Tandem Mass Spectrometry Analysis Research Articles

Efficacy of BRAF/MEK-inhibitor therapy for epithelioid glioblastoma with a novel BRAFV600 mutation

Epithelioid glioblastoma (eGB), a very aggressive and rare brain tumour, is associated with a dismal median overall survival. Effective therapies for patients with eGB, particularly with leptomeningeal dissemination, are still lacking. Here, we describe a case of a 25-year-old male diagnosed with an intramedullary cervical tumour with subsequent leptomeningeal disease. Histopathology identified a highly necrotising, epithelioid-type tumour with high cell density, most compatible with the diagnosis of an eGB. DNA analysis revealed an unprecedented B-Raf protooncogene, serine/threonine kinase (BRAF) gene variant in exon 15 (ENST00000288602.6, c.1799_1810delinsATG, p.(V600_W604delinsDG)), triggering activation of the mitogen-activated protein kinase (MAPK) pathway. Consequently, we initiated MAPK inhibitor (MAPKi) therapy, utilizing a combination of BRAF and mitogen-activated protein kinase kinase (MEK) inhibitors. Liquid chromatography–tandem mass spectrometry analysis confirmed the drugs’ presence in the patient’s cerebrospinal fluid, indicating their capacity to cross the blood-brain barrier. Remarkably, the patient responded very well to therapy and transitioned from a near-comatose state to significantly improved health, sustained for over three months. This study highlights that MAPKi, particularly targeted towards novel BRAFV600 mutations, might offer promising advancements in eGB treatment strategies.

Targeted Metabolomic Analysis of Serum Free Fatty Acids: Lipidomics Disturbance in Patients with Superior Limbic Keratoconjunctivitis

This study aimed to identify and quantify free fatty acids (FFAs), secretory phospholipase A2 group IIa (sPLA2-IIa) and cytosolic phospholipase A2 (cPLA2) in serum of superior limbic keratoconjunctivitis (SLK) patients and explored the association between FFAs, sPLA2-IIa and cPLA2 variations and SLK. Targeted metabolomic analysis of FFAs in serum was performed by gas chromatography tandem mass spectrometry (GC-MS/MS) analysis on 16 SLK patients (43.88±7.88 years; female: 62.50%) and 25 healthy controls (43.12±7.88 years; female: 64.00%). Qualitative and absolute quantitative results of FFAs were obtained and classified according to gender and thyroid tests. Differential lipid metabolites, metabolomic pathways and biomarkers were further evaluated. The serum sPLA2-IIa and cPLA2 were determined by enzyme linked immunosorbent assay (ELISA).Among 40 FFAs identified, 6 FFAs showed significant changes (P<0.05) in SLK patients, including 4 decreased and 2 increased. They were mainly related to unsaturated fatty acid biosynthesis, α-linolenic acid and linoleic acid metabolism, and fatty acid biosynthesis. When dividing the data by gender or abnormal thyroid tests, some comparable FFAs alterations displayed in SLK patients. The ROC analysis revealed that the AUC values of linoleic acid, γ-linolenic acid, cis-8,11,14-eicosatrienoic acid, stearic acid, and palmitic acid, were all greater than 0.8. The serum concentrations of sPLA2-IIa and cPLA2 in patients with SLK were significantly higher than that in healthy controls. Lipidomics disturbance might be the potential mechanism of SLK. Serum FFA biomarkers associated with SLK have potential for the diagnosis and treatment of the disease.

Novel Fe2O3/Bi2O3/CeO2 heterojunction photocatalyst for wastewater treatment: Characterization, photocatalytic performance and mechanism study

A novel ternary Fe2O3/Bi2O3/CeO2 (FeB/CeO2) heterojunction nanocomposite with different Fe2O3/Bi2O3 amounts on CeO2 (5, 10, 20, 30 and 40 wt%) was synthesized through a simple wet impregnation process. The properties of the synthesized nanocomposite were validated thorough XPS, XRD, FE-SEM, TEM, and EDS-Mapping analyses. This photocatalyst exhibited exceptional efficiency in the of Tetracycline’s (TC) photodegradation in comparison to individual Fe2O3, Bi2O3, and CeO2. The effect of Fe2O3/Bi2O3 loading on CeO2 and the influence of operational factors on TC decomposition including photocatalyst dosage (0.05–0.2 g/L), TC concentration (10–40 mg/L), and pH (1–11) were examined with the optimal conditions yielding an experimental degradation efficiency of 99.35 %. Moreover, scavenger tests showed that O2− and OH radicals served as the main oxidative radical species. The study further delved into proposing potential degradation pathways of TC in accordance with liquid chromatography tandem-mass spectrometry analysis. Notably, the stability of the synthesized photocatalyst was assessed through five degradation cycles, affirming its sustained effectiveness.

