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Ontiveros-Rodríguez, Julio C., José I. Serrano-Contreras, José Roberto Villagómez-Ibarra, Hugo A. García-Gutiérrez e L. Gerardo Zepeda-Vallejo. "A semi-targeted NMR-based chemical profiling of retail samples of Mexican gordolobo". Journal of Pharmaceutical and Biomedical Analysis 212 (abril de 2022): 114651. http://dx.doi.org/10.1016/j.jpba.2022.114651.
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Protti, Michele, Marco Cirrincione, Sarah Palano, Eleonora Poeta, Giorgia Babini, Maria Chiara Magnifico, Simona Nicole Barile et al. "Targeted quantitative metabolic profiling of brain-derived cell cultures by semi-automated MEPS and LC-MS/MS". Journal of Pharmaceutical and Biomedical Analysis 236 (novembro de 2023): 115757. http://dx.doi.org/10.1016/j.jpba.2023.115757.
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Chatterjee, Niladri S., Akanksha Singh, K. V. Vishnu, K. K. Ajeeshkumar, R. Anandan, K. Ashok Kumar e Suseela Mathew. "Authentication of Two Bio-Active Fish Oils by Qualitative Lipid Profiling Using Semi-Targeted Approach: An Exploratory Study". Journal of AOAC INTERNATIONAL 103, n.º 1 (1 de janeiro de 2020): 78–82. http://dx.doi.org/10.5740/jaoacint.19-0208.
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Abstract Background: Fish oils, which are rich in health-promoting polyunsaturated fatty acids (PUFA), have emerged as promising functional foods in the global health and wellness food market. Their source regarding the fish type, season, and location of harvesting might influence the nutritional value of such bioactive oils and determine their market price. The differences in price among such oils often lead to economically motivated mislabeling and adulteration. Objective: In this study, our objective was to demonstrate how a qualitative targeted shotgun lipid profile workflow using an electrospray ionization–quadrupole-linear ion trap MS (QTrap) could differentiate fish oils originating from two different species. Methods: Five samples each of sardine (Sardinella longiceps) oil and shark (Echinorhinus brucus) liver oil were diluted to a concentration of 80 µg/mL in chloroform–methanol (1 + 2, v/v) with 5 mM ammonium acetate. These samples were directly infused into a QTrap MS. The data were acquired for 23 precursor ion and 4 neutral loss scan experiments in the positive ionization mode and compared. Results: We identified the following major lipid classes: cholesteryl ester, diacyl glycerol, triacylglycerol, monoalkyldiacylglycerol, and phophatydyl choline. The relative peak areas of the identified lipid species, when subjected to supervised multivariate analysis, could effectively distinguish the sardine oil and shark liver oil. Conclusions: The approach will be useful in establishing authenticity of fish oil and to support the regulatory agencies in dispute resolution. It can also be extended to establish authenticity in other agricultural and food commodities. Highlights: This paper reports a proof of concept for authenticating PUFA-rich fish supplements. A shotgun targeted lipidomics profile and chemometrics modeling successfully discriminated sardine oil and shark liver oil.
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Ciubotaru, Ramona Mihaela, Mar Garcia-Aloy, Domenico Masuero, Pietro Franceschi, Luca Zulini, Marco Stefanini, Michael Oberhuber, Peter Robatscher, Giulia Chitarrini e Urska Vrhovsek. "Semi-Targeted Profiling of the Lipidome Changes Induced by Erysiphe Necator in Disease-Resistant and Vitis vinifera L. Varieties". International Journal of Molecular Sciences 24, n.º 4 (17 de fevereiro de 2023): 4072. http://dx.doi.org/10.3390/ijms24044072.
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The ascomycete Erysiphe necator is a serious pathogen in viticulture. Despite the fact that some grapevine genotypes exhibit mono-locus or pyramided resistance to this fungus, the lipidomics basis of these genotypes’ defense mechanisms remains unknown. Lipid molecules have critical functions in plant defenses, acting as structural barriers in the cell wall that limit pathogen access or as signaling molecules after stress responses that may regulate innate plant immunity. To unravel and better understand their involvement in plant defense, we used a novel approach of ultra-high performance liquid chromatography (UHPLC)-MS/MS to study how E. necator infection changes the lipid profile of genotypes with different sources of resistance, including BC4 (Run1), “Kishmish vatkhana” (Ren1), F26P92 (Ren3; Ren9), and “Teroldego” (a susceptible genotype), at 0, 24, and 48 hpi. The lipidome alterations were most visible at 24 hpi for BC4 and F26P92, and at 48 hpi for “Kishmish vatkhana”. Among the most abundant lipids in grapevine leaves were the extra-plastidial lipids: glycerophosphocholine (PCs), glycerophosphoethanolamine (PEs) and the signaling lipids: glycerophosphates (Pas) and glycerophosphoinositols (PIs), followed by the plastid lipids: glycerophosphoglycerols (PGs), monogalactosyldiacylglycerols (MGDGs), and digalactosyldiacylglycerols (DGDGs) and, in lower amounts lyso-glycerophosphocholines (LPCs), lyso-glycerophosphoglycerols (LPGs), lyso-glycerophosphoinositols (LPIs), and lyso-glycerophosphoethanolamine (LPEs). Furthermore, the three resistant genotypes had the most prevalent down-accumulated lipid classes, while the susceptible genotype had the most prevalent up-accumulated lipid classes.
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Mireault, Myriam, Vivaldy Prinville, Leanne Ohlund e Lekha Sleno. "Semi-Targeted Profiling of Bile Acids by High-Resolution Mass Spectrometry in a Rat Model of Drug-Induced Liver Injury". International Journal of Molecular Sciences 24, n.º 3 (27 de janeiro de 2023): 2489. http://dx.doi.org/10.3390/ijms24032489.
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Using a semi-targeted approach, we have investigated the effect of acetaminophen on circulating bile acid profiles in rats, including many known bile acids and potential isomeric structures, as well as glucuronide and sulfate conjugates. The chromatographic separation was based on an optimized reverse-phase method exhibiting excellent resolution for a complex mix of bile acids using a solid-core C18 column, coupled to a high-resolution quadrupole time-of-flight system. The semi-targeted workflow consisted of first assigning all peaks detectable in samples from 46 known bile acids contained in a standard mix, as well as additional peaks for other bile acid isomers. The presence of glucuronide and sulfate conjugates was also examined based on their elemental formulae and detectable peaks with matching exact masses were added to the list of features for statistical analysis. In this study, rats were administered acetaminophen at four different doses, from 75 to 600 mg/kg, with the highest dose being a good model of drug-induced liver injury. Statistically significant changes were found by comparing bile acid profiles between dosing levels. Some tentatively assigned conjugates were further elucidated using in vitro metabolism incubations with rat liver fractions and standard bile acids. Overall, 13 identified bile acids, 23 tentatively assigned bile acid isomers, and 9 sulfate conjugates were found to increase significantly at the highest acetaminophen dose, and thus could be linked to drug-induced liver injury.
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Viejo-Boyano, Iris, Marta Isabel Roca-Marugán, María Peris-Fernández, Julián Luis Amengual, Ángel Balaguer-Timor, Marta Moreno-Espinosa, María Felipe-Barrera et al. "Early Metabolomic Profiling as a Predictor of Renal Function Six Months After Kidney Transplantation". Biomedicines 12, n.º 11 (22 de outubro de 2024): 2424. http://dx.doi.org/10.3390/biomedicines12112424.
