Metabolomics profiling and chemoresistance mechanisms in ovarian cancer cell lines: Implications for targeting glutathione pathway.

Ovarian cancer presents a significant challenge due to its high rate of chemoresistance, which complicates the effectiveness of drug-response therapy. This study provides a comprehensive metabolomic analysis of ovarian cancer cell lines OVCAR-3 and SK-OV-3, characterizing their distinct metabolic landscapes. Metabolomics coupled with chemometric analysis enabled us to discriminate between the metabolic profiles of these two cell lines. The OVCAR-3 cells, which are sensitive to doxorubicin (DOX), exhibited a preference for biosynthetic pathways associated with cell proliferation. Conversely, DOX-resistant SK-OV-3 cells favored fatty acid oxidation for energy maintenance. Notably, a marked difference in glutathione (GSH) metabolism was observed between these cell lines. Our investigations further revealed that GSH depletion led to a profound change in drug sensitivity, inducing a shift from a cytostatic to a cytotoxic response. The results derived from this comprehensive metabolomic analysis offer potential targets for novel therapeutic strategies to overcome drug resistance. Our study suggests that targeting the GSH pathway could potentially enhance chemotherapy's efficacy in treating ovarian cancer.

Lipidomics analysis reveals the effect of Sirex noctilio infestation on the lipid metabolism in Pinus radiata needles.

The Sirex noctilio's climatic adaption and rapid proliferation have caused Pinus mortality worldwide. The infestation combines the early effect of female S. noctilio gland secretion and the spreading symbiotic fungus Amylostereum areolatum. 'Lipidomics' is the study of all non-water-soluble components of the metabolome. Most of these non-water-soluble compounds correspond to lipids which can provide information about a biological activity, an organelle, an organism, or a disease. Using HPLC-MS/MS based lipidomics, 122 lipids were identified in P. radiata needles during S. noctilio infestation. Phosphatidic acids, N-acylethanolamines, and phosphatidylinositol-ceramides accumulated in infested trees could suggest a high level of phospholipases activities. The phosphatidylcholines were the most down-regulated species during infection, which could also suggest that they may be used as a substrate for up-regulated lipids. The accumulation of very long-chain fatty acids and long-chain fatty acids during the infestation could imply the tree defense response to create a barrier in the drilled zone to avoid larvae development and fungus proliferation. Also, the growth arrest phase of the trees during the prolonged infestation suggests a resistance response, regulated by the accumulation of NAE, which potentially shifts the tree energy to respond to the infestation.

Metabolome expression in Eucryphia cordifolia populations: Role of seasonality and ecological niche centrality hypothesis.

The ecological niche centrality hypothesis states that population abundance is determined by the position in the ecological niche, expecting higher abundances towards the center of the niche and lower at the periphery. However, the variations in the conditions that favor the persistence of populations between the center and the periphery of the niche can be a surrogate of stress factors that are reflected in the production of metabolites in plants. In this study we tested if metabolomic similarity and diversity in populations of the tree species Eucryphia cordifolia Cav. vary according to their position with respect to the structure of the ecological niche. We hypothesize that populations growing near the centroid should exhibit lower metabolites diversity than plants growing at the periphery of the niche. The ecological niche of the species was modeled using correlative approaches and bioclimatic variables to define central and peripheral localities from which we chose four populations to obtain their metabolomic information using UHPLC-DAD-QTOF-MS. We observed that populations farther away from the centroid tend to have higher metabolome diversity, thus supporting our expectation of the niche centrality hypothesis. Nonetheless, the Shannon index showed a marked variation in metabolome diversity at the seasonal level, with summer and autumn being the periods with higher metabolite diversity compared to winter and spring. We conclude that both the environmental variation throughout the year in combination with the structure of the ecological niche are relevant to understand the variation in expression of metabolites in plants.

Berberis microphylla G. Forst Intake Reduces the Cardiovascular Disease Plasmatic Markers Associated with a High-Fat Diet in a Mice Model.