Combining SDS-PAGE to capillary zone electrophoresis-tandem mass spectrometry for high-resolution top-down proteomics analysis of intact histone proteoforms.

Mass spectrometry (MS)-based top-down proteomics (TDP) analysis of histone proteoforms provides critical information about combinatorial post-translational modifications (PTMs), which is vital for pursuing a better understanding of epigenetic regulation of gene expression. It requires high-resolution separations of histone proteoforms before MS and tandem MS (MS/MS) analysis. In this work, for the first time, we combined SDS-PAGE-based protein fractionation (passively eluting proteins from polyacrylamide gels as intact species for mass spectrometry, PEPPI-MS) with capillary zone electrophoresis (CZE)-MS/MS for high-resolution characterization of histone proteoforms. We systematically studied the histone proteoform extraction from SDS-PAGE gel and follow-up cleanup as well as CZE-MS/MS, to determine an optimal procedure. The optimal procedure showed reproducible and high-resolution separation and characterization of histone proteoforms. SDS-PAGE separated histone proteins (H1, H2, H3, and H4) based on their molecular weight and CZE provided additional separations of proteoforms of each histone protein based on their electrophoretic mobility, which was affected by PTMs, for example, acetylation and phosphorylation. Using the technique, we identified over 200 histone proteoforms from a commercial calf thymus histone sample with good reproducibility. The orthogonal and high-resolution separations of SDS-PAGE and CZE made our technique attractive for the delineation of histone proteoforms extracted from complex biological systems.

Xylitol is prothrombotic and associated with cardiovascular risk.

The pathways and metabolites that contribute to residual cardiovascular disease risks are unclear. Low-calorie sweeteners are widely used sugar substitutes in processed foods with presumed health benefits. Many low-calorie sweeteners are sugar alcohols that also are produced endogenously, albeit at levels over 1000-fold lower than observed following consumption as a sugar substitute. Untargeted metabolomics studies were performed on overnight fasting plasma samples in a discovery cohort (n = 1157) of sequential stable subjects undergoing elective diagnostic cardiac evaluations; subsequent stable isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses were performed on an independent, non-overlapping validation cohort (n = 2149). Complementary isolated human platelet, platelet-rich plasma, whole blood, and animal model studies examined the effect of xylitol on platelet responsiveness and thrombus formation in vivo. Finally, an intervention study was performed to assess the effects of xylitol consumption on platelet function in healthy volunteers (n = 10). In initial untargeted metabolomics studies (discovery cohort), circulating levels of a polyol tentatively assigned as xylitol were associated with incident (3-year) major adverse cardiovascular event (MACE) risk. Subsequent stable isotope dilution LC-MS/MS analyses (validation cohort) specific for xylitol (and not its structural isomers) confirmed its association with incident MACE risk [third vs. first tertile adjusted hazard ratio (95% confidence interval), 1.57 (1.12-2.21), P < .01]. Complementary mechanistic studies showed xylitol-enhanced multiple indices of platelet reactivity and in vivo thrombosis formation at levels observed in fasting plasma. In interventional studies, consumption of a xylitol-sweetened drink markedly raised plasma levels and enhanced multiple functional measures of platelet responsiveness in all subjects. Xylitol is associated with incident MACE risk. Moreover, xylitol both enhanced platelet reactivity and thrombosis potential in vivo. Further studies examining the cardiovascular safety of xylitol are warranted.

