Tandem Mass Spectrometric Analysis Research Articles

Characterization of naphthoquinones as inhibitors of glutathione reductase and inducers of intracellular oxidative stress

ABSTRACT Glutathione reductase (GR), one of the most important antioxidant enzymes in maintaining intracellular redox homeostasis, has become a novel target to suppress cancer cell growth and metastasis. In this work, we evaluated a series of naphthoquinones (NQs) as potential GR inhibitors and elucidated the mechanism of inhibition. NQ-6, one of the most potent compounds among this series, inhibited GR in vitro and in vivo and was identified as a competitive and irreversible inhibitor. The Ki and kinact values of NQ-6 were determined to be 17.30 ± 3.63 μM and 0.0136 ± 0.0005 min-1, respectively. The tandem mass spectrometric analysis revealed that the two substrate binding sites Cys61 and Cys66 of yeast GR were modified simultaneously through arylation or only Cys66 was covalently modified by NQ-6. Intracellular reactive oxygen species, collapsing of mitochondrial membrane potential and protein S-glutathionylation elevation were induced by NQ-6. NQs can be valuable compounds in GR inhibition and oxidative stress-related research.

Mentha piperita silver nanoparticle-loaded hydrocolloid film for enhanced diabetic wound healing in rats.

To investigate the role of Mentha piperita silver nanoparticle-loaded carbopol gel for enhanced wound healing in a diabetic rat model. This research further aims to explore bioactive compounds derived from Mentha piperita obtained from high altitude. Methanolic extracts of Mentha piperita (MP), Mentha spicata (MS) and Mentha longifolia (ML) were used to synthesise silver nanoparticles (AgNP). AgNP synthesis was confirmed by ultraviolet-visible (UV-Vis) spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The antioxidant activity was assessed by 2, 2-diphenyl-1-picrylhydrazyl (DDPH) assay. Antiglycation potential was determined by measuring the fluorescent advanced glycation end products. The bioactive compound identified in the Mentha piperita methanolic (MPM) fraction through electrospray ionisation tandem mass spectrometric analysis (ESI-MS) was responsible for the highest antiglycation. The effects of MPM and MPM.AgNP-loaded Carbopol (Sanare Lab, India) on wound healing were compared in male, alloxan-induced, diabetic albino rats (200-250g), divided into control and treated groups. Effects on wound healing were assessed via histopathology. UV-Vis and FTIR confirmed NP synthesis with peaks for flavonoids and polyphenols. SEM and XRD explored the cubical, 30-63nm crystalline NP. The maximum antioxidant and antiglycation potential was observed in order of; MP.AgNP>MS.AgNP>ML.AgNP. The highest antioxidant activity was observed by methanolic and aqueous MP.AgNPs (88.55% and 83.63%, respectively) at 2mg.ml-1, and (75.16% and 69.73%, respectively) at 1mg.ml-1, compared to ascorbic acid (acting as a positive control, 90.01%). MPM.AgNPs demonstrated the best antiglycation potential of 75.2% and 83.3% at 1mg.ml-1 and 2mg.ml-1, respectively, comparable to positive control (rutin: 88.1%) at 14 days post-incubation. A similar trend was observed for antimicrobial activity against Bacillus subtilis, Micrococcus luteus and Escherichia coli with an inhibition zone of 21mm, 21.6mm and 24.6mm. Rosmarinic acid was the active compound present in Mentha piperita, as identified by ESI-MS. MPM.AgNP-loaded Carbopol resulted in 100% wound closure compared with control at 20 days post-wounding. In the treatment group, re-epithelialisation was achieved by day 18, compared with 25 days for the positive control group. MPM.AgNP-loaded Carbopol demonstrated safer and more effective biological properties, hence accelerating the diabetic excision wound healing process in alloxan-induced diabetic rats.

