have adverse effects in treated patients. Spectrofluorometric and atomic force microscopy analyses showed poor binding of venom with commercial ASAs. The percent of antibodies raised against the venom toxins in commercial ASAs was determined at the range of 5.3–6.3%, which is a reason for their poor efficacy. This study advocates the importance of in vitro laboratory analyses for assessing commercial antivenom's quality and safety parameters before their pre-clinical research and clinical use to treat Indian red scorpion sting. [Display omitted] • Commercial anti-scorpion antivenoms (ASAs) manufactured in India contain F(ab') 2 with a trace quantity of immunoglobulin G. • Study showed that the ASAs were prepared according to the guidelines of the World Health Organization. • The ASAs were devoid of aggregate content and virus particles. • Biophysical analyses demonstrated the poor binding of venom with commercial ASAs. • Commercial ASAs contain less proportion of scorpion venom toxins-specific antibodies. [ABSTRACT FROM AUTHOR]

Manure is a major source of soil and plant contamination with veterinary drugs residues. The aim of this study was to evaluate the uptake of 14 veterinary pharmaceuticals by parsley from soil fertilized with manure. Pharmaceutical content was determined in roots and leaves. Liquid chromatography coupled with tandem mass spectrometry was used for targeted analysis. Screening analysis was performed to identify transformation products in the parsley tissues. A solid-liquid extraction procedure was developed combined with solid-phase extraction, providing recoveries of 61.9–97.1% for leaves and 51.7–95.6% for roots. Four analytes were detected in parsley: enrofloxacin, tylosin, sulfamethoxazole, and doxycycline. Enrofloxacin was detected at the highest concentrations (13.4–26.3 ng g−1). Doxycycline accumulated mainly in the roots, tylosin in the leaves, and sulfamethoxazole was found in both tissues. 14 transformation products were identified and their distribution were determined. This study provides important data on the uptake and transformation of pharmaceuticals in plant tissues. [ABSTRACT FROM AUTHOR]

The Indian monocled cobra (Naja kaouthia) is one of the most prevalent venomous snakes in northeast India (NEI) and is the cause of many fatalities. The composition of NEI N. kaouthia venom (NkV) was deciphered using two different proteomic approaches: (i) 1D SDS-PAGE coupled to label-free quantification of protein bands using stringent identification criteria and (ii) reversed-phase high-performance liquid chromatography (RP-HPLC) followed by quantification based on area under the RP-HPLC peaks. The proteomic data from both strategies were compared. Proteomic analyses from both workflows identified 32 proteins (toxins) distributed over 10–14 snake venom protein families in NEI NkV. The relative abundances of the venom proteins determined from the analytical workflows coincided with the densitometry band intensities of the NEI NkV. Phospholipase A 2 (13.1–16.0%) and three-finger toxins (58.5–64.2%) represented the most abundant enzymatic and non-enzymatic proteins in NEI NkV, respectively. Immuno-cross-reactivity studies by enzyme-linked immunoassay and immunoblot analyses pointed to the poor efficacy of commercial PAVs in recognizing the low molecular mass (<15 kDa) toxins of NEI NkV. Spectrofluorometric titration determined the presence of NEI NkV-specific antibodies in commercial PAV, at a level that was higher than that previously reported for eastern India NkV-specific antibodies in commercial antivenom. • Each method of venom de-complexation for protein identification and quantification has its pros and cons • The venom proteome composition of N. kaouthia from North-East India was deciphered using two proteomic workflows and compared • The study demonstrated that both analytical workflows can be considered reliable approaches for analyzing snake venom proteome • The 3FTx and PLA 2 represented the most abundant non-enzymatic and enzymatic proteins in the tested venom • Immunoprofiling demonstrated poor efficacy of commercial PAVs against the low molecular weight (<15 kDa) NEI NKVtoxins [ABSTRACT FROM AUTHOR]