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Background: Kidney transplantation is the therapy of choice for patients with advanced chronic kidney disease; however, predicting graft outcomes remains a significant challenge. Early identification of reliable biomarkers could enhance post-transplant management and improve long-term outcomes. This study aimed to identify metabolomic biomarkers within the first week after kidney transplantation that predict renal function at six months. Methods: We conducted a prospective study involving 50 adult patients who received deceased donor kidney transplants. Plasma samples collected one week after transplant were analyzed using liquid chromatography–mass spectrometry in a semi-targeted metabolomic approach. A Partial Least Squares-Discriminant Analysis (PLS-DA) model identified metabolites associated with serum creatinine > 1.5 mg/dL at six months. Metabolites were selected based on a Variable Importance in Projection (VIP) score > 1.5, which was used to optimize model performance. Results: The PLS-DA model demonstrated strong predictive performance with an area under the curve (AUC) of 0.958. The metabolites negatively associated with serum creatinine > 1.5 mg/dL were 3-methylindole, guaiacol, histidine, 3-indolepropionic acid, and α-lipoic acid. Conversely, the metabolites positively associated with worse kidney graft outcomes included homocarnosine, 5-methylcytosine, xanthosine, choline, phenylalanine, kynurenic acid, and L-kynurenine. Conclusions: Early metabolomic profiling after transplantation shows promise in predicting renal function. Identifying metabolites with antioxidant and anti-inflammatory properties, as well as those that are harmful and could be targeted therapeutically, underscores their potential clinical significance. The link between several metabolites and the tryptophan pathway suggests that further specific evaluation of this pathway is warranted. These biomarkers can enhance patient management and graft survival.
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Karnachuk, Olga V., Inna A. Panova, Vasilii L. Panov, Olga P. Ikkert, Vitaly V. Kadnikov, Igor I. Rusanov, Marat R. Avakyan et al. "Active Sulfate-Reducing Bacterial Community in the Camel Gut". Microorganisms 11, n.º 2 (4 de fevereiro de 2023): 401. http://dx.doi.org/10.3390/microorganisms11020401.
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The diversity and activity of sulfate-reducing bacteria (SRB) in the camel gut remains largely unexplored. An abundant SRB community has been previously revealed in the feces of Bactrian camels (Camelus bactrianus). This study aims to combine the 16S rRNA gene profiling, sulfate reduction rate (SRR) measurement with a radioactive tracer, and targeted cultivation to shed light on SRB activity in the camel gut. Fresh feces of 55 domestic Bactrian camels grazing freely on semi-arid mountain pastures in the Kosh-Agach district of the Russian Altai area were analyzed. Feces were sampled in early winter at an ambient temperature of −15 °C, which prevented possible contamination. SRR values measured with a radioactive tracer in feces were relatively high and ranged from 0.018 to 0.168 nmol S cm−3 day−1. The 16S rRNA gene profiles revealed the presence of Gram-negative Desulfovibrionaceae and spore-forming Desulfotomaculaceae. Targeted isolation allowed us to obtain four pure culture isolates belonging to Desulfovibrio and Desulforamulus. An active SRB community may affect the iron and copper availability in the camel intestine due to metal ions precipitation in the form of sparingly soluble sulfides. The copper-iron sulfide, chalcopyrite (CuFeS2), was detected by X-ray diffraction in 36 out of 55 analyzed camel feces. In semi-arid areas, gypsum, like other evaporite sulfates, can be used as a solid-phase electron acceptor for sulfate reduction in the camel gastrointestinal tract.
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Funke, Sebastian, Carsten Schmelter, Sascha D. Markowitsch, Natarajan Perumal, Janis C. Heyne, Katharina Bell, Norbert Pfeiffer e Franz H. Grus. "Comparative Quantitative Analysis of Porcine Optic Nerve Head and Retina Subproteomes". International Journal of Molecular Sciences 20, n.º 17 (29 de agosto de 2019): 4229. http://dx.doi.org/10.3390/ijms20174229.
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Optic nerve head (ONH) and retina (RET) are the main sites of damage in neurodegenerative optic neuropathies including glaucoma. Up to date, little is known about the molecular interplay between these two adjoining ocular components in terms of proteomics. To close this gap, we investigated ONH and RET protein extracts derived from porcine eyes (n = 12) (Sus scrofa domestica Linnaeus 1758) using semi-quantitative mass spectrometry (MS)-based proteomics comprising bottom-up LC–ESI MS/MS and targeted SPE-MALDI-TOF MS analysis. In summary, more than 1600 proteins could be identified from the ONH/RET tissue complex. Moreover, ONH and RET displayed tissue-specific characteristics regarding their qualitative and semi-quantitative protein compositions. Gene ontology (GO)-based functional and protein–protein interaction analyses supported a close functional connection between the metabolic-related RET and the structural-associated ONH subproteomes, which could be affected under disease conditions. Inferred from the MS findings, stress-associated proteins including clusterin, ceruloplasmin, and endoplasmin can be proposed as extracellular mediators of the ONH/ RET proteome interface. In conclusion, ONH and RET show obvious proteomic differences reflecting characteristic functional features which have to be considered for future protein biomarker profiling studies.
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Altomare, Alessandra, Giovanna Baron, Marta Balbinot, Alessandro Pedretti, Beatrice Zoanni, Maura Brioschi, Piergiuseppe Agostoni, Marina Carini, Cristina Banfi e Giancarlo Aldini. "In-Depth AGE and ALE Profiling of Human Albumin in Heart Failure: Ex Vivo Studies". Antioxidants 10, n.º 3 (27 de fevereiro de 2021): 358. http://dx.doi.org/10.3390/antiox10030358.
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Advanced glycation end-products (AGEs) and advanced lipoxidation end-products (ALEs), particularly carboxymethyl-lysine (CML), have been largely proposed as factors involved in the establishment and progression of heart failure (HF). Despite this evidence, the current literature lacks the comprehensive identification and characterization of the plasma AGEs/ALEs involved in HF (untargeted approach). This work provides the first ex vivo high-resolution mass spectrometry (HR-MS) profiling of AGEs/ALEs occurring in human serum albumin (HSA), the most abundant protein in plasma, characterized by several nucleophilic sites and thus representing the main protein substrate for AGE/ALE formation. A set of AGE/ALE adducts in pooled HF-HSA samples was defined, and a semi-quantitative analysis was carried out in order to finally select those presenting in increased amounts in the HF samples with respect to the control condition. These adducts were statistically confirmed by monitoring their content in individual HF samples by applying a targeted approach. Selected AGEs/ALEs proved to be mostly CML derivatives on Lys residues (i.e., CML-Lys12, CML-Lys378, CML-Lys402), and one deoxy-fructosyl derivative on the Lys 389 (DFK-Lys 389). The nature of CML adducts was finally confirmed using immunological methods and in vitro production of such adducts further confirmed by mass spectrometry.
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Derveaux, Elien, Michiel Thomeer, Liesbet Mesotten, Gunter Reekmans e Peter Adriaensens. "Detection of Lung Cancer via Blood Plasma and 1H-NMR Metabolomics: Validation by a Semi-Targeted and Quantitative Approach Using a Protein-Binding Competitor". Metabolites 11, n.º 8 (12 de agosto de 2021): 537. http://dx.doi.org/10.3390/metabo11080537.