The adsorption of dragon fruit (Hylocereus polyrhizus) pigments on chitin for food colorants

AbstractBACKGROUNDGrowing concern over the potential adverse health effects of synthetic colorants has led to a rising utilization of plant‐derived pigments within the food industry. Purple betacyanins from red dragon fruits (Hylocereus polyrhizus) have been subjected to an environmentally sustainable purification process for natural colorants.RESULTSThe violet‐hued pigments were absorbed onto preconditioned chitin material, a biodegraded polymer derived from shrimp shells. Liquid chromatographic–electrospray ionization–tandem mass spectrometric analysis was employed to analyze the betalain pigment composition in the raw fruit extract, making comparisons between the adsorbed supernatant and desorbed solutions. Fourier transform infrared analysis, optical microscopy, and scanning electron microscopy were conducted on the resin pre‐ and post‐adsorption stages, revealing distinctive infrared bands (RNHR, CO, CO, OH) indicative of betacyanin functional groups in the pigmented materials. The uniform adsorption of the colorant onto the adsorbents causes the intense purple color of the resin, with a maximum removal efficiency of 0.29 mg g−1. Adsorption data were subjected to various kinetic models, with the pseudo‐second‐order model demonstrating the optimal fit to the kinetic data (R2 = 0.97), while the Freundlich model proved most suitable for describing the sorption isotherm (R2 = 0.98). The adsorption energy, as determined by the Dubinin–Radushkevich equation, was 0.23 kJ mol−1, implying a physical adsorption mechanism on a heterogeneous surface.CONCLUSIONBetacyanins derived from red dragon fruits could be recovered for food colorants through chitin adsorption. This recovery mechanism is attributed to electrostatic interactions among the functional groups, including hydroxyl and carboxyl groups, and hydrogen bonds. © 2024 Society of Chemical Industry (SCI).

Gas chromatography tandem mass spectrometric analysis of alkylphosphonofluoridic acids as verification targets of nerve agents

Alkylphosphonofluoridic Acids (APFA) are the major thermal degradation products of G- and A-series nerve agents and thus play a vital role in the verification analysis of Chemical Weapons Convention. Present study focuses on the development of sample clean-up, derivatization procedures and gas chromatography tandem mass spectrometric analysis of APFA in aqueous samples. APFA were found to be much more delicate than the corresponding alkylphosphonic acids and thus required subtle optimizations. Retention of analytes on silica and polymer-based anion exchangers followed by elution under alkaline conditions yielded best recoveries. Elution under acidic conditions led to partial or complete degradation of the analytes to alkylphosphonic acids. Silylation reactions, particularly with MTBSTFA were found the best in terms of chromatographic responses and resolution of the derivative peaks. Methylations with diazomethane, which requires acidic reaction media, failed to produce desired yields of the derivatives. Under optimized conditions, the analytes produced the recoveries ranging from 76.9 to 94.5% with RSD ≤9.2%. The best LOD's in the tandem mass spectrometric analysis ranged from 13 to 56 ng/ml. The applicability of the method was tested by spiking the analytes in the retained aqueous samples received for the 52nd proficiency test conducted by the Organization for the Prohibition of Chemical Weapons (OPCW).

Flipped C-Terminal Ends of APOA1 Promote ABCA1-Dependent Cholesterol Efflux by Small HDLs.