Comprehensive Identification of the Chemical Substances in Effective Component Groups of Polygalae Radix Based on Targeted Tandem Mass Spectrometry Analysis Strategy

ABSTRACTPolygalae Radix is a commonly used traditional Chinese medicine with multiple pharmacological activities. Modern research have revealed that saponins, xanthones, and oligosaccharide esters are its effective component groups. However, up to date the chemical substances in these effective component groups are still unclear, which has become a fundamental scientific issue needing to be addressed. In our study, an analysis strategy based on targeted tandem mass spectrometry (MS/MS) analysis was proposed to comprehensively identify the chemical substances in effective component groups. First, the extract of the Polygalae Radix was detected by ultra‐performance liquid chromatography coupled with electrospray ionization–orbitrap mass spectrometry in a full MS scan mode to minimize the losses of signals; second, the precursor ions of all compounds were extracted from the obtained full MS scan data using Compound Discoverer software; third, the interested precursor ions were locked based on their molecular features and constructed in a precursor ion list; finally, each precursor ion in the list was subjected to targeted MS/MS analysis for structure characterization. Using our analysis strategy, a total of 565 components were successfully identified.

Characterization of the covalent binding of cyanidin-3-glucoside to bovine serum albumin and its inhibition mechanism for advanced nonenzymatic glycosylation reactions.

Nonenzymatic glycosylation of proteins can generate advanced glycosylation end products, which are closely associated with the pathogenesis of certain chronic physiological diseases and aging. In this study, we characterized the covalent binding of cyanidin-3-glucoside (C3G) to bovine serum albumin (BSA) and investigated the mechanism by which this covalent binding inhibits the nonenzymatic glycosylation of BSA. The results indicated that the covalent interaction between C3G and BSA stabilized the protein's secondary structure. Through liquid chromatography-electrospray ionization tandem mass spectrometry analysis, we identified the covalent binding sites of C3G on BSA as lysine, arginine, asparagine, glutamine, and cysteine residues. This covalent interaction significantly suppressed the nonenzymatic glycosylation of BSA, consequently reducing the formation of nonenzymatic glycosylation products. C3G competitively binds to nonenzymatic glycosylation sites (e.g., lysine and arginine) on BSA, thereby impeding the glycosylation process and preventing the misfolding and structural alterations of BSA induced by fructose. Furthermore, the covalent attachment of C3G to BSA preserves the secondary structure of BSA and hinders subsequent nonenzymatic glycosylation events.

Integrated UHPLC-Q-exactive-orbitrap MS/MS and ABTS analyses screen antioxidant and anti‑counterfeiting marker of Eucommiae Cortex (Duzhong) for Pharmacopoeias

Eucommiae Cortex (Duzhong) has been collected in several Pharmacopoeias. However, its Pharmacopoeia quality-marker is outdated and unreasonable. Therefore, the study used database-aided ultra-high-performance liquid chromatography coupled with hybrid quadrupole-orbitrap tandem mass spectrometry (UHPLC-Q-Exactive-Orbitrap MS/MS) analysis to putatively identify 33 compounds from Eucommiae Cortex. Especially, 14 “unexcavated” compounds were detected in Eucommiae Cortex for the first time; Meanwhile, all monocaffeoylquinic acid isomers (i.e., 1-O-, 3-O-, 4-O-, and 5-O- caffeoylquinic acids) have been strictly distinguished from each other, and three dicaffeoylquinic acid isomers (1,3-O-, 3,5-O-, and 4,5-O- dicaffeoylquinic acids) have been distinguished as well. All identified compounds were further subjected to a semi-quantification analysis, through which cholesteryl acetate (4.11 ± 0.46 %), sucrose (3.53 ± 0.063 %), and (−)-syringaresinol (2.81 ± 1.31 %) were found to be highly abundant. Furthermore, the semi-quantification results were integrated with ABTS+• antioxidant assay to individually calculate the antioxidant contribution of all compounds. (−)-Syringaresinol, neochlorogenic acid, vanillic acid, chlorogenic acid, and (−) pinoresinol were proven to be the top five antioxidant contributors, and their contribution values were 97.50, 46.31, 40.57, 35.05, and 30.90. Of the top contributors, chlorogenic acid was proposed as an additional pharmacopoeia Q-marker for its good testability and traceability. The additional Q-marker not only enhances the pharmacological relevance, but also effectively recognizes Eucommiae Cortex from its counterfeiting via liquid chromatography analysis. Thus, these findings will help Pharmacopoeias to update its Q-markers.