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Metabolite profiling of blood plasma, by proton nuclear magnetic resonance (1H-NMR) spectroscopy, offers great potential for early cancer diagnosis and unraveling disruptions in cancer metabolism. Despite the essential attempts to standardize pre-analytical and external conditions, such as pH or temperature, the donor-intrinsic plasma protein concentration is highly overlooked. However, this is of utmost importance, since several metabolites bind to these proteins, resulting in an underestimation of signal intensities. This paper describes a novel 1H-NMR approach to avoid metabolite binding by adding 4 mM trimethylsilyl-2,2,3,3-tetradeuteropropionic acid (TSP) as a strong binding competitor. In addition, it is demonstrated, for the first time, that maleic acid is a reliable internal standard to quantify the human plasma metabolites without the need for protein precipitation. Metabolite spiking is further used to identify the peaks of 62 plasma metabolites and to divide the 1H-NMR spectrum into 237 well-defined integration regions, representing these 62 metabolites. A supervised multivariate classification model, trained using the intensities of these integration regions (areas under the peaks), was able to differentiate between lung cancer patients and healthy controls in a large patient cohort (n = 160), with a specificity, sensitivity, and area under the curve of 93%, 85%, and 0.95, respectively. The robustness of the classification model is shown by validation in an independent patient cohort (n = 72).
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Da Costa, Maria Vera Jesus, Venkategowda Ramegowda, Sheshshayee Sreeman e Karaba N. Nataraja. "Targeted Phytohormone Profiling Identifies Potential Regulators of Spikelet Sterility in Rice under Combined Drought and Heat Stress". International Journal of Molecular Sciences 22, n.º 21 (28 de outubro de 2021): 11690. http://dx.doi.org/10.3390/ijms222111690.
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Rice cultivated under rainfed or semi-irrigated ecosystems is frequently exposed to a combination of drought and heat stress. As a sensitive crop at the reproductive stage, exposure to combined drought and heat stress will have a deleterious effect on yield. In this study, two rice cultivars with contrasting spikelet sterility, AVT2-5315 (low sterility) and AC35027 (high sterility), under combined stress were selected for physiological characterization and phytohormonal profiling at anthesis. Under combined stress, both cultivars did not differ in the physiological parameters such as relative water content, photosynthetic rate, light-adapted chlorophyll fluorescence and biomass, suggesting a similar source activity under stress. However, AVT2-5315 showed better yield due to better pollen and spikelet fertility than AC35027, suggesting its intrinsic tolerance ability under combined stress. Targeted profiling of 15 phytohormones from drought, heat and combined stress-treated flag leaf and spikelet tissues using LC–MS/MS showed increased accumulation of auxins (indole 3-acetic acid and indole 3-butyric acid) in flag leaves and jasmonic acid in spikelets of AVT2-5315, while there was increased accumulation of ethylene in flag leaves and methyl-jasmonate in spikelets of AC35027. Increased accumulation of these hormones correlated with key biosynthetic pathway genes. In the flag leaves, increased accumulation of auxins was correlated with increased transcript levels of YUCCA-like gene 1 (OsYUCCA1) and fish bone (OsFIB), in AVT2-5315 under combined stress. In AC35027, increased ethylene content was correlated with expression of 1-aminocyclopropane-1-carboxylate synthase 1 (OsASC1) and aminocyclopropane-1-carboxylic acid oxidase 2 (OsACO2). Similarly, in spikelets, increased accumulation of jasmonic acid in AVT2-5315 was correlated with expression of allene oxide cyclase (OsAOC) and 12-oxophytodienoic acid reductase 1 (OsOPR1). The mechanism of regulating spikelet sterility by these hormones needs further investigation towards improving rice tolerance to combined stress.
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Karimi, Ali, Torsten Meiners e Christoph Böttcher. "Metabolite Profiling and Bioassay-Guided Fractionation of Zataria multiflora Boiss. Hydroethanolic Leaf Extracts for Identification of Broad-Spectrum Pre and Postharvest Antifungal Agents". Molecules 27, n.º 24 (14 de dezembro de 2022): 8903. http://dx.doi.org/10.3390/molecules27248903.
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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.
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Farsijani, Samaneh, Megan M. Marron, Iva Miljkovic, Mary E. Baugh, Stephen Kritchevsky e Anne B. Newman. "METABOLOMICS PROFILING OF MUSCLE FAT DEPOSITION IN AGING: RESULTS FROM THE HEALTH ABC STUDY". Innovation in Aging 3, Supplement_1 (novembro de 2019): S952. http://dx.doi.org/10.1093/geroni/igz038.3457.
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Abstract Background: Age-related inter-muscular fat (IMF) deposition is associated with poor physical function in those with preserved/high muscle mass. However, the heterogeneity of IMF in aging is poorly understood. We used a semi-targeted metabolomics approach to: 1) determine the metabolites associated with IMF infiltration in aging; and 2) establish a model to predict IMF using the metabolome. Methods: We performed a cross-sectional analysis of 314 African-American men (age: 69-79 years) from the Health ABC study at baseline. Mid-thigh IMF area (cm2, by CT) and 350 plasma metabolites (by liquid-chromatography/mass spectrometry) were measured. Correlation analysis was performed to determine metabolites associated with IMF. An IMF prediction model was calculated, using regression analysis with 10-fold cross-validations on random halves of the population with metabolites, age and weight as predictors. Results: Of 161 metabolites correlated with IMF (P<0.05), 34 remained significant after adjusting for age, weight, physical activity, medications, smoking and multiple comparisons (false discovery rate ≤0.25). IMF-associated metabolites were primarily lipids (76%) and amino acids (15%). Most metabolites were positively correlated with IMF (94%), with the exception of mevalonic acid (from fatty acids sub-class) and glutamine (from organic-acids) which were negatively associated with IMF. IMF-associated metabolites predicted 49% of the variability in the actual IMF in the test set of the random half of the population (50%, n= 144). Conclusion: Identification of the unique metabolomics features associated with IMF may improve our understanding of key biological alterations of muscle during aging.
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Yang, Zhaocong, Yanfeng Zhang, Tingting Tang, Qinhua Zhu, Wanyue Shi, Xin Yin, Yun Xing, Yumeng Shen, Yi Pan e Liang Jin. "Transcriptome Profiling of Panc-1 Spheroid Cells with Pancreatic Cancer Stem Cells Properties Cultured by a Novel 3D Semi-Solid System". Cellular Physiology and Biochemistry 47, n.º 5 (2018): 2109–25. http://dx.doi.org/10.1159/000491479.
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Background/Aims: Pancreatic cancer remains one of the deadliest human malignancies, the lethality of which may be attributed to the presence of pancreatic cancer stem cells (PCSCs), a small subpopulation of cells existing within pancreatic tumor with high carcinogenesis. Therefore, it is crucial to establish an efficient enrichment and culture system of PCSCs and identify the key genes involved in the regulation of PCSCs. The three-dimensional (3D) liquid suspension mammosphere culture system has been established for enrichment and culture of PCSCs in vitro as the cell spheres are likely to originate from individual cell clone, but it has been challenged because the cell spheroids could be a result of cell aggregation. Methods: We optimized the existing culture system by adding methylcellulose to create a 3D semi-solid system which prevented the non-specific aggregation. Then we identified the CSC properties of Panc-1 spheroid cells cultured by this system by detecting the genes associated with stemness and by evaluation of the tumorigenicity in vitro and in vivo through invasion, migration and xenograft experiments methods. Subsequently, we performed high-throughput sequencing (HTS) of the Panc-1 spheroid cells. Results: We confirmed the PCSCs properties and high malignancy of the Panc-1 spheroid cells enriched by our novel 3D semi-solid system both in vitro and in vivo. Hundreds of mRNA, microRNA (miRNA) and dozens of long non-coding RNA (LncRNA) were identified to be differentially regulated in PCSCs-like Panc-1 spheroid cells compared with their parental cells by HTS. Conclusions: Our results demonstrate an efficient enrichment and culture system for Panc-1 spheroid cells with the PCSCs properties. The differentially expressed genes and their targets identified by the HTS of the Panc-1 spheroid cells can serve as new potential biomarkers for pancreatic cancer diagnosis and targeted therapy.