Cholesterol efflux capacity (CEC) predicts cardiovascular disease independently of high-density lipoprotein (HDL) cholesterol levels. Isolated small HDL particles are potent promoters of macrophage CEC by the ABCA1 (ATP-binding cassette transporter A1) pathway, but the underlying mechanisms are unclear. We used model system studies of reconstituted HDL and plasma from control and lecithin-cholesterol acyltransferase (LCAT)-deficient subjects to investigate the relationships among the sizes of HDL particles, the structure of APOA1 (apolipoprotein A1) in the different particles, and the CECs of plasma and isolated HDLs. We quantified macrophage and ABCA1 CEC of 4 distinct sizes of reconstituted HDL. CEC increased as particle size decreased. Tandem mass spectrometric analysis of chemically cross-linked peptides and molecular dynamics simulations of APOA1, the major protein of HDL, indicated that the mobility of C-terminus of that protein was markedly higher and flipped off the surface in the smallest particles. To explore the physiological relevance of the model system studies, we isolated HDL from LCAT-deficient subjects, whose small HDLs (like reconstituted HDLs) are discoidal and composed of APOA1, cholesterol, and phospholipid. Despite their very low plasma levels of HDL particles, these subjects had normal CEC. In both the LCAT-deficient subjects and control subjects, the CEC of isolated extra-small HDL (a mixture of extra-small and small HDL by calibrated ion mobility analysis) was 3- to 5-fold greater than that of the larger sizes of isolated HDL. Incubating LCAT-deficient plasma and control plasma with human LCAT converted extra-small and small HDL particles into larger particles, and it markedly inhibited CEC. We present a mechanism for the enhanced CEC of small HDLs. In smaller particles, the C-termini of the 2 antiparallel molecules of APOA1 are "flipped" off the lipid surface of HDL. This extended conformation allows them to engage with ABCA1. In contrast, the C-termini of larger HDLs are unable to interact productively with ABCA1 because they form a helical bundle that strongly adheres to the lipid on the particle. Enhanced CEC, as seen with the smaller particles, predicts decreased cardiovascular disease risk. Thus, extra-small and small HDLs may be key mediators and indicators of the cardioprotective effects of HDL.

Tandem mass spectrometric analysis of residual persistence, decontamination and dehydration on cyantraniliprole residues in Chilli

The dissipation behavior and persistence of cyantraniliprole residues in chilli and soil were assessed using high performance liquid chromatography and tandem mass spectrometry. Analytical methodology was suitably validated for recovery, precision, accuracy and sensitivity Limit of quantification (LOQ) and limit of detection (LOD) were found to be 0.01 and 0.003 mg kg-1. After two applications of cyantraniliprole @ 60, 75, 120 g a.i ha-1, the average initial deposits were found to be 0.69–0.72, 0.88–0.92, 1.40–1.42 mg kg-1, respectively, years 2021 & 2022. Half-life values (T1/2) for cyantraniliprole in chilli were 3.24, 3.53 and 3.89 days in 2021 and 3.78, 3.62 and 3.79 days in 2022, respectively for 60, 75, 120 g a.i ha-1 dose. Household processing methods were evaluated for decontamination of residues such as tap water wash, luke warm water wash, baking soda, common salt, acetic acid, potassium permanganate and boiling water. Among these methods, washing with boiling water was found to be most effective in reducing cyantraniliprole residues (up to 86%) followed by soaking in potassium permanganate (up to 72.27%) and soaking in baking soda (up to 69.37%). After dehydration of red chilli fruits, highest processing factor was 2.40 and 2.45 after application of cyantraniliprole @ 75 and 120 g a.i ha-1 in the test years.

Proteomic Analysis of the Cold Stress Response of Ammopiptanthus mongolicus Reveals the Role of AmCHIA in Its Cold Tolerance

Ammopiptanthus mongolicus, a traditional ethnic medicinal herb, is a rare broad-leaved evergreen shrub in the arid region of central Asia that can survive under extremely low temperatures during winter. In this study, we investigated the cold stress response of A. mongolicus leaves using physiological and proteomic approaches. Cold stress treatment increased the relative electrolyte leakage, proline, and soluble sugar levels and decreased the chlorophyll content in A. mongolicus leaves. Moreover, 93 differentially abundant proteins (DAPs) were identified using two-dimensional electrophoresis, of which 79 were further confirmed via tandem mass spectrometric analysis. The predicted functions of DAPs were mainly associated with photosynthesis in chloroplasts, reactive oxygen species scavenging, defense, and protein synthesis, folding, and degradation. A. mongolicus chitinase A (AmCHIA) is a cold-induced apoplast protein whose transcription is upregulated under cold, osmotic, high-salinity, and mechanical stresses. Recombinant AmCHIA expressed in Escherichia coli exhibits chitinase activity. Here, AmCHIA expression enhanced the cold tolerance of E. coli cells, suggesting that it may contribute to the cold adaptation of A. mongolicus after cold treatment. The present study not only provides important data for understanding the cold stress responses in plants but also serves as a basis for further exploration of the biological functions of cold-induced proteins in A. mongolicus using genetic approaches.