Mycotoxin contamination profiling in coffee bean by targeted LC-HRMS

Abstract Indonesia is the fourth coffee producer in the world which is 95% produced by smallholder plantations with various ways of processing and storage in the form of green beans and roasted beans. This study aims to analyse the presence of mycotoxin contamination of Menoreh coffee in various storage levels using Liquid Chromatography tandem High-Resolution Mass Spectrometry (LC-HRMS) analysis. The analysis of mycotoxin contamination in green beans and roasted beans is crucial to ensure the safety and quality of the coffee for consumption. This study used a random sampling method. The samples tested were Menoreh Robusta coffee bean from two processors in Samigaluh, Kulon Progo, Yogyakarta, Indonesia. The results showed that green beans stored in the dryer house for more than 6 months were contaminated with aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), ochratoxin, and patulin with 24.48; 10.98; 57.18 and 16.92 of area max (106), respectively. Samples of broken green beans stored in contaminated warehouses were contaminated with patulin. However, all roasted bean and roasted bean samples that have been mashed do not contain aflatoxin B1, B2, or patulin. Targeted metabolomic using LC-HRMS profiling is a powerful tool for the rapid detection of mycotoxin in coffee bean. This method could be developed for quantitative analysis to provide accurate concentration.

Determining nine fasciolicides and three metabolite residues in milk and infant formula using solid-phase extraction and liquid chromatography–tandem mass spectrometry

A new sensitive method of liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis for nine fasciolicides (closantel, rafoxanide, oxyclozanide, niclosamide, nitroxinil, ioxynil, 4-nitro-3-(trifluoromethyl)phenol, salicylanilide, and triclabendazole) and three metabolite residues (ketotriclabnedazole, triclabendazole sulfone, and triclabendazole sulfoxide) in milk and infant formula was established. The samples were extracted and purified through solid-phase extraction and analyzed using LC–MS/MS. The proposed method demonstrated high accuracy (the average recoveries ranged from 70.5 % to 107.4 %) and high sensitivity (the limits of quantification ranged from 1.0 to 25.0 µg/kg). This method was successfully applied to determine nine fasciolicides and three metabolite residues in 45 milk and infant formula, providing technical support for the safety and quality evaluation of dairy products.

Mass-Spectrometry-Based Assay at Single-Base Resolution for Simultaneously Detecting m6A and m6Am in RNA.

The methylation modifications of adenosine, especially N6-methyladenosine (m6A) and N6, 2'-odimethyladenosine (m6Am), play vital roles in various biological, physiological, and pathological processes. However, current methods for detecting these modifications at single-base resolution have limitations. Mass spectrometry (MS), a highly accurate and sensitive technique, can be utilized to differentiate between m6A and m6Am by analyzing the molecular weight differences in their fragments during tandem MS analysis. In this study, we present an MS-based method that allows for the simultaneous determination of m6A and m6Am sites in targeted RNA fragments at single-nucleotide resolution. The approach involves the utilization of tandem MS in conjunction with targeted RNA enrichment and enzymatic digestion, eliminating the need for PCR amplification. By employing this strategy, we can accurately identify m6A and m6Am sites in targeted RNA fragments with high confidence. To evaluate the effectiveness of our method, we applied it to detect m6A and m6Am sites in cell and tissue samples. Furthermore, we verified the accuracy of our approach by performing CRISPR/Cas9-mediated knockout of the corresponding methyltransferases. Overall, our MS-based method offers a reliable and precise means for the simultaneous detection of m6A and m6Am modifications in targeted RNA fragments, providing valuable insights into the functional characterization of these modifications in various biological contexts.

Removal of antibiotic residue from aqueous solution by advanced oxidation process

Advanced oxidation processes are low-cost, highly efficient, and eco-friendly technologies in the removal of organic pollutants in wastewater using hydroxyl radicals for oxidation. Hydroxyl radicals possess high oxidation potential and can react with organic compounds, resulting in the complete mineralization of these compounds into carbon dioxide, water, and inorganic salt or their conversion into other compounds. The present investigation deals with the removal of tetracycline from water using simulated ultraviolet radiation and hydrogen peroxide, assessing the effect of operational parameters like the solution's initial pH, retention time, hydrogen peroxide dosage in terms of chemical oxygen demand (COD) removal from the standard aqueous solution of tetracycline. Results indicate that alkaline conditions and larger hydrogen peroxide dosage negatively affect the degradation. The removal efficiency of 68 % was achieved at 150 min of batch reaction under optimum conditions: pH = 4, and a dose of hydrogen peroxide of 0.3 ml per 100 ml of the solution to be treated. At optimum conditions, LC-MS/MS (liquid chromatography tandem mass spectrometry) analysis results showed a reduction in initial concentration of aqueous solution of tetracycline. Photocatalytic degradation followed pseudo-first-order kinetics with the rate constant (k) of 0.0061 min-1. Photocatalysis based on hydrogen peroxide is effective in the degradation of tetracycline in an aqueous solution and can be applied as a pretreatment of hospital wastewater containing tetracycline residues.