Considering medicinal plants as an inexhaustible source of active ingredients that may be easily isolated using simple and inexpensive techniques, phytotherapy is becoming increasingly popular. Various experimental approaches and analytical methods have been used to demonstrate that the genus Calendula (Asteraceae) has a particular richness in active ingredients, especially phenolic compounds, which justifies the growing interest in scientific studies on this genus' species. From a chemical and biological viewpoint, Calendula aegyptiaca is a little-studied plant. For the first time, high-performance liquid chromatography combined with negative electrospray ionization mass spectrometry (HPLC-HESI-MS) was used to analyze methanolic extracts of Calendula aegyptiaca (C. aegyptiaca) fruits. Thirty-five molecules were identified. Flavonoids (47.87%), phenolic acids (5.18%), and saponins (6.47%) formed the majority of these chemicals. Rutin, caffeic acid hexoside, and Soyasaponin βg' were the most abundant molecules in the fruit methanolic extract, accounting for 17.49% of total flavonoids, 2.32 % of total phenolic acids, and 0.95% of total saponins, respectively. The antioxidant activity of the fruit extracts of C. aegyptiaca was investigated using FRAP, TAC, and DPPH as well as flavonoids and total phenols content. Because the phenolic components were more extractable using polar solvents, the antioxidant activity of the methanolic extract was found to be higher than that of the dichloromethane and hexane extracts. The IC50 value for DPPH of methanolic extract was found to be 0.041 mg·mL−1. Our findings showed that C. aegyptiaca is an important source of physiologically active compounds. [ABSTRACT FROM AUTHOR]

Calophyllum soulattri (Sulatri), a plant from Clusiaceae family, has been empirically used as traditional medicine. In the present study, C. soulattri stem bark extract and fractions were evaluated for their toxicity against MCF-7 breast cancer cells. The chemical contents of the extract and fractions were analyzed using =liquid chromatography-mass spectrometry (LC-MS/MS). The results showed that methanol extract and n-hexane fractions had a strong cytotoxic activity with IC50 values of 93.6 and 36 µg/mL, respectively. Meanwhile, as the positive control, ethyl acetate and cisplatin fractions have IC50 values of 233 and 16.2 µg/mL, respectively. The LC-MS/MS analysis showed that the extract and fractions contained polyporusterone A, poricoic acid D, polyporusterone F, esculentagenin, and 1-acetyl-3-(methoxy-carbonyl)-_-carboline. Therefore, C. soulattri stem bark extract and fractions have potential activity as an anticancer agent that can inhibit MCF-7 breast cancer cell growth. [ABSTRACT FROM AUTHOR]

Endophytes are bacteria and fungi in the inner parts of the plant tissue which other than their benefits for the plants, they contain effective intracellular and extracellular bioactive compounds.The aim of this study was to investigate the antifungal activity of the intracellular content of endophytic bacteria isolated from some medicinal plants. Endophytic bacteria were isolated from 12 medicinal plants and their antifungal activities of intracellular content was observed against Aspergillus flavus, Aspergillus fumigatus, Penicillium citrinum, Fusarium oxysporum and Rhizopus stolonifer by disk diffusion method. The bacteria having intracellular antifungal activity against all five fungi were detected by 16S rRNA. LC–MS/MS analysis was perfomed to identify the probable proteins responsible for the antifungal action. The intracellular content of 12 endophytic bacteria had antifungal activity. Six of the bacterial intracellular contents' showed antifungal activity towards all five fungi. PCR analysis identified all 6 strains as Bacillus pumilus. LC–MS/MS analysis revealed the precence of cysteine rich proteins along with glyceraldehyde-3-phosphate dehydrogenase, proteinase inhibitors and alcohol dehydrogenase which may be responsible for the intracellular antifungal activities. [ABSTRACT FROM AUTHOR]

Summary: The prevalent pesticide carbofuran was banned in the European Union (EU) in 2008; however, the extent of its actual elimination from the environment has been little studied. The presence of this pesticide in the livers of the protected raptors the white-tailed eagle (Haliaeetus albicilla) and the common buzzard (Buteo buteo) was monitored in Poland from 2008 to 2019 using liquid chromatography with tandem mass spectrometry analysis and data from government institutions. Carbofuran residues were detected in the liver samples of the analysed raptors throughout the period studied. In total, carbofuran was detected in the livers of 33% of the eagles and 54% of the buzzards; concentrations were in the ranges of 11–699 and 14–1890 μg kg–1 of dry matter, respectively. Effective measures to eliminate banned pesticides from the market more efficiently are required. [ABSTRACT FROM AUTHOR]