Polyphenols are bioactive substances that participate in the prevention of chronic illnesses. High content has been described in Berberis microphylla G. Forst (calafate), a wild berry extensively distributed in Chilean-Argentine Patagonia. We evaluated its beneficial effect through the study of mouse plasma metabolome changes after chronic consumption of this fruit. Characterized calafate extract was administered in water, for four months, to a group of mice fed with a high-fat diet and compared with a control diet. Metabolome changes were studied using UHPLC-DAD-QTOF-based untargeted metabolomics. The study was complemented by the analysis of protein biomarkers determined using Luminex technology, and quantification of OH radicals by electron paramagnetic resonance spectroscopy. Thirteen features were identified with a maximum annotation level-A, revealing an increase in succinic acid, activation of tricarboxylic acid and reduction of carnitine accumulation. Changes in plasma biomarkers were related to inflammation and cardiovascular disease, with changes in thrombomodulin (-24%), adiponectin (+68%), sE-selectin (-34%), sICAM-1 (-24%) and proMMP-9 (-31%) levels. The production of OH radicals in plasma was reduced after calafate intake (-17%), especially for the group fed with a high-fat diet. These changes could be associated with protection against atherosclerosis due to calafate consumption, which is discussed from a holistic and integrative point of view.

Steroidal Saponins with Plant Growth Stimulation Effects; Yucca schidigera as a Commercial Source.

Plant growth-stimulation bioactivity of triterpenoid saponins is well known, especially for oleanane-type compounds. Nevertheless, a few phytotoxicity bioassays performed on some steroidal saponins have shown hormesis profiles and growth stimulation on Lactuca sativa roots. The focus of the work described here was on the use of the wheat coleoptile bioassay to evaluate plant growth stimulation, and on the search for a commercially available source of active saponins by bio-guided fractionation strategy. Selected saponins were tested and a cluster analysis showed that those saponins with a sugar chain of more than five units had a hormesis profile, while saponins with growth enhancement had fewer sugar residues. Two saponins showed similar activity to the positive control, namely the phytohormone indole-3-butyric acid (IBA). As a potential source of these metabolites, a commercial extract of Yucca schidigera used as a fertilizer was selected. Bio-guided fractionation led to the identification of two fractions of defined composition and these showed stimulation values similar to the positive control. It was observed that the presence of a carbonyl group at C-12 on the aglycone skeleton led to improved activity. A saponin-rich fraction from Y. schidigera could be proposed to enhance crop quality and production.

Metabolic profile and antioxidant capacity of five Berberis leaves species: A comprehensive study to determine their potential as natural food or ingredient.

A comprehensive study of bioactive compounds was carried out in the leaves of the main Berberis species growing in Chile (Berberis microphylla, Berberis darwinii, Berberis empetrifolia, Berberis trigona, and the introduced Berberis vulgaris). We identified 117 compounds, by a detailed analysis of each molecule, including phenolic acids, alkaloids, flavonols, and other compounds, using high-performance liquid chromatography and high-resolution mass spectrometry. Quantitative analysis of main compound was also included for all species. Hydroxycinnamic acid derivatives were the main compounds in all the studied leaves, with the highest concentration in Berberis microphylla. Quercetin derivatives were the most relevant flavonols in all species, except in Berberis vulgaris, in which isorhamnetin-3-rutinoside was the most concentrated. The fatty acid profile, determined by gas chromatography mass spectrometry revealed the presence of linoleic and linolenic acids in all species studied. Berberis vulgaris showed higher levels of these fatty acids. The antioxidant capacity, explored by three in vitro methods, showed high values for all studied Berberis species. The obtained levels are higher than those of other prominent foods. The findings will inform novel approaches for the valorization of these leaves as natural food or ingredient.

Sirex noctilio infestation led to inevitable pine death despite activating pathways involved in tolerance.

Defense-related metabolome traits in pine species after infestation by Sirex noctilio are largely unknown, despite, in most cases, trees being overwhelmed. Using LC-MS-based untargeted metabolomics, we revealed the systemic metabolic changes induced by this insect in 14-year-old Pinus radiata trees, the most affected species worldwide. An immediate metabolome alteration was expressed in needles after infestation, including the up-regulation of flavonols, flavan-3-ols, oxyneolignans, auxins, proline, and tryptophan, among others. The flavan-3-ols (catechin and procyanidin B1) suggested a rapidly induced photoprotection mechanism aided by diverting proline as an alternative substrate for respiration to compensate for the progressive chlorosis that degrades photosystems. Meanwhile, glutathione, glutamate, and ascorbate levels significantly dropped in needles, which may indicate the critical oxidative stress that trees had to face since the onset of the infestation. They were not fully replenished after long-term infestation, and redox homeostasis was probably not achieved, compromising tree survival. Nevertheless, a huge auxins overexpression detected in needles throughout the infestation may reflect tolerance against the premature senescence caused by the woodwasp venom. In contrast, the metabolome of wood tissues remained initially unchanged, although it seems to collapse after three months. Overall, the metabolomics strategy adopted in this work evidenced its usefulness in uncovering the fundamental roles of plants' chemical defense that govern interactions with specific stressors.