Altered landscape of total RNA, tRNA and sncRNA modifications in the liver and spleen of mice infected by Toxoplasma gondii.

Pathogens can impact host RNA modification machinery to establish a favorable cellular environment for their replication. In the present study, we investigated the effect of Toxoplasma gondii infection on host RNA modification profiles and explored how these modifications may influence the host-parasite interaction. We analyzed the modification levels of ∼ 80 nt tRNA and 17-50 nt sncRNAs in mouse liver, spleen, and serum using liquid chromatography and tandem mass spectrometry analysis. The results revealed alterations in RNA modification profiles, particularly during acute infection. The liver exhibited more differentially abundant RNA modifications than the spleen. RNA modification levels in serum were mostly downregulated during acute infection compared to control mice. Correlations were detected between different RNA modifications in the liver and spleen during infection and between several RNA modifications and many cytokines. Alterations in RNA modifications affected tRNA stability and protein translation. These findings provide new insight into the role of RNA modifications in mediating the murine host response to T. gondii infection.

Characterization of HphA: The First Enzyme in the Homologation Pathway of l-Phenylalanine and l-Tyrosine.

Homologation of amino acids is the insertion or deletion of a methylene group to their side chain, which is a relatively uncommon chemical transformation observed in peptide natural product (NP) structure. Homologated amino acids can potentially make the NP more stable in a biological system, but its biosynthesis is yet to be understood. This study biochemically characterized the first of three unexplored enzymes in the homologation pathway of l-phenylalanine and l-tyrosine. Previously proposed reactions catalyzed by HphA were confirmed by reversed-phase high-performance liquid chromatography and tandem mass spectrometry analysis. The substrate profile and kinetic parameters showed high selectivity for the natural substrates and their close analogs. The comparability of HphA to homologous enzymes in primary metabolic pathways, 2-isopropylmate synthase and homocitrate synthase which are involved in l-leucine and l-lysine biosynthesis, respectively, was validated by bioinformatical and site-directed mutagenesis studies. The knowledge obtained from this study has deepened the understanding of the homologation of amino acids, which can lead to future combinatorial biosynthesis and metabolic engineering studies.

Cytotoxicity of Physalis minima Linn (Solanaceae) fruit against HCT116 and HT29 colorectal cancer cell lines

The pantropical Physalis minima are traditionally used for the prevention and treatment of various illnesses, diseases, and cancers. While most earlier studies on the species have focused on the phytochemistry of the leaf and stem extracts, recent studies have indicated that its fruit may contain bioactive compounds of medical interest. In this study, we investigated the cytotoxicity of extracts from the fruit of P. minima against colorectal cancer cell lines and revealed its phytochemical profile via high-resolution tandem mass spectrometry analysis. Following a 24-h treatment with the fruit extract, cytoplasm shrinkage and nucleus condensation were observed in the colorectal cancer cell lines HCT116 and HT29, indicating the induction of programmed cell death. Phytochemically, 71 putative metabolites were identified. Some of these metabolites have been reported to inhibit cancers to varying degrees, further supporting the correlation of the putative metabolites with the cytotoxicity against colorectal cancer cells demonstrated in this study.