Dereplication of crude plant extracts through liquid chromatography-mass spectrometry is a powerful technique for the discovery of novel natural products. Unfortunately, this technique is often plagued by a low level of confidence in natural product identification. This is mainly due to the lack of extensive chromatographic and mass spectrometric optimizations that result in improper and incomplete MS/MS fragmentation data. This study proposes a solution to this problem by the optimization of chromatographic separation and mass spectrometry parameters. We report herein a direct and high-throughput strategy for natural product dereplication in five Salvia species using high-resolution ESI-QTOF-MS/MS data. In the present study, we were able to identify a total of forty-seven natural products in crude extracts of five Salvia species using MS/MS fragmentation data. In addition to dereplication of Salvia species, quantitative profiling of twenty-one bioactive constituents of the genus was also performed on an ion trap mass spectrometer. For the quantitation study, method development focused on chromatographic optimizations to achieve maximum sensitivity. The developed dereplication and quantitation strategy can be extended to develop comprehensive metabolic profiles of other plant genera and species and thus can prove useful in the field of drug discovery from plants. [ABSTRACT FROM AUTHOR]

Goji berry (Lycium barbarum L.) is a fruit that has many beneficial effects (such as antidiabetic, antioxidant, anticancer, antidepressant, and immunomodulatory) on human health because of several dietary constituents such as phenolics, vitamins, carotenoids, and polysaccharides. In order to develop in vitro culture protocols for callus induction and plant regeneration from different explants of Goji plants and to compare the phenolic composition in calli of different origin, various combinations of plant growth regulators (PGRs) were applied. Various types of explants (leaf, petiole, root, hypocotyl, and node) were cultured on MS medium containing 28 different concentrations and combinations of PGRs [thidiazuron (TDZ) and naphthalene acetic acid (NAA), TDZ and indole-3-acetic acid (IAA), benzyl adenine (BA) and NAA, alone TDZ, and alone BA]. The highest mean number of shoots (23.33 ± 1.86) and percentage of shoot formation (100%) were obtained from nodal explants on medium containing 0.5 mg/l BA alone. The highest mean callus diameter was obtained from hypocotyl explants on medium containing 0.25 mg/l TDZ and 0.1 mg/l IAA (21.40 ± 0.71 mm). The amounts of selected phenolic substances were significantly different in the callus obtained from different PGRs or combinations, individually. The combination of BA/NAA significantly increased the production and accumulation of chlorogenic acid and caffeic acid. The TDZ/IAA combination, TDZ alone, and TDZ/NAA combination significantly increased vanillic acid and rutin, gallic acid, and quercetin synthesis, respectively. These outcomes indicate that different PGRs lead to the production of different kinds of secondary metabolites and affect/accelerate accumulation in the callus of goji berry. The culture protocol described in this paper could be employed for the development of novel methods for the commercial production of goji secondary metabolites. • New seed sterilization, callus induction, and regeneration protocols of goji berry (Lycium barbarum L.) were established. • Effects of different combinations and concentrations of PGRs on secondary metabolites production and accumulation of goji berry callus cultures were detected by LC-ESI-MS/MS analysis. • Phenolic biosynthesis of goji berry was accelerated by PGRs. • BA + NAA, TDZ + IAA, alone TDZ, and TDZ + NAA treatments significantly enhanced the production and accumulation of chlorogenic acid and caffeic acid, vanillic acid and rutin hydrate, gallic acid, and quercetin in goji berry callus cultures, respectively. [ABSTRACT FROM AUTHOR]

One of the major problems with the formulation of herbal medicines is the quality control of plant material to ensure its efficacy and safety. Quality control of medicinal plants requires analysis of many bioactive compounds present in the plant. C-alkylated flavonoids are an important bioactive subclass of flavonoids. A simple, rapid, sensitive and selective method is presented here for the quantification of bioactive C-alkylated flavonoids. This is the first quantitative method for analysis of C-alkylated flavonoids based on the multiple reaction monitoring (MRM) approach so far. This study focuses on method development for quantification of bioactive C-alkylated flavonoids. Quantification of a total of five C-alkylated flavonoids was done employing the MRM approach on an HPLC-QqQ-MS instrument. LODs and LOQs for quantified flavonoids were in the range of 0.41–1.32 and 1.23–3.96 ng/mL, respectively. Linear calibration curves between 25 and 1500 ng/mL were obtained with the regression coefficients of ≥0.996. Accuracy (% bias) and precision (% RSD) of the analyses were found to be less than 5%. Developed HPLC-ESI-MS/MS can be employed as a quality control method of plant raw materials. [ABSTRACT FROM AUTHOR]