Distinctive mechanisms of epilepsy-causing mutants discovered by measuring S4 movement in KCNQ2 channels.

Neuronal KCNQ channels mediate the M-current, a key regulator of membrane excitability in the central and peripheral nervous systems. Mutations in KCNQ2 channels cause severe neurodevelopmental disorders, including epileptic encephalopathies. However, the impact that different mutations have on channel function remains poorly defined, largely because of our limited understanding of the voltage-sensing mechanisms that trigger channel gating. Here, we define the parameters of voltage sensor movements in wt-KCNQ2 and channels bearing epilepsy-associated mutations using cysteine accessibility and voltage clamp fluorometry (VCF). Cysteine modification reveals that a stretch of eight to nine amino acids in the S4 becomes exposed upon voltage sensing domain activation of KCNQ2 channels. VCF shows that the voltage dependence and the time course of S4 movement and channel opening/closing closely correlate. VCF reveals different mechanisms by which different epilepsy-associated mutations affect KCNQ2 channel voltage-dependent gating. This study provides insight into KCNQ2 channel function, which will aid in uncovering the mechanisms underlying channelopathies.

Special Issue "Natural Plant Substances-Structural and Application Aspects: A Theme Issue in Honor of Professor Wieslaw Oleszek".

Dear Colleagues, [...].

Experimental protocol for the study of One-pot amination of Cyclohexanone-to-secondary amines over Carbon-supported Pd.

The validation of protocols for carrying out the experimental analysis of amination reactions is of paramount importance to enhance the scientific knowledge and reproducibility of results. Accordingly, in the present paper, a protocol has been proposed for the study of the amination of cyclohexanone-to-secondary amines (Diphenylamine and N-Cyclohexylaniline) over heterogeneous catalysts. The results of activity and selectivity, and the elucidation of a plausible reaction pathway were described in a parent paper. Therefore, the purpose of this document is to inform about the details of the experimental setups, the methods, and the analytical techniques to identify and quantify the reaction products. Finally, some practical and safety considerations are also included.•One-pot catalytic amination of cyclohexanone with aniline was performed efficiently in liquid phase on Pd/C.•Stirring, He atmosphere and temperature control were critical to achieve reproducible activity results.•Ultra-High Performance Liquid Chromatography allows identifying products and reaction intermediates, while nonane performed well as internal standard for GC-FID quantification.

Mechanisms of disease-modifying effect of saracatinib (AZD0530), a Src/Fyn tyrosine kinase inhibitor, in the rat kainate model of temporal lobe epilepsy.

We have recently demonstrated the role of the Fyn-PKCδ signaling pathway in status epilepticus (SE)-induced neuroinflammation and epileptogenesis in experimental models of temporal lobe epilepsy (TLE). In this study, we show a significant disease-modifying effect and the mechanisms of a Fyn/Src tyrosine kinase inhibitor, saracatinib (SAR, also known as AZD0530), in the rat kainate (KA) model of TLE. SAR treatment for a week, starting the first dose (25 mg/kg, oral) 4 h after the onset of SE, significantly reduced spontaneously recurring seizures and epileptiform spikes during the four months of continuous video-EEG monitoring. Immunohistochemistry of brain sections and Western blot analyses of hippocampal lysates at 8-day (8d) and 4-month post-SE revealed a significant reduction of SE-induced astrogliosis, microgliosis, neurodegeneration, phosphorylated Fyn/Src-419 and PKCδ-tyr311, in SAR-treated group when compared with the vehicle control. We also found the suppression of nitroxidative stress markers such as iNOS, 3-NT, 4-HNE, and gp91phox in the hippocampus, and nitrite and ROS levels in the serum of the SAR-treated group at 8d post-SE. The qRT-PCR (hippocampus) and ELISA (serum) revealed a significant reduction of key proinflammatory cytokines TNFα and IL-1ß mRNA in the hippocampus and their protein levels in serum, in addition to IL-6 and IL-12, in the SAR-treated group at 8d in contrast to the vehicle-treated group. These findings suggest that SAR targets some of the key biomarkers of epileptogenesis and modulates neuroinflammatory and nitroxidative pathways that mediate the development of epilepsy. Therefore, SAR can be developed as a potential disease-modifying agent to prevent the development and progression of TLE.