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Ziegler, Jörg, Stephan Schmidt, Ranju Chutia, Jens Müller, Christoph Böttcher, Nadine Strehmel, Dierk Scheel e Steffen Abel. "Non-targeted profiling of semi-polar metabolites in Arabidopsis root exudates uncovers a role for coumarin secretion and lignification during the local response to phosphate limitation". Journal of Experimental Botany 67, n.º 5 (17 de dezembro de 2015): 1421–32. http://dx.doi.org/10.1093/jxb/erv539.
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Fontana, Ariel, Isaac Rodríguez e Rafael Cela. "Dispersive liquid–liquid microextraction and gas chromatography accurate mass spectrometry for extraction and non-targeted profiling of volatile and semi-volatile compounds in grape marc distillates". Journal of Chromatography A 1546 (abril de 2018): 36–45. http://dx.doi.org/10.1016/j.chroma.2018.03.003.
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Silva, Priscila O., Diego S. Batista, João Henrique F. Cavalcanti, Andréa D. Koehler, Lorena M. Vieira, Amanda M. Fernandes, Carlos Hernan Barrera-Rojas, Dimas M. Ribeiro, Fabio T. S. Nogueira e Wagner C. Otoni. "Leaf heteroblasty in Passiflora edulis as revealed by metabolic profiling and expression analyses of the microRNAs miR156 and miR172". Annals of Botany 123, n.º 7 (12 de março de 2019): 1191–203. http://dx.doi.org/10.1093/aob/mcz025.
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Abstract Background and Aims Juvenile-to-adult phase transition is marked by changes in leaf morphology, mostly due to the temporal development of the shoot apical meristem, a phenomenon known as heteroblasty. Sugars and microRNA-controlled modules are components of the heteroblastic process in Arabidopsis thaliana leaves. However, our understanding about their roles during phase-changing in other species, such as Passiflora edulis, remains limited. Unlike Arabidopsis, P. edulis (a semi-woody perennial climbing vine) undergoes remarkable changes in leaf morphology throughout juvenile-to-adult transition. Nonetheless, the underlying molecular mechanisms are unknown. Methods Here we evaluated the molecular mechanisms underlying the heteroblastic process by analysing the temporal expression of microRNAs and targets in leaves as well as the leaf metabolome during P. edulis development. Key Results Metabolic profiling revealed a unique composition of metabolites associated with leaf heteroblasty. Increasing levels of glucose and α-trehalose were observed during juvenile-to-adult phase transition. Accumulation of microRNA156 (miR156) correlated with juvenile leaf traits, whilst miR172 transcript accumulation was associated with leaf adult traits. Importantly, glucose may mediate adult leaf characteristics during de novo shoot organogenesis by modulating miR156-targeted PeSPL9 expression levels at early stages of shoot development. Conclusions Altogether, our results suggest that specific sugars may act as co-regulators, along with two microRNAs, leading to leaf morphological modifications throughout juvenile-to-adult phase transition in P. edulis.
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Yang, Dong-Sik, Zhentian Lei, Mohamed Bedair e Lloyd W. Sumner. "An Optimized SPME-GC-MS Method for Volatile Metabolite Profiling of Different Alfalfa (Medicago sativa L.) Tissues". Molecules 26, n.º 21 (27 de outubro de 2021): 6473. http://dx.doi.org/10.3390/molecules26216473.
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Solid-phase microextraction (SPME) was coupled to gas chromatography mass spectrometry (GC-MS) and a method optimized to quantitatively and qualitatively measure a large array of volatile metabolites in alfalfa glandular trichomes isolated from stems, trichome-free stems, and leaves as part of a non-targeted metabolomics approach. Major SPME extraction parameters optimized included SPME fiber composition, extraction temperature, and extraction time. The optimized SPME method provided the most chemically diverse coverage of alfalfa volatile and semi-volatile metabolites using a DVB/CAR/PDMS fiber, extraction temperature of 60 °C, and an extraction time of 20 min. Alfalfa SPME-GC-MS profiles were processed using automated peak deconvolution and identification (AMDIS) and quantitative data extraction software (MET-IDEA). A total of 87 trichome, 59 stem, and 99 leaf volatile metabolites were detected after background subtraction which removed contaminants present in ambient air and associated with the fibers and NaOH/EDTA buffer solution containing CaCl2. Thirty-seven volatile metabolites were detected in all samples, while 15 volatile metabolites were uniquely detected only in glandular trichomes, 9 only in stems, and 33 specifically in leaves as tissue specific volatile metabolites. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) of glandular trichomes, stems, and leaves showed that the volatile metabolic profiles obtained from the optimized SPME-GC-MS method clearly differentiated the three tissues (glandular trichomes, stems, and leaves), and the biochemical basis for this differentiation is discussed. Although optimized using plant tissues, the method can be applied to other types of samples including fruits and other foods.
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Akyol, Sumeyya, Zafer Ugur, Ali Yilmaz, Ilyas Ustun, Santosh Kapil Kumar Gorti, Kyungjoon Oh, Bernadette McGuinness et al. "Lipid Profiling of Alzheimer’s Disease Brain Highlights Enrichment in Glycerol(phospho)lipid, and Sphingolipid Metabolism". Cells 10, n.º 10 (29 de setembro de 2021): 2591. http://dx.doi.org/10.3390/cells10102591.
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Alzheimer’s disease (AD) is reported to be closely linked with abnormal lipid metabolism. To gain a more comprehensive understanding of what causes AD and its subsequent development, we profiled the lipidome of postmortem (PM) human brains (neocortex) of people with a range of AD pathology (Braak 0–6). Using high-resolution mass spectrometry, we employed a semi-targeted, fully quantitative lipidomics profiling method (Lipidyzer) to compare the biochemical profiles of brain tissues from persons with mild AD (n = 15) and severe AD (AD; n = 16), and compared them with age-matched, cognitively normal controls (n = 16). Univariate analysis revealed that the concentrations of 420 lipid metabolites significantly (p < 0.05; q < 0.05) differed between AD and controls. A total of 49 lipid metabolites differed between mild AD and controls, and 439 differed between severe AD and mild AD. Interestingly, 13 different subclasses of lipids were significantly perturbed, including neutral lipids, glycerolipids, glycerophospholipids, and sphingolipids. Diacylglycerol (DAG) (14:0/14:0), triacylglycerol (TAG) (58:10/FA20:5), and TAG (48:4/FA18:3) were the most notably altered lipids when AD and control brains were compared (p < 0.05). When we compare mild AD and control brains, phosphatidylethanolamine (PE) (p-18:0/18:1), phosphatidylserine (PS) (18:1/18:2), and PS (14:0/22:6) differed the most (p < 0.05). PE (p-18:0/18:1), DAG (14:0/14:0), and PS (18:1/20:4) were identified as the most significantly perturbed lipids when AD and mild AD brains were compared (p < 0.05). Our analysis provides the most extensive lipid profiling yet undertaken in AD brain tissue and reveals the cumulative perturbation of several lipid pathways with progressive disease pathology. Lipidomics has considerable potential for studying AD etiology and identifying early diagnostic biomarkers.
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Nguyen, Ha, e Wendy V. Wismer. "Temporal Sensory Profiles of Regular and Sodium-Reduced Foods Elicited by Temporal Dominance of Sensations (TDS) and Temporal Check-All-That-Apply (TCATA)". Foods 11, n.º 3 (3 de fevereiro de 2022): 457. http://dx.doi.org/10.3390/foods11030457.