To understand phycotoxin contamination in shellfish in the sub-Arctic and Arctic areas, scanning for the presence of 13 hydrophilic and lipophilic toxin components each was by liquid chromatography tandem quadrupole mass spectrometry analysis in shellfish samples collected from the Northern Bering Sea and the Chukchi Sea in 2014. The results showed that shellfish collected in both areas werecontaminated to different extents. Saxitoxin (STX), decarbamoylsaxitoxin (dcSTX) and decarbamoylneosaxitoxin (dcNEO) were the most frequently detected hydrophilic components, with maximum concentrations of 90.1 μg/kg, 112.25 μg/kg and 23.09 μg/kg, respectively. Although gonyautoxins (GTXs) were only detected in 3 samples, they were the main contributors to overall toxicity of high-latitude samples, especially GTX1. For lipophilic toxins, spirolide-1 (SPX1) and yessotoxin (YTX) were present in all samples at low levels (< 7 μg/kg and < 50 μg/kg, respectively). Only 5 samples showed evidence of okadaic acid (OA) and dinophysistoxin-2 (DTX-2) at low concentrations, ranging from 0.42 μg/kg to 7.23 μg/kg and 3.03 μg/kg to 30.59 μg/kg, respectively. Notably, a high level of pectenotoxin-1 (PTX-1) at 467.40 μg/kg was found in the shellfish collected at the northernmost station, exceeding the safety regulation standard by nearly 3 times. For both lipophilic and hydrophilic toxins, contamination in shellfish in the sub-Arctic and the Arctic area may be much more widespread and severe than was previously thought. This study highlighted the need to monitor toxins in a wider variety of shellfish, especially economic or commercial species, and across a wider range of sub-Arctic and Arctic waters, as well as the potential sources of these toxins. • STX, dcSTX and GTXs were the dominant hydrophilic toxins in shellfish from the Northern Bering Sea and the Chukchi Sea. • SPX1 and YTX were detected in all samples from both sampling areas. • Shellfish from high latitude locations showed higher total toxicity and variety of toxin components. • A high level of PTX-1 at 467 μg kg−1 was found at the northernmost station, exceeding 3 times the safety regulation standard. [ABSTRACT FROM AUTHOR]

Protein hydrolyzates of Stripped weakfish (Cynoscion guatucupa) industrial byproducts were prepared, and their antimicrobial and antioxidant activities, as well as bioaccessibility were evaluated. Byproducts were hydrolyzed by Alcalase (HA) and Protamex (HP) to achieve degrees of hydrolysis (DH) of 5, 10 and 15%, respectively. Resulting hydrolyzates were enzymatically digested with pepsin and pancreatin to determine the in vitro bioaccessibility. The highest antimicrobial activity was verified for HA with DH 5% (HA5) against Escherichia coli O157:H7 (5.50 ± 0.17 mm). Whereas HP with DH 5% (HP5) showed the highest antioxidant activity for the tested assays. After simulated in vitro gastrointestinal digestion, all samples were bioaccessible and showed an increase for the ABTS and hydroxyl radical scavenging with the highest activities for HA5 (87.7 ± 0.21%) and HP5 (87.5 ± 0.34%) and HP5 (96.7 ± 0.86%) and HP15 (97.5 ± 0.15%), respectively. We then submitted HA5 and HP5 to peptide sequences analysis, the bioactivities were attributed to the presence of amino acid such Phe, Leu, and Trp in the peptide sequences and peptide sequences like WDDMEK (HP5). The developed peptides present a potential for use as natural antimicrobial and antioxidant preservatives in food. • Hydrolyzates of C. guatucupa byproducts were prepared using Alcalase and Protamex. • Inhibition of E. coli and S. aureus growth by protein hydrolyzates with 5% DH. • Hydrolyzates with 5% DH by Protamex showed higher antioxidant activity. • The hydrolyzates were bioaccessible with antioxidant activity. • Bioactivities were attributed to the presence of specific amino acids or fragments. [ABSTRACT FROM AUTHOR]