Cysteine Counting via Isotopic Chemical Labeling for Intact Mass Proteoform Identifications in Tissue.

Top-down proteomics, the tandem mass spectrometric analysis of intact proteoforms, is the dominant method for proteoform characterization in complex mixtures. While this strategy produces detailed molecular information, it also requires extensive instrument time per mass spectrum obtained and thus compromises the depth of proteoform coverage that is accessible on liquid chromatography time scales. Such a top-down analysis is necessary for making original proteoform identifications, but once a proteoform has been confidently identified, the extensive characterization it provides may no longer be required for a subsequent identification of the same proteoform. We present a strategy to identify proteoforms in tissue samples on the basis of the combination of an intact mass determination with a measured count of the number of cysteine residues present in each proteoform. We developed and characterized a cysteine tagging chemistry suitable for the efficient and specific labeling of cysteine residues within intact proteoforms and for providing a count of the cysteine amino acids present. On simple protein mixtures, the tagging chemistry yields greater than 98% labeling of all cysteine residues, with a labeling specificity of greater than 95%. Similar results are observed on more complex samples. In a proof-of-principle study, proteoforms present in a human prostate tumor biopsy were characterized. Observed proteoforms, each characterized by an intact mass and a cysteine count, were grouped into proteoform families (groups of proteoforms originating from the same gene). We observed 2190 unique experimental proteoforms, 703 of which were grouped into 275 proteoform families.

Intra-specific pharmacognostic biochemical screening of various populations of Clitoria ternatea L. using liquid chromatography/tandem mass spectrometry product ion scanning (LC-MS2 PIS)

Purpose of the researchWhile many inferences have been made about the low degree of genetic variation among intra specific populations of Clitoria ternatea L. (CT), pharmacognostic biochemical investigations using tandem mass spectrometry have never been conducted. Methanol leaf extract of Clitoria ternatea L., populations collected from 13 different agro-climatic zones of India was assessed for identification and characterization of targeted nootropic metabolites, using liquid chromatography tandem mass spectrometry product ion scanning (LC-MS2 PIS). Based on the RI values derived from LC-MS2 (PIS) superior population(s), in terms of the presence of the presence of nootropic metabolites, were identified. The intra-specific correlation among different populations, was analyzed through PCA (principal component analysis), scree plot, correlation matrix and hierarchical cluster analysis. Principal resultsThe targeted mass spectrum obtained by tandem mass spectrometric analysis revealed the presence of acetylcholine, choline, caffeic acid, gallic acid, ferulic acid, kaempferol and D-L valine mass fragments in the leaf of CT. These mass groups are belonging to four classes viz., phenols, flavonoids, neurotransmitters and amino acids. Based on the high degree of relative intensities of targeted nootropic metabolites, the Odisha population, collected from Cuttack, outperformed the rest of twelve populations. Correlation matrix data also revealed that populations habituated in similar climatic conditions show significant correlation with each other. Hierarchical cluster analysis classified the 13 populations in two groups. The first group contained 11 populations and the second group is restrained into two populations; one from Cuttack, Odisha and second one from Udaipur, Rajasthan. Major conclusionThis is the first report to identify the superior population of Clitoria ternatea L. across India, through multivariate statistical analysis of LC-MS2(PIS) for gallic acid, ferulic acid, caffeic acid, kaempferol, D-L-valine, acetylcholine, and choline. Even though earlier studies reported an enhancement of acetylcholine content in the central nervous system. The present work may explain that the plant is a producer of acetylcholine and choline itself so it can directly affect the memory and cognition of the brain. Simultaneously, the study confirms the presence of abovementioned seven nootropic metabolites in the leaf of CT. The Odisha population (collected from Cuttack) transcends over all other 12 populations, with highest RI values of seven nootropic metabolites. According to hierarchical cluster analysis, two populations, viz., the one collected from Cuttack, Odisha and the other collected from Udaipur, Rajasthan were distantly clustered from other eleven populations. Hence, both these populations should be given priority for pharmaceutical supply and value chain management systems.