Features in the NMR spectra of the aglycones of Agave spp. saponins. HMBC method for aglycone identification (HMAI).

INTRODUCTION: The analysis and detection of steroidal saponins is mainly performed using chromatographic techniques coupled with mass spectrometry. However, nuclear magnetic resonance (NMR) spectroscopy is a potential tool that can be combined with these techniques to obtain unambiguous structural characterisation. OBJECTIVE: This work provides a review of the carbon-13 (13 C)- and proton (1 H)-NMR spectroscopic data of aglycones from Agave saponins reported in the literature and also the development of an easy identification method for these natural products. METHODS: The database Scifinder was used for spectroscopic data collection in addition to data obtained from the Cadiz Allelopathy research group. The keywords used were Agave, spirostanic, furostanic, and saponin. RESULTS: The shielding variations produced by functional groups on the aglycone core and the structural features of the most representative aglycones from Agave species are described. The effects are additive for up to four long-range connectivities. A method for the identification of aglycones (HMAI) is proposed to classify aglycones from Agave spp. through the use of 1 H-NMR and heteronuclear multiple bond correlation (HMBC) experiments. CONCLUSIONS: The HMBC spectrum is representative of the structural features of aglycones from Agave spp. The HMBC method for aglycone identification (HMAI) method allowed the identification of pure saponins or mixtures thereof and this method can be used in combination with chromatographic techniques coupled with mass spectrometry to provide a more thorough analysis of Agave samples that contain aglycones.

Berberis microphylla G. Forst (Calafate) Berry Extract Reduces Oxidative Stress and Lipid Peroxidation of Human LDL.

Calafate (Berberis microphylla G. Forst) is a Patagonian barberry very rich in phenolic compounds. Our aim was to demonstrate, through in vitro models, that a comprehensive characterized calafate extract has a protective role against oxidative processes associated to cardiovascular disease development. Fifty-three phenolic compounds (17 of them not previously reported in calafate), were tentatively identified by Ultra-Liquid Chromatography with Diode Array Detector, coupled to Quadrupole-Time of Fly Mass Spectrometry (UHPLC-DAD-QTOF). Fatty acids profile and metals content were studied for the first time, by Gas Chromatography Mass Spectrometry (GC-MS) and Total X-ray Fluorescence (TXRF), respectively. Linolenic and linoleic acid, and Cu, Zn, and Mn were the main relevant compounds from these groups. The bioactivity of calafate extract associated to the cardiovascular protection was evaluated using Human Umbilical Vein Endothelial Cells (HUVECs) and human low density lipoproteins (LDL) to measure oxidative stress and lipid peroxidation. The results showed that calafate extract reduced intracellular Reactive Oxygen Species (ROS) production (51%) and completely inhibited LDL oxidation and malondialdehyde (MDA) formation. These findings demonstrated the potential of the relevant mix of compounds found in calafate extract on lipoperoxidation and suggest a promising protective effect for reducing the incidence of cardiovascular disease.

Inducible nitric oxide synthase inhibitor, 1400W, mitigates DFP-induced long-term neurotoxicity in the rat model.