In mass spectrometry, compounds that have different ionization properties experience challenges in simultaneous analysis. In the present paper, the authors proposed a polarity switching (+ve and -ve) LC-MS/MS method to analyze oxycodone and topiramate in a single run. The developed method was validated in the range of 5–1000 ng/mL for oxycodone and 20–5000 ng/mL for topiramate as per the US FDA guidelines. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode to analyze oxycodone and topiramate simultaneously using oxycodone-d 6 and topiramate-d 12 as internal standards, respectively. Sample preparation was performed in 96-well protein precipitation plates using acetonitrile. Processed samples were analyzed using a C 18 column with a gradient mobile phase composed of 10 mm ammonium formate with 0.1% formic acid and acetonitrile. The method was validated for selectivity, specificity, linearity, precision and accuracy, dilution integrity and stability. After validation, this method was successfully applied to quantify oxycodone and topiramate in plasma of concomitantly treated Sprague Dawley (SD) rats. • Polarity switching to detect and quantify analytes with different polarities. • High-throughput sample processing using 96-well plate format. • Application to pharmacokinetic interaction study. [ABSTRACT FROM AUTHOR]

Abstract The pseudo-targeted metabolomics approach was developed recently which combined the advantages of untargeted and targeted analysis. However, the current pseudo-targeted analysis method has limitations due to the technical characteristics. In this study, a novel metabolic pathway-based pseudo-targeted approach was proposed for urine metabolomics analysis using an ultra-high-performance liquid chromatography (UPLC)-MS/MS system operated in the multiple reaction monitoring (MRM) mode. MRM ion pairs were acquired from urine samples through untargeted analysis using UPLC-HRMS, as well as by searching for metabolites in related pathways in relevant databases and from previous relevant research, including amino acids, fatty acids, nucleosides, carnitines, glycolysis metabolites, and steroids. This improved pseudo-targeted method exhibited good repeatability and precision, and no complicated peak alignment was required. As a proof of concept, the developed novel method was applied to the discovery of urine biomarkers for patients with esophageal squamous cell carcinoma (ESCC). The results showed that ESCC patients had altered acylcarnitines, amino acids, nucleosides, and steroid derivative levels et al. compared to those of healthy controls. The novelty of this study lies in the fact that it provides an approach for acquiring MRM ion pairs not only from untargeted MS analysis but also from targeted searching for metabolites in related metabolic pathways. By improving the detection limit of low-abundance metabolites, it enlarges the range for the discovery of potential biomarkers. Our work provides a foundation for achieving pseudo-targeted metabolomics analysis on the widely used LC-MS/MS MRM platform. Graphical abstract fx1 Highlights • Ion pairs were from untargeted analysis and targeted searching in metabolic pathways. • It improves the detection limit for low-abundance metabolites. • It enlarges the range for the discovery of potential biomarkers. • A low-abundance potential biomarkers for ESCC was newly discovered by this method. [ABSTRACT FROM AUTHOR]

In the present study, we aimed to establish high-fat diet model for evaluating anti-hyperlipidaemic activity of GBA and to establish the HPLC-MS method for measuring the pharmacokinetics of GBA in rat blood plasma and liver after oral administration. Rats were orally administrated with GBA at the dose of 50 mg/kg body weight. GBA concentrations in blood plasma and liver were detected by HPLC from 0.5–10 h post administrations. The pharmacokinetic parameters were analyzed by the DAS 1.0 Software. The concentration-time curves of the plasma GBA and liver GBA were conformed to two-compartment model. GBA was rapidly absorbed in the gastrointestinal tract and could be detected in plasma and liver 0.5 h after oral administration. [ABSTRACT FROM AUTHOR]