Investigation of the Protective Effect for GcMAF by a Glycosidase Inhibitor and the Glycan Structure of Gc Protein

O-linked α-N-acetylgalactosamine (α-GalNAc) in the Gc protein is essential for macrophage activation; thus, the GalNAc-attached form of Gc protein is called Gc macrophage activating factor (GcMAF). O-linked glycans in Gc proteins from human plasma mainly consist of trisaccharides. GcMAF is produced when glycans on the Gc protein are hydrolyzed by α-Sia-ase and β-Gal-ase, leaving an α-GalNAc. Upon hydrolysis of α-GalNAc present on GcMAF, the protein loses the macrophage-activating effect. In contrast, our synthesized pyrrolidine-type iminocyclitol possessed strong in vitro α-GalNAc-ase inhibitory activity. In this study, we examined the protective effects of iminocyclitol against GcMAF via inhibition of α-GalNAc-ase activity. Detailed mass spectrometric analyses revealed the protective effect of the inhibitor on GcMAF. Furthermore, structural information regarding the glycosylation site and glycan structure was obtained using tandem mass spectrometric (MS/MS) analysis of the glycosylated peptides after tryptic digestion.

Acetyl-CoA carboxylase inhibitor increases LDL-apoB production rate in NASH with cirrhosis: prevention by fenofibrate

Treatment with acetyl-CoA carboxylase inhibitors (ACCi) in nonalcoholic steatohepatitis (NASH) may increase plasma triglycerides (TGs), with variable changes in apoB concentrations. ACC is rate limiting in de novo lipogenesis and regulates fatty acid oxidation, making it an attractive therapeutic target in NASH. Our objectives were to determine the effects of the ACCi, firsocostat, on production rates of plasma LDL-apoB in NASH and the effects of combined therapy with fenofibrate. Metabolic labeling with heavy water and tandem mass spectrometric analysis of LDL-apoB enrichments was performed in 16 NASH patients treated with firsocostat for 12 weeks and in 29 NASH subjects treated with firsocostat and fenofibrate for 12 weeks. In NASH on firsocostat, plasma TG increased significantly by 17% from baseline to week 12 (P = 0.0056). Significant increases were also observed in LDL-apoB fractional replacement rate (baseline to week 12: 31 ± 20.2 to 46 ± 22.6%/day, P = 0.03) and absolute synthesis rate (ASR) (30.4–45.2 mg/dl/day, P = 0.016) but not plasma apoB concentrations. The effect of firsocostat on LDL-apoB ASR was restricted to patients with cirrhosis (21.0 ± 9.6 at baseline and 44.2 ± 17 mg/dl/day at week 12, P = 0.002, N = 8); noncirrhotic patients did not change (39.8 ± 20.8 and 46.3 ± 14.8 mg/dl/day, respectively, P = 0.51, N = 8). Combination treatment with fenofibrate and firsocostat prevented increases in plasma TG, LDL-apoB fractional replacement rate, and ASR. In summary, in NASH with cirrhosis, ACCi treatment increases LDL-apoB100 production rate and this effect can be prevented by concurrent fenofibrate therapy.

Four-year follow-up of surface contamination by antineoplastic drugs in a compounding unit

ObjectivesThis study aimed to monitor the contamination by antineoplastic drugs on work surfaces in a compounding unit 4 years after its implementation.MethodsThis descriptive study was done in a unit performing...

Chemical, Antioxidant and Biological Studies of Brassica incana subsp. raimondoi (Brassicaceae) Leaf Extract.

Brassica incana subsp. raimondoi is an endemic taxon present in a restricted area located on steep limestone cliffs at an altitude of about 500 m a.s.l. in eastern Sicily. In this research, for the first time, studies on the phytochemical profile, the antioxidant properties in cell-free and cell-based systems, the cytotoxicity on normal and cancer cells by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay, and on Artemia salina Leach, were performed. The total phenolic, flavonoid, and condensed tannin contents of the leaf hydroalcoholic extract were spectrophotometrically determined. Ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) analysis highlighted the presence of several phenolic acids, flavonoids, and carotenoids, while High-Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD) identified various kaempferol and isorhamnetin derivatives. The extract exhibited different antioxidant properties according to the five in vitro methods used. Cytotoxicity by MTT assay evidenced no impact on normal human fibroblasts (HFF-1) and prostate cancer cells (DU145), and cytotoxicity accompanied by necrotic cell death for colon cancer cells (CaCo-2) and hepatoma cells (HepG2), starting from 100 μg/mL and 500 μg/mL, respectively. No cytotoxic effects were detected by the A. salina lethality bioassay. In the H2O2-induced oxidative stress cell model, the extract counteracted cellular reactive oxygen species (ROS) production and preserved non-protein thiol groups (RSH) affected by H2O2 exposure in HepG2 cells. Results suggest the potential of B. incana subsp. raimondoi as a source of bioactive molecules.