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Temporal sensory methods can be used to highlight the impact of sodium reduction on the dynamic sensory profile of foods targeted for sodium reduction. Study aims were to compare the temporal sensory attribute profiles of regular and sodium-reduced food products elicited by TDS and TCATA, over single and multiple oral intakes. A total of 20 semi-trained participants evaluated commercially available regular and sodium-reduced canned corn, cooked ham (single intakes), potato chips and cream of mushroom soup (5 intakes) using both TDS and TCATA. Regular and sodium-reduced products differed in not only salty but also other sensory attributes, noticeably dry for chips, sweet for corn, bitter and metallic for ham, thick, creamy, sweet, and starchy for soup. TDS and TCATA provided comparable information for the key sensory attributes characterizing and differentiating the regular and sodium-reduced products. TDS profiled significant differences between samples for a larger number of attributes than TCATA, while TCATA profiles were more consistent across intakes. Multiple intakes changed the duration of attribute dominance but not the number of significantly dominant attributes in TDS profiles. The current findings provide insight for applications of temporal profiling to other food products and development of sodium-reduced foods with attribute profiles acceptable to consumers.
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Salamoun, Yezan M., Kishore Polireddy, Yu Kyoung Cho e Ryan Sol Funk. "Metabolomic Profiling of Red Blood Cells to Identify Molecular Markers of Methotrexate Response in the Collagen Induced Arthritis Mouse Model". Future Pharmacology 2, n.º 4 (8 de dezembro de 2022): 625–41. http://dx.doi.org/10.3390/futurepharmacol2040038.
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Although methotrexate (MTX) is the first line disease-modifying therapy used in the treatment of autoimmune arthritis, it is limited by its unpredictable and variable response profile and lack of therapeutic biomarkers to predict or monitor therapeutic response. The purpose of this work is to evaluate the utility of red blood cell (RBC) metabolite profiles to screen for molecular biomarkers associated with MTX response. Methods: Utilizing the collagen-induced arthritis mouse model, DBA/1J mice were treated with subcutaneous MTX (20 mg/kg/week) and RBC samples were collected and analyzed by semi-targeted global metabolomic profiling and analyzed by univariate analysis. Results: MTX treatment normalized the following RBC metabolite levels that were found to be altered by disease induction: N-methylisoleucine, nudifloramide, phenylacetylglycine, 1-methyl-L-histidine, PC 42:1, PE 36:4e, PC 42:3, PE 36:4e (16:0e/20:4), and SM d34:0. Changes in the RBC metabolome weakly but significantly correlated with changes in the plasma metabolome following MTX treatment (ρ = 0.24, p = 1.1 × 10−13). The RBC metabolome resulted in the detection of nine significant discriminatory biomarkers, whereas the plasma metabolome resulted in two. Overall, the RBC metabolome yielded more highly sensitive and specific biomarkers of MTX response compared to the plasma metabolome. N-methylisoleucine was found to be highly discriminatory in both plasma and RBCs. Conclusions: Our results suggest that RBCs represent a promising biological matrix for metabolomics and future studies should consider the RBC metabolome in their biomarker discovery strategy.
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Hewitt, Ashley N., Eric Beauregard e Garth Davies. "An Empirical Examination of the Victim-Search Methods Utilized by Serial Stranger Sexual Offenders: A Classification Approach". Journal of Interpersonal Violence 34, n.º 21-22 (1 de novembro de 2016): 4522–49. http://dx.doi.org/10.1177/0886260516675921.
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Past research on the spatial mobility of serial offenders has generally found that these individuals make calculated decisions about the ways in which they come into contact with suitable victims. Within the geographic profiling literature, four victim-search methods have been theorized that describe how serial predatory offenders hunt for their victims: hunter, poacher, troller, and trapper. Using latent class analysis, the aim of this study is to test whether this theoretical typology can be empirically derived using data that were collected from both police files and semi-structured interviews with 72 serial sex offenders who committed 361 stranger sexual assaults. Empirical support is found for each of the aforementioned victim-search methods, in addition to two others: indiscriminate opportunist and walking prowler. Chi-square analyses are also conducted to test for associations between this typology and characteristics of the offense such as victim information, environmental factors, and the offender’s modus operandi strategies. Findings from these analyses suggest that the types of victims and environments targeted by the offender, as well as the behaviors that take place both before and during the offense, are dependent upon the offender’s victim-search strategy. Although the theoretical hunter, poacher, troller, and trapper were intended to describe the victim-search methods of serial violent predators more generally, the finding that these strategies exist along with two others in this sample of sexual offenders may indicate that search behavior is specific to certain crime types. Furthermore, these findings may be of assistance in the investigation of stranger sexual assaults by providing law-enforcement officials with possible clues as to the characteristics of the unknown suspect, the times and places likely targeted in any past or future events, and possibly even his base of operations.
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Deinichenko, K. A., G. S. Krasnov, S. P. Radko, K. G. Ptitsyn, V. V. Shapovalova, O. S. Timoshenko, S. A. Khmeleva et al. "Human CHR18: “Stakhanovite” Genes, Missing and uPE1 Proteins in Liver Tissue and HepG2 Cells". Biomedical Chemistry: Research and Methods 4, n.º 1 (janeiro de 2021): e00144. http://dx.doi.org/10.18097/bmcrm00144.
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Missing (MP) and functionally uncharacterized proteins (uPE1) comprise less than 5% of the total number of proteins encoded by human Chr18 genes. Within half a year, since the January 2020 version of NextProt, the number of entries in the MP+uPE1 datasets changed, mainly due to the achievements of antibody-based proteomics. Assuming that the proteome is closely related to the transcriptome scaffold, quantitative PCR, Illumina HiSeq, and Oxford Nanopore Technology were applied to characterize the liver samples of three male donors in comparison with the HepG2 cell line. The data mining of the Expression Atlas (EMBL-EBI) and the profiling of biopsy samples by using orthogonal methods of transcriptome analysis have shown that in HepG2 cells and the liver, the genes encoding functionally uncharacterized proteins (uPE1) are expressed as low as for the missing proteins (less than 1 copy per cell), except the selected cases of HSBP1L1, TMEM241, C18orf21, and KLHL14. The initial expectation that uPE1 genes might be expressed at higher levels than MP genes, was compromised by severe discrepancies in our semi-quantitative gene expression data and in public databanks. Such discrepancy forced us to revisit the transcriptome of Chr18, the target of the Russian C-HPP Consortium. Tanglegram of highly expressed genes and further correlation analysis have shown the severe dependencies on the mRNA extraction method and the analytical platform. Targeted gene expression analysis by quantitative PCR (qPCR) and high-throughput transcriptome profiling (Illumina HiSeq and ONT MinION) for the same set of samples from normal liver tissue and HepG2 cells revealed the detectable expression of 250+ (92%) protein-coding genes of Chr18 (at least one method). The expression of slightly more than 50% protein-coding genes was detected simultaneously by all three methods. Correlation analysis of the gene expression profiles showed that the grouping of the datasets depended almost equally on both the type of biological material and the experimental method, particularly cDNA/mRNA isolation and library preparation.
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Gopalakrishnan, Vancheswaran, Elizabeth Ashley Dozier, Matthew S. Glover, Steven Novick, Michael Ford, Christopher Morehouse, Paul Warrener et al. "Engraftment of Bacteria after Fecal Microbiota Transplantation Is Dependent on Both Frequency of Dosing and Duration of Preparative Antibiotic Regimen". Microorganisms 9, n.º 7 (29 de junho de 2021): 1399. http://dx.doi.org/10.3390/microorganisms9071399.