Abstract: Wastewater treatment plant (WWTP) effluents are major sources of endocrine‐disrupting chemicals (EDCs) and other chemicals of toxicological concern for the aquatic environment. In the present study, we used an integrated strategy combining passive sampling (Chemcatcher®), developmental toxicity, and mechanism‐based in vitro and in vivo bioassays to monitor the impacts of a WWTP on a river. In vitro screening revealed the WWTP effluent as a source of estrogen, glucocorticoid, and aryl hydrocarbon (AhR) receptor‐mediated activities impacting the downstream river site where significant activities were also measured, albeit to a lesser extent than in the effluent. Effect‐directed analysis of the effluent successfully identified the presence of potent estrogens (estrone, 17α‐ethinylestradiol, and 17β‐estradiol) and glucocorticoids (clobetasol propionate and fluticasone propionate) as the major contributors to the observed in vitro activities, even though other unidentified active chemicals were likely present. The impact of the WWTP was also assessed using zebrafish embryo assays, highlighting its ability to induce estrogenic response through up‐regulation of the aromatase promoter‐dependent reporter gene in the transgenic (cyp19a1b–green fluorescent protein [GFP]) zebrafish assay and to generate teratogenic effects at nonlethal concentrations in the zebrafish embryo toxicity test. The present study argues for the use of such an integrated approach, combining passive sampling, bioassays, and effect‐directed analysis, to comprehensively identify endocrine active compounds and associated hazards of WTTP effluents. Environ Toxicol Chem 2018;37:2079–2088. © 2018 SETAC [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the correlation between histories of zolpidem and benzodiazepines use and their concentrations in hair as determined by segmental hair analysis, that is, by analyzing hair samples taken 0-1, 1-2, 2-3, 3-4, 4-5, and 5-6cm etc. and 0-3cm from the scalp, and whole hair. Of the 23 hair samples examined, 18 were collected from patients in a rehabilitation program and five were from patients that had taken zolpidem only once by prescription. All 23 patients provided written informed consent after reviewing the research plan, described their zolpidem and benzodiazepines use histories accurately, and provided hair samples, which were weighed, washed, cut into lengths of <1mm, and extracted in 100% methanol for 16h (diazepam-d5 was used as an internal standard). Extracts were evaporated under reduced pressure and reconstituted with aqueous methanol (1:1 v/v). These extracts (10μL) were analyzed by Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS). The method used was validated by determining LOD, LOQ, calibration curves, intra- and inter-accuracies, precisions, matrix effects, process efficiencies, extraction efficiencies, and processed sample stabilities. Five hundred and ninety-five 1cm hair segments showed 61.59% positive probability and 86.71% negative probability of quality correlation between zolpidem and benzodiazepines use and concentrations in hair. Good qualitative correlations were observed between drug use and detection in hair. False positivity and false negativity were very low. Of the hair samples taken from patients in a rehabilitation program, subject nos. 4, 5, and 12 had correlation coefficients of 0.68, 0.54 and 0.71, respectively, for relationships between zolpidem use and concentration of zolpidem in hair. For the 5 patients taking only a single dose of zolpidem (10mg), the average zolpidem concentrations in hair were 20, 15 and 40pg/mg after 5, 30 and 60 days, respectively. This study shows a relationship between history of zolpidem and benzodiazepines use and their concentrations in 1cm hair segment. [ABSTRACT FROM AUTHOR]

The sample preparation procedure for determination of microcystins from biomass by the LC-MS method was optimized. Treatment of freeze-dried biomass samples with acetonitrile prior to the main extraction was effectively used for decreasing the amount of protein-like matrix compounds in extracts. Use of acetonitrile-water mixtures during the main ultrasound-assisted extraction allowed increasing the total microcystins amount in the resultant extracts compared to the traditional extraction using 75% methanol. [ABSTRACT FROM AUTHOR]