Reaction of human telomeric unit TTAGGG and a photoactivatable Pt(IV) anticancer prodrug.

The interaction of a photoactivatable diazidodihydroxido Pt(IV) prodrug, trans,trans,trans-[Pt(N3)2(OH)2(py)2] (py = pyridine; 1), with a hexamer straight human telomeric DNA unit sequence (5'-T1T2A3G4G5G6-3', I) upon light irradiation was investigated by electrospray ionization mass spectroscopy (ESI-MS). In the primary mass spectrum, two major mono-platinated I adducts with the bound Pt moieties, trans-[PtII(N3)(py)2]+ (1') and trans-[PtII(py)2]2+ (1''), respectively, were detected. It is rare to observe such high abundance and nearly equal intensity platinated DNA adducts formed by these two PtII species because 1' is usually the only major reduced Pt(II) species produced by the photodecomposition of complex 1 in the presence of DNA while 1'' was rarely detected as the major reduced PtII species reported previously. Subsequent tandem mass spectrometric analysis by collision-induced dissociation (CID) showed that in the former adduct {I + 1'}2+, G6 and A3 were the platination sites. While in the latter adduct {I + 1''}2+, a potential intrastrand crosslink was speculated after G4 and G6 sites were identified. Additionally, other minor platinated adducts like di-platinated I adduct by 1' with platination sites at G4 and G6 and mono-platinated I adducts containing base oxidation were also detected by mass spectrometry. Due to the rich guanines and their sensitivity to oxidation, the oxidation induced by 1 most probably occurred at guanine. The oxidation adducts were proposed as 8-hydroxyl guanine, spiroiminodihydantoin (Sp), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), 5-guanidinohydantoin (Gh), and/or dehydroguanidinohydantoin (DGh) referring to previous reports. The obtained results provide useful chemical information about the photoreaction between photoactivatable Pt(IV) anticancer prodrugs and human telomeric DNA. Such special damages of Pt(IV) prodrugs on human telomeric DNA implicate its active role in the mechanism of Pt(IV) prodrugs and further support the unique sequence-dependent photointeraction profile of complex 1 reacting with DNA.

Immune checkpoint therapy-elicited sialylation of IgG antibodies impairs antitumorigenic type I interferon responses in hepatocellular carcinoma

The reinvigoration of anti-tumor Tcells in response to immune checkpoint blockade (ICB) therapy is well established. Whether and how ICB therapy manipulates antibody-mediated immune response in cancer environments, however, remains elusive. Using tandem mass spectrometric analysis of modification of immunoglobulin G (IgG) from hepatoma tissues, we identified a role of ICB therapy in catalyzing IgG sialylation in the Fc region. Effector Tcells triggered sialylation of IgG via an interferon (IFN)-γ-ST6Gal-I-dependent pathway. DC-SIGN+ macrophages represented the main target cells of sialylated IgG. Upon interacting with sialylated IgG, DC-SIGN stimulated Raf-1-elicited elevation of ATF3, which inactivated cGAS-STING pathway and eliminated subsequent type-I-IFN-triggered antitumorigenic immunity. Although enhanced IgG sialylation in tumors predicted improved therapeutic outcomes for patients receiving ICB therapy, impeding IgG sialylation augmented antitumorigenic Tcell immunity after ICB therapy. Thus, targeting antibody-based negative feedback action of ICB therapy has potential for improving efficacy of cancer immunotherapies.