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The gut microbiota has emerged as a key mediator of human physiology, and germ-free mice have been essential in demonstrating a role for the microbiome in disease. Preclinical models using conventional mice offer the advantage of working with a mature immune system. However, optimal protocols for fecal microbiota transplant (FMT) engraftment in conventional mice are yet to be established. Conventional BALB/c mice were randomized to receive 3-day (3d) or 3-week (3w) antibiotic (ABX) regimen in their drinking water followed by 1 or 5-daily FMTs from a human donor. Fecal samples were collected longitudinally and characterized using 16S ribosomal RNA (rRNA) sequencing. Semi-targeted metabolomic profiling of fecal samples was also done with liquid chromatography–mass spectrometry (LC-MS). Lastly, we sought to confirm our findings in BKS mice. Recovery of baseline diversity scores were greatest in the 3d groups, driven by re-emergence of mouse commensal microbiota, whereas the most resemblance to donor microbiota was seen in the 3w + 5-FMT group. Amplicon sequence variants (ASVs) that were linked to the input material (human ASVs) engrafted to a significantly greater extent when compared to mouse ASVs in the 3-week groups but not the 3-day groups. Lastly, comparison of metabolomic profiles revealed distinct functional profiles by ABX regimen. These results indicate successful model optimization and emphasize the importance of ABX duration and frequency of FMT dosing; the most stable and reliable colonization by donor ASVs was seen in the 3wk + 5-FMT group.
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Mukohyama, Junko, Masahiro Shinoda, Osamu Itano, Yoshihiro Kakeji e Yohei Shimono. "Identification of the epigenetic mechanism for colorectal cancer stem cell regulation through the comprehensive microRNA expression profiling of human colorectal cancer tissues." JCO Global Oncology 9, Supplement_1 (agosto de 2023): 42. http://dx.doi.org/10.1200/go.2023.9.supplement_1.42.
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42 Background: MicroRNAs (miRNAs) are key regulators of cancer stem cell (CSC) functions, including self-renewal and differentiation. However, little is known about miRNAs that regulate such properties in human colorectal cancer. Methods: We compared the expression levels of 754 miRNAs between paired samples of EpCAM+/CD44+ cancer cells (enriched in CSCs) and EpCAM+/CD44neg cancer cells (non-tumorigenic cancer cells (NTCs)) sorted in parallel from human primary colorectal cancers by multiplex semi-quantitative PCR. Results: Comparison of the expression levels of miRNAs in the CSCs and NTCs resulted in the identification of 10 miRNAs upregulated (miR-221, miR-218, miR-500 and miR-195) or downregulated (miR-185, miR-19a, miR-337, miR-10a, miR-21 and miR-24) in CSCs compared to NTCs. Among them, miR-221 was most highly expressed in CSCs; and its expression level was very low in NTCs and normal colon epithelial cells (n=6, p<0.05). Kaplan-Myer estimates and univariate and multivariate analyses showed that higher miR-221 expression was an independent prognosis factor for poorer outcome. Constitutive overexpression of miR-221 enhanced organoid-forming capacity of both conventional colorectal carcinoma cell lines and patient-derived xenografts (PDX) in vitro. Importantly, constitutive downregulation of miR-221 suppressed organoid-forming capacity in vitro and substantially reduced the tumorigenic capacity of CSC populations from PDX lines in vivo. miR-221 directly targeted Quaking (QKI) 5, the most abundant splicing isoform of the human QKI gene, and suppressed its protein level. Knockdown of miR-221 suppressed the clonogenicity of colorectal CSCs in vitro in a QKI-expression dependent manner and ability to form tumors in vivo. Conclusions: Our study reveals a mechanistic link between miR-221 and QKI and highlights their key role in regulating CSC properties in human colorectal cancer and proposes that miR-221 will be a promising marker and a specific therapeutic target for human colorectal CSCs.
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Ludovico, Nuccio, Federica Dessi e Marino Bonaiuto. "Stakeholders Mapping for Sustainable Biofuels: An Innovative Procedure Based on Computational Text Analysis and Social Network Analysis". Sustainability 12, n.º 24 (10 de dezembro de 2020): 10317. http://dx.doi.org/10.3390/su122410317.
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The identification and engagement of stakeholders is a challenge whose outcomes have a strong impact on a project’s success. This is even more relevant when the project concerns the introduction of sustainable technologies; these technologies are often less competitive on the market than traditional ones, both in terms of development complexity and production costs. This paper presents a stakeholder identification and mapping procedure, based on an Interest x Influence model, that emphasizes a quantitative methodological approach. The method has been applied on publicly available online data to identify and map potential stakeholders of a European research project aiming at creating a new biomass-derived biofuel. A semi-supervised procedure, built by combining computational text analysis and social network analysis techniques, has been used to calculate Interest and Influence scores for each potential stakeholder toward the project. The results show that stakeholders can be ranked on both dimensions and mapped on a bi-dimensional space according to their level of Interest and Influence. Within projects aiming at developing technologies for sustainability in which a wide range of stakeholders are involved at a transnational level, this stakeholder mapping technique provides a useful tool that can be adopted even with little knowledge on specific fields of application. A further asset of this approach lies in the possibility of profiling stakeholders on the basis of their Interest in the target project: this allows us to know the contents of a stakeholder (or stakeholders category) Interest, and therefore to have useful information for addressing the targeted stakeholder by means of a content design which is based on specific content categories, substantiating the stakeholder(s) Interest in the specific project.
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Yeh, Chen, Shu-Ti Lin e Hung-Chih Lai. "A Transformative Technology Linking Patient’s mRNA Expression Profile to Anticancer Drug Efficacy". Onco 4, n.º 3 (14 de julho de 2024): 143–62. http://dx.doi.org/10.3390/onco4030012.
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As precision medicine such as targeted therapy and immunotherapy often have limited accessibility, low response rate, and evolved resistance, it is urgent to develop simple, low-cost, and quick-turnaround personalized diagnostic technologies for drug response prediction with high sensitivity, speed, and accuracy. The major challenges of drug response prediction strategies employing digital database modeling are the scarcity of labeled clinical data, applicability only to a few classes of drugs, and losing the resolution at the individual patient level. Although these challenges have been partially addressed by large-scale cancer cell line datasets and more patient-relevant cell-based systems, the integration of different data types and data translation from pre-clinical to clinical utilities are still far-fetched. To overcome the current limitations of precision medicine with a clinically proven drug response prediction assay, we have developed an innovative and proprietary technology based on in vitro patient testing and in silico data analytics. First, a patient-derived gene expression signature was established via the transcriptomic profiling of cell-free mRNA (cfmRNA) from the patient’s blood. Second, a gene-to-drug data fusion and overlaying mechanism to transfer data were performed. Finally, a semi-supervised method was used for the database searching, matching, annotation, and ranking of drug efficacies from a pool of ~700 approved, investigational, or clinical trial drug candidates. A personalized drug response report can be delivered to inform clinical decisions within a week. The PGA (patient-derived gene expression-informed anticancer drug efficacy) test has significantly improved patient outcomes when compared to the treatment plans without PGA support. The implementation of PGA, which combines patient-unique cfmRNA fingerprints with drug mapping power, has the potential to identify treatment options when patients are no longer responding to therapy and when standard-of-care is exhausted.
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Mazzara, Saveria, Laura L. Travaini, Francesca Botta, Chiara Granata, Giovanna Motta, Federica Melle, Stefano Fiori et al. "Integration of Targeted Gene Expression Profiling and FDG-PET Radiomics Uncovers Radiometabolic Signatures Associated with Outcome in Diffuse Large B-Cell Lymphoma". Blood 138, Supplement 1 (5 de novembro de 2021): 3496. http://dx.doi.org/10.1182/blood-2021-152679.