Fast and eco-friendly analytical method to determine bisphenols, parabens, benzophenone-3 and triclosan in human urine by ultra-performance liquid chromatography coupled to mass spectrometry

Bisphenols, parabens, benzophenone-3 and triclosan are synthetic chemicals commonly used in various products such as cosmetics, pharmaceuticals and food items, leading to human exposure through ingestion, dermal contact and inhalation. Once entering the body, they are rapidly absorbed, metabolised, and excreted in urine due to their short half-lives. Recognized as potential endocrine-disrupting chemicals (EDCs), they have raised concerns among researchers and regulatory bodies. In response to the concerns regarding environmental harm and risks, innovative analytical strategies are required based on the new criteria focused on the twelve principles of Green Analytical Chemistry. Therefore, a new methodology for the environmentally friendly, fast, efficient and simultaneous determination of nine potential EDCs in human urine, including three bisphenols (A, F and S), four parabens (methyl paraben, ethyl paraben, propyl paraben and butyl paraben), benzophenone-3 and triclosan, has been developed and validated. It is based on a dilute-and-shoot method and ultra-performance liquid chromatography coupled to tandem mass spectrometry analysis. The relative recoveries ranged from 80 % to 120 % with a precision lower than 20 % for all analytes. The concentration ranges obtained encompass the published levels of the compounds in urine and the limits of quantification were between 0.2 and 0.5 ng·mL−1. The proposed method is the most environmentally friendly and practical so far and it was successfully applied for the determination of these compounds in 20 human urine samples from adults residing in the Valencian Region (Spain), and initial findings suggest that methyl paraben, benzophenone-3 and ethyl paraben were the predominant compounds.

POF-based biosensors for cortisol detection in seawater as a tool for aquaculture systems

A surface plasmon resonance (SPR) phenomenon implemented via D-shaped polymer optical fiber (POF) is exploited to realize cortisol biosensors. In this work, two immonosensors are designed and developed for the qualitative as well as quantitative measurement of cortisol in artificial and real samples. The performances of the POF-based biosensors in cortisol recognition are achieved using different functionalization protocols to make the same antibody receptor layer over the SPR surface via cysteamine and lipoic acid, achieving a limit of detection (LOD) of 0.8 pg/mL and 0.2 pg/mL, respectively. More specifically, the use of cysteamine or lipoic acid changes the distance between the receptor layer and the SPR surface, improving the sensitivity at low concentrations of about one order of magnitude in the configuration based on lipoic acid. The LODs of both cortisol biosensors are achieved well competitively with other sensor systems but without the need for amplification or sample treatments. In order to obtain the selectivity tests, cholesterol and testosterone were used as interfering substances. Moreover, tests in simulated seawater were performed for the same cortisol concentration range achieved in buffer solution to assess the immunosensor response to the complex matrix. Finally, the developed cortisol biosensor was used in a real seawater sample to estimate the cortisol concentration value. The gold standard method has confirmed the estimated cortisol concentration value in real seawater samples. Liquid–liquid extraction was implemented to maximize the response of cortisol in liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) analysis.

Assessment of false discovery rate control in tandem mass spectrometry analysis using entrapment.

A pressing statistical challenge in the field of mass spectrometry proteomics is how to assess whether a given software tool provides accurate error control. Each software tool for searching such data uses its own internally implemented methodology for reporting and controlling the error. Many of these software tools are closed source, with incompletely documented methodology, and the strategies for validating the error are inconsistent across tools. In this work, we identify three different methods for validating false discovery rate (FDR) control in use in the field, one of which is invalid, one of which can only provide a lower bound rather than an upper bound, and one of which is valid but under-powered. The result is that the field has a very poor understanding of how well we are doing with respect to FDR control, particularly for the analysis of data-independent acquisition (DIA) data. We therefore propose a new, more powerful method for evaluating FDR control in this setting, and we then employ that method, along with an existing lower bounding technique, to characterize a variety of popular search tools. We find that the search tools for analysis of data-dependent acquisition (DDA) data generally seem to control the FDR at the peptide level, whereas none of the DIA search tools consistently controls the FDR at the peptide level across all the datasets we investigated. Furthermore, this problem becomes much worse when the latter tools are evaluated at the protein level. These results may have significant implications for various downstream analyses, since proper FDR control has the potential to reduce noise in discovery lists and thereby boost statistical power.

Chemical and Biological Characterization of the Ethyl Acetate Fraction from the Red Sea Marine Sponge Hymedesmia sp.