Metabolite Profiling and Bioassay-Guided Fractionation of Zataria multiflora Boiss. Hydroethanolic Leaf Extracts for Identification of Broad-Spectrum Pre and Postharvest Antifungal Agents

Hydroethanolic leaf extracts of 14 Iranian Zataria multiflora Boiss. populations were screened for their antifungal activity against five plant pathogenic fungi and metabolically profiled using a non-targeted workflow based on UHPLC/ESI-QTOFMS. Detailed tandem mass-spectrometric analyses of one of the most active hydroethanolic leaf extracts led to the annotation of 68 non-volatile semi-polar secondary metabolites, including 33 flavonoids, 9 hydroxycinnamic acid derivatives, 14 terpenoids, and 12 other metabolites. Rank correlation analyses using the abundances of the annotated metabolites in crude leaf extracts and their antifungal activity revealed four O-methylated flavones, two flavanones, two dihydroflavonols, five thymohydroquinone glycoconjugates, and five putative phenolic diterpenoids as putative antifungal metabolites. After bioassay-guided fractionation, a number of mono-, di- and tri-O-methylated flavones, as well as three of unidentified phenolic diterpenoids, were found in the most active subfractions. These metabolites are promising candidates for the development of new natural fungicides for the protection of agro-food crops.

Fentanyl in the labor epidural impacts the results of intrapartum and postpartum maternal and neonatal toxicology tests

A positive urine fentanyl toxicology test may have considerable consequences for peripartum individuals, yet the extent to which fentanyl administration in a labor epidural may lead to such a positive test is poorly characterized. This study aimed to quantify the extent to which neuraxial fentanyl in labor neuraxial analgesia can lead to a positive peripartum maternal or neonatal urine toxicology test. We performed a prospective cohort study of pregnant participants planning a vaginal delivery with neuraxial analgesia. Participants with a history of substance use disorder, hypertension, or renal or liver disease were excluded. A urine sample was collected before initiation of neuraxial analgesia, each time the bladder was emptied during labor, and up to 4 times postpartum. Neonatal urine was collected once. Urine fentanyl testing was performed using 2 common toxicology testing methods, namely immunoassay and liquid chromatography with tandem mass spectrometric detection. A total of 33 maternal-infant dyads yielded a total of 178 urine specimens. All maternal specimens were negative for fentanyl using liquid chromatography with tandem mass spectrometric analysis and immunoassay before initiation of neuraxial analgesia. Intrapartum, 26 of 30 (76.7%) participants had positive liquid chromatography with tandem mass spectrometry results for fentanyl or its metabolites, and 12 of 30 (40%) participants had positive immunoassay results. Postpartum, 19 of 21 (90.5%) participants had positive liquid chromatograph with tandem mass spectrometric results, and 13 of 21 (61.9%) had a positive immunoassay result. Of the 13 neonatal specimens collected, 10 (76.9%) were positive on liquid chromatography with tandem mass spectrometry analysis, the last of which remained positive 29 hours and 50 minutes after delivery. Neuraxial fentanyl for labor analgesia may lead to positive maternal and neonatal toxicology tests at various times after epidural initiation and cessation and at different rates depending on the testing method used. Caution should be used in interpreting toxicology test results of individuals who received neuraxial analgesia to avoid false assumptions about nonprescribed use.

Proteomic changes in human spermatozoa during in vitro capacitation and acrosome reaction in normozoospermia and asthenozoospermia.