Chloroplast, the energy organelle unique to plant cells, is a dynamic entity which integrates an array of metabolic pathways and serves as first level for energy conversion for the entire ecological hierarchy. Increasing amount of sequence data and evolution of mass spectrometric approaches has opened up new avenues for opportune exploration of the global proteome of this organelle. In our study, we aimed at generation of a comprehensive catalogue of chloroplast proteins in a grain legume, chickpea and provided a reference proteome map. To accurately assign the identified proteins, purity of chloroplast-enriched fraction was stringently monitored by multiple chemical and immunological indexes, besides pigment and enzyme analyses. The proteome analysis led to the identification of 2451 proteins, including 27 isoforms, which include predicted and novel chloroplast constituents. The identified proteins were validated through their sequence analysis. Extensive sequence based localization prediction revealed more than 50% proteins to be chloroplast resident by at least two different algorithms. Chromosomal distribution of identified proteins across nuclear and chloroplast genome unveiled the presence of 55 chloroplast encoded gene. In depth comparison of our dataset with the non-redundant set of chloroplast proteins identified so far across other species revealed novel as well as overlapping candidates. Biological significance Pulses add large amount of nitrogen to the soil and has very low water footprint and therefore, contributes to fortification of sustainable agriculture. Chickpea is one of the earliest cultivated legumes and serves as an energy and protein source for humans and animals. Chloroplasts are the unique organelles which conduct photosynthesis. Investigation on chloroplast proteome is of particular significance, especially to plant biologists, as it would allow a better understanding of chloroplast function in plants. Generation of a saturated proteome map would not only validate the proteome inventory from its genome sequencing, but also serve as a comprehensive catalogue for future studies. We identified 2451 proteins, encoded by both the nuclear as well as chloroplast genomes, presumably involved in multivariate metabolic processes. The chloroplast deduced proteome and putative chloroplast proteins identified in this study would provide a foundation for future investigation of the expression and function of the chloroplast proteins of chickpea in specific and other crops species in general. [ABSTRACT FROM AUTHOR]

Chemically unique environment of endophytes makes them to have various adaptive mechanisms for survival. One of such mechanisms involves the production of pharmacologically significant plant-specific metabolites. In the present study, 26 endophytic fungi were isolated from stem of Bacopa monnieri (L.) Wettst. plants. All the isolates were screened for bacopaside production property by HPLC. Among these, the fungal isolate BmF 16 which was identified as Aspergillus sp. was confirmed for bacopaside N1 production ( m/z 796) by LC-MS/MS analysis. As the extract of BMF16 used in the study was prepared from the fifth generation of culture, the obtained result can be confirmed as due to fungal production of bacopaside. In addition, this property was identified only for one among the 26 fungi screened. As bacopaside N1 production in fungi has not yet been reported, the results of the study are novel. [ABSTRACT FROM AUTHOR]

A simple, fast, sensitive and accurate methodology based on a LLE followed by liquid chromatography–tandem mass spectrometry for simultaneous determination of four regioisomers (8- iso prostaglandin F 2α , 8- iso -15(R)-prostaglandin F 2α , 11β-prostaglandin F 2α , 15(R)-prostaglandin F 2α ) in routine analysis of human plasma samples was developed. Isoprostanes are stable products of arachidonic acid peroxidation and are regarded as the most reliable markers of oxidative stress in vivo . Validation of method was performed by evaluation of the key analytical parameters such as: matrix effect, analytical curve, trueness, precision, limits of detection and limits of quantification. As a homoscedasticity was not met for analytical data, weighted linear regression was applied in order to improve the accuracy at the lower end points of calibration curve. The detection limits (LODs) ranged from 1.0 to 2.1 pg/mL. For plasma samples spiked with the isoprostanes at the level of 50 pg/mL, intra-and interday repeatability ranged from 2.1 to 3.5% and 0.1 to 5.1%, respectively. The applicability of the proposed approach has been verified by monitoring of isoprostane isomers level in plasma samples collected from young patients ( n = 8) subjected to hyperbaric hyperoxia (100% oxygen at 280 kPa(a) for 30 min) in a multiplace hyperbaric chamber. [ABSTRACT FROM AUTHOR]

Phoenix dactylifera L. plays an important role in social, economic, and ecological Tunisian sectors. Some date palms produce parthenocarpic fruit named Sish. The aqueous ethanolic extract from P. dactylifera parthenocarpic dates demonstrated a potent inhibition of the enzymes related to type II diabetes. In this work, extraction optimization of amylase inhibitors was carried out using Box-Behnken Design. Bioactivity-guided fractionation of the 70% aqueous ethanol extract was performed to identify the active compounds. The physicochemical results by liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis showed the presence of 13 phenolic compounds. The in vitro study showed that the extract exhibited a more specific inhibitor of α-glucosidase than α-amylase with an IC 50 value of 0.6 and 2.5 mg/mL, respectively. The in vivo study of this extract effect on the postprandial hyperglycemia activity showed a decrease in plasma glucose levels after 30 min stronger than the Acarbose effect. These results confirmed the anti-postprandial hyperglycemia activity of the aqueous ethanolic extract from P. dactylifera parthenocarpic dates, which could lend support for its pharmaceutical use. [ABSTRACT FROM AUTHOR]