Hymedesmiidae is one of the largest families of marine sponges and stands out as an exceptional source of variable metabolites with diverse biological activities. In this study, the ethyl acetate fraction (HE) of a Hymedesmia sp. marine sponge from the Red Sea, Egypt, was analyzed for the first time using Ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) analysis. The analysis tentatively identified 29 compounds in this fraction, including the isolation and identification of six compounds (two pyrimidine nucleosides, one purine, and two pyrimidine bases in addition to one cerebroside) for the first time. The structures of the isolated compounds were established by 1D and 2D NMR (nuclear magnetic resonance), MS (mass spectrometry), and IR (infrared) spectroscopy. Furthermore, the cytotoxic, antioxidant, and antimicrobial activities of the ethyl acetate fraction were evaluated in vitro. The fraction exhibited strong DPPH scavenging activity with an IC50 of 78.7 µg/mL, compared to ascorbic acid as a positive control with an IC50 of 10.6 µg/mL. It also demonstrated significant cytotoxic activity with IC50 values of 13.5 µg/mL and 25.3 µg/mL against HCT-116 and HEP-2 cell lines, respectively, compared to vinblastine as a positive control with IC50 values of 2.34 µg/mL and 6.61 µg/mL against HCT-116 and HEP-2, respectively. Additionally, the ethyl acetate fraction displayed promising antibacterial activity against S. aureus with a MIC value of 62.5 µg/mL, compared to ciprofloxacin as a positive control with MIC values of 1.56 µg/mL for Gram-positive bacteria and 3.125 µg/mL for Gram-negative bacteria. It also exhibited activity against E. coli and P. aeruginosa with MIC values of 250 µg/mL and 500 µg/mL, respectively. Briefly, this is the first report on the biological activities and secondary metabolite content of the ethyl acetate fraction of Hymedesmia sp. marine sponge, emphasizing the potential for further research against resistant bacterial and fungal strains, as well as different cancer cell lines. The ethyl acetate fraction of Hymedesmia sp. is a promising source of safe and unique natural drugs with potential therapeutic and pharmaceutical benefits.

PRM1201 effectively inhibits colorectal cancer metastasis via shaping gut microbiota and short- chain fatty acids

BackgroundPRM1201 is a traditional medicine with beneficial effects against colorectal cancer (CRC) metastasis. However, the underlying mechanism of this action remains to be determined. HypothesisRemodeling microbiota and short-chain fatty acids (SCFAs) metabolism might be a potential mechanism to explain the anti-metastatic action of PRM1201, as this gut-microbiota dependent effect involves downregulation of histone deacetylation and EMT. MethodsTo investigate this possibility, clinical specimens were sequenced and the correlation between the anti-metastatic efficacy of PRM1201 and the restoration of SCFA-producing bacteria was studied. To obtain solid causal evidence, a mouse metastasis model was established to detect the influence of PRM1201 on cancer metastasis. Specifically, 16S amplicon sequencing, ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) analysis, and bacterial manipulation were used to examine the gut microbiota-driven anti-metastatic action of PRM1201. ResultsClinical data showed that PRM1201 increased both the number of SCFA-producing bacteria and generation of SCFAs in the feces of CRC patients. A positive correlation between the anti-metastatic efficacy of PRM1201 and the restoration of SCFAs observed. The animal experiments demonstrated that PRM1201 effectively blocked CRC metastasis in a dose-dependent manner. PRM1201 treatment modulated the composition of gut microbiota, and promoted the proliferation of beneficial SCFAs producers such as Akkermansia, Lachnospiraceae_NK4A136_group and Blautia, while simultaneously reducing the abundance of pathogenic bacteria like Escherichia-Shigella. In addition, PRM1201 led to augmentation of SCFAs content. Further results indicated that the anti-cancer metastatic mechanism of PRM1201 was linked to inhibition of histone deacetylation and suppression of epithelial-to-mesenchymal transition (EMT) in metastatic lesions. Microbiota depletion treatment and fecal microbiota transplantation (FMT) underscored the microbiota-dependent nature of this phenomenon. Moreover, this anti-colorectal cancer metastatic effect and mechanism of total SCFAs and single SCFA were also confirmed. ConclusionIn summary, PRM1201 exerts its anti-metastatic effects by modulating SCFA-producing bacteria and enhancing the production of SCFAs. Furthermore, the prebiotic-like actions of PRM1201, along with the PRM1201-treated bacteria, function as inhibitors of histone deacetylases (DHACs) thereby effectively suppressing EMT events.