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Abstract Metabolic rewiring is a hallmark of cancer and a predominant feature of aggressive lymphoproliferative disorders such as diffuse large B-cell lymphomas (DLBCL), which need a reshaped metabolism in order to meet the increased demands related to rapid cell proliferation. Emerging evidence indicates that chemoresistance is closely related to altered metabolism in cancer. However, the relationship between metabolic rewiring and chemoresistance in lymphoma is yet to be elucidated. Radiomic analysis applied to functional imaging with fluoroedoxyglucose positron emission tomography (FDG-PET) provides a unique opportunity to explore DLBCL metabolism. In this study we hypothesized that distinct gene expression (GEP) signatures might be correlated with specific FDG-PET radiomics signatures, which in turn could be associated with resistance to standard chemoimmunotherapy and DLBCL outcome. First, we retrospectively analyzed a discovery cohort of 48 consecutive DLBCL patients (pts) treated at our center with standard first line R-CHOP/R-CHOP-like chemoimmunotherapy from 2010 to 2018, with available formalin-fixed paraffin embedded (FFPE) tissue from the initial diagnostic biopsy and FDG-PET radiomics data extracted from the same target lesion. Median follow-up was 55 months (range 18-110). We profiled this cohort with targeted-GEP (T-GEP) (NanoString platform), using a custom panel to define the cell of origin (COO) and MYC/BCL-2 levels, and a dedicated panel comprising 180 genes encompassing the most relevant cancer metabolism pathways. By applying the maxstat package we found that a 6-gene metabolic signature was strongly associated with outcome and outperformed the COO, the MYC/BCL-2 status and the International Prognostic Index (IPI) score for progression free survival (PFS) and overall survival (OS) in multivariate analysis. The 6-gene metabolic signature included genes regulating oxidative metabolism and fatty acid oxidation (SCL25A1, PDK4, PDPR) which were upregulated, and was inversely associated with genes involved in glycolytic pathways (MAP2K1, HIF1A, GBE1) which were downregulated. Notably 5-year PFS and OS were 100% and 95% in metabolic signature (met-Sig) low pts vs 24% and 45% in met-Sig high pts respectively (p<0.0001 for PFS and OS). There was no significant association between the COO, MYC/BCL-2 levels, standardized uptake value (SUV), and the 6-gene signature. The prognostic value of the 6-gene signature for OS was validated in 2 large publicly available cohorts of 469 (Sha et al. J Clin Oncol 2019) and 233 (Lenz et al. N Eng J Med 2005) pts. Next, we integrated PET radiomics and T-GEP data. Radiomics analysis (LifeX package) was performed by applying regions of interest semi-automatically, using a 25% SUV max threshold for segmentation. Fifty-five radiomic features (RFs) were extracted and 10 RFs significantly correlated either positively or negatively with the T-GEP metabolic signature (Spearman). After stability evaluation, applying a stepwise feature selection procedure, 4 RFs (Histo Curtosis, Histo Energy, Shape Sphericity, NGLDM Contrast) were used to generate a radiomic signature (hereafter called radiometabolic signature) characterized by the most significant correlation with both the metabolic T-GEP signature (r=0.43, p=0.0027) and PFS (p=0.004). These results (obtained analyzing the lesion of the initial diagnostic biopsy), were confirmed using different target lesions (i.e. the most FDG-avid and the largest lesion), and were validated in a second independent cohort of 64 patients (validation cohort) treated at our center in the same period of time (with no FFPE tissue available). A multivariate analysis performed in the whole cohort of 112 pts (discovery + validation) indicated that the radiometabolic signature retained independent prognostic value in relation to the IPI score and metabolic tumor volume. The robustness of the radiometabolic signature was further confirmed by using a second segmentation method (fixed 2.5 SUV max threshold). These data indicate that oxidative metabolic rewiring could be a powerful adverse prognostic predictor, suggesting the possibility of targeting oxidative metabolism to overcome chemorefractoriness in DLBCL. This study provides the proof of principle for the use of FDG-PET radiomics as a tool for non-invasive assessment of cancer metabolism, and for predicting metabolic vulnerabilities in DLBCL. Figure 1 Figure 1. Disclosures Tarella: ADC-THERAPEUTICS: Other: ADVISORY BOARD; Abbvie: Other: ADVISORY BOARD. Pileri: CELGENE: Other: ADVISORY BOARD; ROCHE: Other: ADVISORY-BOARD; NANOSTRING: Other: ADVISORY BOARD. Derenzini: TAKEDA: Research Funding; BEIGENE: Other: ADVISORY BOARD; ASTRA-ZENECA: Consultancy, Other: ADVISORY-BOARD; TG-THERAPEUTICS: Research Funding; ADC-THERAPEUTICS: Research Funding.
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Lohoff, Caroline, Thi Huong Lan Do, Ferris Jung, Sandra Kummer, Vladimir Benes, Wolfgang Huber, Thorsten Zenz e Junyan Lu. "Unraveling Molecular Subgroup-Specific Pathway Dependencies in Chronic Lymphocytic Leukemia through Drug-Perturbed Transcriptomic Profiling and Statistical Modeling". Blood 144, Supplement 1 (5 de novembro de 2024): 276. https://doi.org/10.1182/blood-2024-210844.
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Gene expression profiling following targeted pathway perturbations on primary patient samples provides important information for understanding heterogeneous pathway dependencies in cancer, thereby supporting the design of personalized therapies. However, analyzing such high-dimensional data poses a challenge. In this study, we developed a novel computational framework utilizing semi-supervised factor analysis, regularized multiple linear regression, and causal inference to analyze and interpret drug-perturbed transcriptomic profiles from patient samples. We applied our framework to RNA sequencing data obtained after ex-vivo perturbation with ten different small-molecule inhibitors in primary tumor cells from 108 chronic lymphocytic leukemia (CLL) patients, yielding 1010 transcriptional drug response profiles (Do et al., ASH2023 abstract 4633). To capture and quantify the shared and unique dimensions of gene expression changes induced by different pathway inhibitors, we employed guided sparse factor analysis (GSFA), originally designed for analyzing single-cell CRISPR screening (Zhou et al., Nat Methods, 2023). We identified perturbations uniquely associated with single factors, such as XPO1 inhibition by selinexor, MDM2 inhibition by nutlin-3a, and MEK1/2 inhibition by trametinib, suggesting the induction of distinct downstream signatures not shared with other perturbations. Conversely, perturbations like BTK inhibition by ibrutinib, PI3K inhibition by duvelisib, and mTOR inhibition by everolimus were linked to multiple factors shared across perturbations, suggesting extensive downstream pathway cross-talks. Notably, PI3K inhibition was significantly associated with two factors, one shared with BTK inhibition, and the other with PI3K and mTOR inhibition. Pathway enrichment analysis revealed that the factor shared between BTK and PI3K was enriched for the B-cell receptor signaling pathway, while the factor shared among PI3K, ATK, and mTOR inhibitions was enriched for MAP kinase pathway genes. This highlights the complex role of PI3K signaling in CLL and provides novel insights into the hierarchical pathway structure in CLL biology. We further examined the heterogeneity of pathway activity changes upon perturbation among patients with different driver mutations. Based on the GSFA model, we derived a per-patient perturbation score for each factor and associated them with major molecular subgroups in CLL using regularized multiple linear regression. We observed a strong association between TP53 mutational status and the factor representing MDM2 inhibition, reflecting the known dependencies of DNA damage response signaling on TP53 mutational status. Furthermore, this approach uncovered that IGHV mutational status strongly determines the downstream MAP kinase activity change upon PI3K/mTOR/AKT inhibition but not the BCR signaling activity change upon PI3K and BTK inhibition. By testing for interaction in linear models, we further investigated the dependence of individual gene expression changes upon pathway perturbation and the presence of driver mutations. Thanks to the high-dimensional nature of gene expression profiling, we could infer causal interactions between mutations and targets of perturbation using the method developed by Fischer et al., eLife, 2015. We successfully recaptured the known signaling structure of the BCR-PI3K cascade and TP53-MDM2 interaction. Moreover, we unveiled other novel interactions, such as the activation of BTK and MEK signaling by trisomy12, explaining our previous observations of increased sensitivity to BTK and MEK inhibitors in CLL samples with trisomy12 (Dietrich et al., J Clin Invest, 2018). This study underscores the value of perturbed transcriptomic profiling of primary cancer patient samples for mapping detailed pathway structures and dependencies on disease drivers. Our comprehensive computational workflow serves as a guide for mining and interpreting complex high-dimensional data, advancing our understanding of disease biology, and facilitating the rational design of novel personalized therapies, including combinational treatments.