The cellular and molecular mechanisms of the events that help spermatozoa acquire their fertilizing capability during capacitation and acrosome reaction are not completely understood. This study was performed with a postulation that the identification of sperm proteins and their changes during in vitro capacitation and acrosome reaction will unravel unknown molecular aspects of fertilization that impact male fertility. Spermatozoa collected from sequential conditions, that is, separation of ejaculated spermatozoa by Percoll gradient centrifugation, in vitro capacitation, and acrosome reaction were processed for tandem mass spectrometric analysis, followed by protein identification, label-free quantitation, and statistical analysis. Collectively, a total of 1088 sperm proteins were identified. In comparison to ejaculated spermatozoa, 44 and 141 proteins were differentially expressed in capacitated and acrosome reacted spermatozoa, respectively. A large number of proteins were found downregulated, including clusterin, pyruvate dehydrogenase E1 component, semenogelin-1 and 2, heat shock protein 90, beta-microseminoprotein, and keratin. It was expected as sperm-membrane-associated proteins are removed during capacitation. There were significant proteomic alterations in asthenozoospermia compared to normozoospermia; however, variation was more noticeable among proteins of acrosome reacted spermatozoa and those released during the acrosome reaction. The processes enriched among downregulated proteins in asthenozoospermia included acrosome assembly, binding of spermatozoa to zona pellucida, nucleosome assembly, flagellated sperm motility, protein folding, oxidative phosphorylation, tricarboxylic acid cycle, chromatin silencing, gluconeogenesis, glycolytic process, and glycolysis. The dynamic information generated about proteomic alterations in spermatozoa during capacitation and acrosome reaction and their variability in asthenozoospermia will contribute not only to enhancing our understanding of processes that prepare spermatozoa to acquire fertilization capability but also help in deciphering novel factors of male infertility.

Identification and characterization of the in vitro metabolites of batatasin III by liquid chromatography in combination with Orbitrap mass spectrometry.

Batatasin III is a biologically active ingredient extracted from Dendrobium scabrilingue, which has been demonstrated to have anticancer activity. To fully understand its action, the present study was performed to investigate the in vitro metabolism of batatasin III using rat and human liver microsomes and hepatocytes. Batatasin III (20 μM) was incubated in the presence of NADPH-supplemented rat and human liver microsomes (0.5mg protein/mL) and hepatocytes (1 million cells/mL) followed by liquid chromatography in combination with hybrid quadrupole Orbitrap tandem mass spectrometric analysis to detect and identify the generated metabolites. The structures of the metabolites were characterized by comparing the accurate masses, elemental compositions as well as indicative fragment ions with those of the parent. A total of 15 metabolites were detected and identified, including 4 phase I and 11 phase II metabolites. Batatasin III is subjected to bioactivation to form reactive quinoid intermediates, which subsequently react with glutathione (GSH) via Michael addition. Glucuronidation and GSH conjugation appear to be the primary elimination pathways in rat hepatocytes, while in human hepatocytes, GSH conjugates are formed to a lesser extent. Phase I metabolic pathways include hydroxylation and demethylation. The present study sheds light on the in vitro metabolic fates of batatasin III, which is indispensable for an understanding of its efficacy and safety.

Investigation of the Captopril-Insulin Interaction by Mass Spectrometry and Computational Approaches Reveals that Captopril Induces Structural Changes in Insulin.

Post-translational modifications remarkably regulate proteins’ biological function. Small molecules such as reactive thiols, metabolites, and drugs may covalently modify the proteins and cause structural changes. This study reports the covalent modification and noncovalent interaction of insulin and captopril, an FDA-approved antihypertensive drug, through mass spectrometric and computation-based approaches. Mass spectrometric analysis shows that captopril modifies intact insulin, reduces it into its “A” and “B” chains, and covalently modifies them by forming adducts. Since captopril has a reactive thiol group, it might reduce the insulin dimer or modify it by reacting with cysteine residues. This was proven with dithiothreitol treatment, which reduced the abundance of captopril adducts of insulin A and B chains and intact Insulin. Liquid chromatography tandem mass spectrometric analysis identified the modification of a total of four cysteine residues, two in each of the A and B chains of insulin. These modifications were identified to be Cys6 and Cys7 of the A chain and Cys7 and Cys19 of the B chain. Mass spectrometric analysis indicated that captopril may simultaneously modify the cysteine residues of intact insulin or its subunits A and B chains. Biophysical studies involving light scattering and thioflavin T assay suggested that the binding of captopril to the protein leads to the formation of aggregates. Docking and molecular dynamics studies provided insights into the noncovalent interactions and associated structural changes in insulin. This work is a maiden attempt to understand the detailed molecular interactions between captopril and insulin. These findings suggest that further investigations are required to understand the long-term effect of drugs like captopril.