Chemical nerve agents (CNA) are increasingly becoming a threat to both civilians and military personnel. CNA-induced acute effects on the nervous system have been known for some time and the long-term consequences are beginning to emerge. In this study, we used diisopropylfluorophosphate (DFP), a seizurogenic CNA to investigate the long-term impact of its acute exposure on the brain and its mitigation by an inducible nitric oxide synthase (iNOS) inhibitor, 1400W as a neuroprotectant in the rat model. Several experimental studies have demonstrated that DFP-induced seizures and/or status epilepticus (SE) causes permanent brain injury, even after the countermeasure medication (atropine, oxime, and diazepam). In the present study, DFP-induced SE caused a significant increase in iNOS and 3-nitrotyrosine (3-NT) at 24 h, 48 h, 7d, and persisted for a long-term (12 weeks post-exposure), which led to the hypothesis that iNOS is a potential therapeutic target in DFP-induced brain injury. To test the hypothesis, we administered 1400W (20 mg/kg, i.m.) or the vehicle twice daily for the first three days of post-exposure. 1400W significantly reduced DFP-induced iNOS and 3-NT upregulation in the hippocampus and piriform cortex, and the serum nitrite levels at 24 h post-exposure. 1400W also prevented DFP-induced mortality in <24 h. The brain immunohistochemistry (IHC) at 7d post-exposure revealed a significant reduction in gliosis and neurodegeneration (NeuN+ FJB positive cells) in the 1400W-treated group. 1400W, in contrast to the vehicle, caused a significant reduction in the epileptiform spiking and spontaneous recurrent seizures (SRS) during 12 weeks of continuous video-EEG study. IHC of brain sections from the same animals revealed a significant reduction in reactive gliosis (both microgliosis and astrogliosis) and neurodegeneration across various brain regions in the 1400W-treated group when compared to the vehicle-treated group. A multiplex assay from hippocampal lysates at 6 weeks post-exposure showed a significant increase in several key pro-inflammatory cytokines/chemokines such as IL-1α, TNFα, IL-1ß, IL-2, IL-6, IL-12, IL-17a, MCP-1, LIX, and Eotaxin, and a growth factor, VEGF in the vehicle-treated animals. 1400W significantly suppressed IL-1α, TNFα, IL-2, IL-12, and MCP-1 levels. It also suppressed DFP-induced serum nitrite levels at 6 weeks post-exposure. In the Morris water maze, the vehicle-treated animals spent significantly less time in the target quadrant in a probe trial at 9d post-exposure compared to their time spent in the same quadrant 11 days previously (i.e., 2 days prior to DFP exposure). Such a difference was not observed in the 1400W and control groups. However, learning and short-term memory were unaffected when tested at 10-16d and 28-34d post-exposure. Accelerated rotarod, horizontal bar test, and the forced swim test revealed no significant changes between groups. Overall, the findings from this study suggest that 1400W may be considered as a potential therapeutic agent as a follow-on therapy for CNA exposure, after controlling the acute symptoms, to prevent mortality and some of the long-term neurotoxicity parameters such as epileptiform spiking, SRS, neurodegeneration, reactive gliosis in some brain regions, and certain key proinflammatory cytokines and chemokine.

Is Eating Raisins Healthy?

Raisins are dried grapes consumed worldwide that contain beneficial components for human health. They are rich in fiber and phytochemicals such as phenolic compounds. Despite a 60% sugar content, several studies have reported health-promoting properties for raisins and this review compiles the intervention studies, as well as the cell line and animal model studies carried out to date. It has been demonstrated that raisins possess a low-to-moderate glycemic index, which makes them a healthy snack. They seem to contribute to a better diet quality and may reduce appetite. Their antioxidant capacity has been correlated to the phenolic content and this may be involved in the improvement of cardiovascular health. In addition, raisins maintain a good oral health due to their antibacterial activity, low adherence to teeth and an optimum oral pH. Raisin consumption also seems to be favorable for colon function, although more studies should be done to conclude this benefit. Moreover, gut microbiota could be affected by the prebiotic content of raisins. Cell line and animal model studies show other potential benefits in specific diseases, such as cancer and Alzheimer's disease. However, deeper research is required and future intervention studies with humans are needed. Overall, incorporating an 80-90 g portion of raisins (half a cup) into the daily diet may be favorable for human health.

Evaluation of a medication intensity screening tool used in malignant hematology and bone marrow transplant services to identify patients at risk for medication-related problems.