Oxylipins are bioactive lipid mediators that participate in a wide range of processes, from blood flow to inflammation. In recent years, their exhaustive characterization as potential biomarkers has been actively pursued. However, several analytical challenges and sources of variability, like the low abundance of oxylipins in samples, their structural diversity, and the lack of harmonized and validated protocols for sample selection, collection, storage, and preparation have hampered their translation into clinical settings. In this review, these sources of variability are addressed and are exemplified with their application in recent clinical studies in different diseases. Our overall analysis highlights the need to achieve harmonized protocols for generating reliable and reproducible results that could be integrated into clinical practice to reach their full diagnostic and prognostic potential. [Display omitted] • Concept of biomarker and stages of their process until being used in clinics. • Analytical considerations for the analysis of oxylipins. • Relevant analytical solutions for isomeric oxylipins resolution. • Discussion of the method validation parameters for oxylipins. • Recent clinical studies of oxylipins. [ABSTRACT FROM AUTHOR]

Livistona jenkinsiana Griff., an endangered multi-purpose palm, is mostly confined to north-eastern India, cultivated and conserved for its edible fruit and other parts by tribal farmers of the state of Arunachal Pradesh. However, the nutritional composition of fruit is not known. Therefore, proximate estimation and metabolite profiling of fruits of this palm were carried out using spectrophotometry, gas chromatography-tandem mass spectrometry (GC-MS/MS), and liquid chromatography-mass spectrophotometry (LC-MS/MS). The major fatty acids were palmitic acid and oleic acid (approximately 45% and 33%, respectively). The purplish peel was due to malvidin (2032.6 μg/g), an anthocyanin. In the fruit pulp, the predominant sugar was fructose (158.7 μg/g); organic acid was citric acid (3856.2 μg/g); phenol was gentisic acid (660.8 μg/g); and flavonoid was rutin (115.8 μg/g). The bioactive compounds present in the fruit confer several health benefits, making L. jenkinsiana a potentially important species in terms of both nutrition and financial returns. [Display omitted] • Biochemical profile of Livistona jenkinsiana fruit assembled for the first time. • Fruit pulp was rich in oleic acid (32.86%, 18:1). • The predominant sugar in the fruit was fructose (0.15 mg/g). • The copper blue colour of the fruits is mainly due to malvidin (2 mg/g). • The antioxidant value of the fruit is mainly due to anthocyanins. [ABSTRACT FROM AUTHOR]

Among various chronic disorders, cancer and diabetes mellitus are the most common disorders. This study was designed to evaluate the effectiveness of hydroalcoholic extract of Phoenix dactylifera L. leaves (HEPdL) in animal models of type II diabetes in vitro / in vivo and in a human melanoma-derived cell line (IG R -39). A liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis was also performed to determine the amount of phenolic and flavonoid compounds in this plant. The physicochemical results by LC–MS/MS analysis of HEPdL showed the presence of 10 phenolic compounds. The in vitro study showed that the extract exhibited a more specific and potent inhibitor of α-glucosidase than α-amylase with an IC 50 value of 20 ± 1 μg/mL and 30 ± 0.8 μg/mL, respectively. More importantly, the in vivo study of the postprandial hyperglycemia activity with (20 mg/kg) of HEPdL showed a decrease in plasma glucose levels after 60 min in resemblance to the glucor (acarbose) (50 mg/kg) effect. The oral administration of HEPdL (20 mg/kg) in alloxan-induced diabetic mices for 28 days showed a more significant anti-diabetic activity than that of the drug (50 mg/kg). Moreover, cytotoxicity effects of HEPdL in IG R -39 cancer cell lines were tested by MTT assay. This extract was effective in inhibiting cancer cells growth (IGR-39) at dose 35 and 75 μg/mL. These results confirm ethnopharmacological significance of the plant and could be taken further for the development of an effective pharmaceutical drug against diabetes and cancer. [ABSTRACT FROM AUTHOR]