Background In 2014, a screening tool was implemented at Medical University of South Carolina (MUSC) Health to identify patients who are at risk for medication-related events. Patients are classified as high-risk if they meet one of the following criteria: receiving anticoagulation therapy, taking more than 10 scheduled medications upon admission, or readmission within the past 30 days. The goal of this study was to determine risk criteria specific to the malignant hematology (MH) and bone marrow transplant (BMT) patients. Methods A retrospective chart review of 114 patients admitted and discharged from the MH/BMT services between 1 September 2015 and 31 October 2015 was performed. A pharmacist-conducted medication history was completed and documented, and all interventions at admission and throughout hospitalization were categorized by severity and by value of service. The primary objective was to evaluate if patients in the MH/BMT services have more medication-related interventions documented upon admission compared with patients who are not screened as high risk. The secondary objectives were to evaluate the different types and severities of interventions made by pharmacists during the entire hospital stay, and to determine if there are certain characteristics that can help identify hematology/oncology high-risk patients. Results More interventions documented upon admission in the high-risk group as a whole when compared with the not high-risk group (73 vs. 31), but when normalized per patients in each group, there was an equal number of interventions (1.0). The most common interventions were to modify regimen (36%) and discontinue therapy (16%). The patient characteristics associated with high-risk included neutropenia, lower average platelet counts on admission, and longer length of stay. Conclusion The screening tool does not further differentiate an already complex MH/BMT patient population. Pharmacists may be more useful at capturing errors or changes during a patient's hospital stay instead of upon admission. Thrombocytopenia, neutropenia, and active infections may correlate with higher-risk status.

Triterpenoid Components from Oak Heartwood (Quercus robur) and Their Potential Health Benefits.

For centuries oak wood (Quercus robur) has been used in aging of wines and spirits, which is based on pleasant flavors given to beverages by phenolics transferred to the liquid during the maturation process. Other metabolites, such as triterpenoids, can also be released. Searching for extractable triterpenoids in oak heartwood, 12 new, 1-12, and five known, 13-17, oleanane types were isolated and characterized. Their cytotoxicities were tested against cancer cells (PC3 and MCF-7) and lymphocytes. Breast cancer cells (MCF-7) were the most affected by triterpenoids, with roburgenic acid, 4, being the most active compound (IC50 = 19.7 µM). Selectivity was observed for compounds 1-3, 8, 9, and 16, exhibiting an IC50 > 200 µM against lymphocytes, while active against cancer cells. A galloyl unit attached to the triterpenoid moiety was established as the key feature for such effect. These results highlight the occurrence of triterpenoids in oak heartwood and their relevance for chemoprevention of breast cancer.

Cytotoxic triterpenoids isolated from sweet chestnut heartwood (Castanea sativa) and their health benefits implication.

For centuries wood containers have been used in aging of wines and spirits, due to the pleasant flavors they give to the beverages. Together with oak, sweet chestnut wood (Castanea sativa) have been often used for such purpose. The maturation process involves the transfer of secondary metabolites, mainly phenolics, from the wood to the liquid. At the same time, other metabolites, such as triterpenoids and their glycosides, can also be released. Searching for the extractable triterpenoids from sweet chestnut heartwood (C. sativa), two new ursane-type triterpenoid saponins named chestnoside A (1) and chestnoside B (2), together with two known oleanen-type analogs (3 and 4) were isolated and characterized. The cytotoxicity of isolated compounds was tested against two cancer cell lines (PC3 and MCF-7), and normal lymphocytes. Breast cancer cells (MCF-7) were more affected by tested compounds than prostate cancer cells (PC3). Chestnoside B (2) exhibited the strongest cytotoxicity with an IC50 of 12.3 µM against MCF-7 cells, lower than those of positive controls, while it was moderately active against normal lymphocytes (IC50 = 67.2 µM). These results highlight the occurrence of triterpenoid saponins in sweet chestnut heartwood and their potential for the chemoprevention of breast cancer.

Fingerprinting of strong spermatogenesis steroidal saponins in male flowers of Phoenix dactylifera (Date Palm) by LC-ESI-MS.

A fingerprint of steroid saponins, the major constituent in 80% methanolic fraction from the male flowers of Phoenix dactylifera has been established. Under ESI-MS/MS conditions, the fragmentation patterns of [M - H]- ions exclusively displayed signals corresponding to the cleavage of the glycosidic bonds, thus allowing a rapid identification of 21 steroidal saponins. Moreover, two unique among them conjugated with histidine were detected by LC-ESI (-)-MS and DFT and were given tentative names of 3-o-histidine-26-o-hexosyl-dioscin and 3-o-histidine-26-o-dihexosyl-hydroxydioscin. Their steroidal saponins exhibited a significant improvement of the sperm cells count, motility and viability in male rats. These effects could be attributed to enhancing the levels of sex hormones.