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Shimizu, Toshio, Anthony W. Tolcher, Kyriakos P. Papadopoulos, Muralidhar Beeram, Drew Warren Rasco, Lon S. Smith, Ronald L. Drengler et al. "Personalizing cancer treatment of combined targeting of PI3K/AKT and MAPK pathways in advanced cancer patients harboring simultaneous oncogenic alterations in both signaling pathways." Journal of Clinical Oncology 30, n.º 15_suppl (20 de maio de 2012): e13025-e13025. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.e13025.
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e13025 Background: Frequent coactivation of both PI3K/AKT and MAPK pathways has been seen in a number of different tumor types. Preclinical studies also provide a clear rationale for the co-inhibition of these, "semi-parallel" pathways. Methods: We reviewed the records of 1,672 consecutive advanced cancer patients (pts) in our Phase I Clinical Trials Program and investigated the clinical impact of simultaneous blockade of both PI3K/AKT and MAPK pathways in patients with oncogenic alterations in both signaling pathways from 317 pts who received PI3K pathway inhibitor and/or MAPK pathway inhibitor. Results: 163 of 317 pts (51.4%) were tested for comprehensive tumor genomic analysis using DNA array-based CGH/PCR-based DNA sequencing. PI3K pathway genetic alterations (PIK3CA mutation, n=3; PTEN deletion, n=18; AKT amplification, n=10) were detected in 29 of the 163 (17.8%) pts. RAS/RAF pathway genetic alterations (KRAS mutation, n=33; HRAS mutation, n=4; BRAF mutation, n=6) were detected in 43 of the 163 (26.4%) pts. Simultaneous oncogenic alterations in both PI3K/AKT and MAPK pathways were detected in 12 pts (colorectal cancer, n=7; pancreatic cancer, n=2; melanoma, n=2; non-seminomatous germ cell tumor, n=1). Six of 8 (75%) pts treated with personalized treatment based on dual pathways inhibition had tumor regression with prolonged duration of time to treatment failure compared to that of pts treated with single pathway inhibition. A pt with pancreatic cancer harboring simultaneous KRAS mutation and AKT2 amplification treated with combination MEK1/2 inhibitor and AKT inhibitor showed central cavitations of numerous lung metastatic lesions along with a decrease in CA19-9, which may reflect treatment effect. Conclusions: These data could serve as a platform for therapeutic decision making. The strategy of targeting parallel pathways may be especially important in pts with coexisting PI3K/AKT and MAPK pathway genetic alterations. The data also suggest the need to further investigate the role of molecular profiling and matching pts with targeted drugs for personalized treatment.
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Chapman-Arvedson, Tara L. "Antibody Drug Conjugates with a Novel Translation Inhibitor Payload Demonstrate Cytotoxicity and Tumor Growth Reduction in Multiple Myeloma Models". Blood 144, Supplement 1 (5 de novembro de 2024): 7465. https://doi.org/10.1182/blood-2024-212081.
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Background Antibody drug conjugates (ADCs) are a validated modality for the targeted delivery of cytotoxic payloads to myeloma cells while sparing healthy tissue. To achieve the full potential of ADCs in multiple myeloma patients, it will be important to have ADCs that incorporate payloads with non-overlapping mechanisms of action (MOA) as compared to existing therapies. By mining a proprietary collection of small molecule natural products (NPs), Hexagon Bio has identified a novel protein translation inhibitor, HB-06510, which exhibits favorable physicochemical properties and significant sensitivity in myeloma cell lines. HB-06510 has been conjugated to antibodies that bind myeloma antigens and has been shown to induce myeloma cell cytotoxicity in vitro and in vivo. Methods and Results Hexagon Bio has established a platform to identify new NPs and determine their MOA. This platform uses a proprietary algorithm integrated with machine learning, genetic screening, and synthetic biology to identify, produce, and define the MOAs of new NPs, primarily isolated from our fungal library comprising > 100,000 strains. Using this platform, we identified a NP that was potently cytotoxic across a panel of cancer cell lines, including multiple myeloma (≤ 3.2 nM average EC50 and 100% killing across 10 cell lines in a 72 hour assay). To determine the mechanism of action, we used genetic screening and found that this NP inhibited protein translation. Physicochemical evaluation of the NP revealed it had good properties for a potential ADC payload (good solubility, low lipophilicity, good stability in human plasma and lysosomal preparations, no reactivity with glutathione), it has a site for linker attachment and is amenable to semi-synthesis and medicinal chemistry. Further cytotoxicity profiling demonstrated that HB-06510 is cytotoxic to multi-drug resistant cells, including those expressing high levels of efflux pumps such as Pgp and ABCG2, indicating that HB-06510 is not a substrate for these pumps. We also demonstrated that HB-06510 is cytotoxic to both fast- and slow-cycling cells and induces immunogenic cell death. To evaluate the potential for HB-06510 to be an ADC payload, we conjugated HB-06510 to antibodies that bind myeloma antigens BCMA, CS1 and CD38 and have demonstrated anti-myeloma cell cytotoxicity in vitro and in vivo. These results will be presented. Conclusions We have identified a novel protein translation inhibitor payload that is highly potent, has good drug-like properties and shows effective anti-myeloma cell activity in vitro and in vivo when conjugated to anti-myeloma antibodies. ADCs with this MOA are distinct from both currently-available targeted therapeutics and chemotherapy and have the potential to provide benefit to patients.
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Shapiro, Ilja E., Marco Tognetti, Tikira Temu, Oliver M. Bernhardt, Daniel Redfern, Yuehan Feng, Roland Bruderer e Lukas Reiter. "Abstract 5376: Quantitative profiling of HLA class I and class II antigens and neoantigens in tissue biopsy and PBMC samples using an optimized mass spectrometry-based workflow". Cancer Research 84, n.º 6_Supplement (22 de março de 2024): 5376. http://dx.doi.org/10.1158/1538-7445.am2024-5376.
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Abstract Major histocompatibility complex (MHC) molecules play a central role in orchestrating immune responses by presenting antigenic peptides derived from both self and foreign proteins. In the context of cancer, understanding the repertoire of tumor-associated antigens (TAAs) presented by MHC molecules (or HLA molecules in human) is crucial for deciphering how the immune system recognizes and responds to malignant cells. The identification of neoantigens, unique to individual tumors due to somatic mutations, has become a focal point in immunopeptidomic studies. One significant hurdle in systematic immunopeptidomics analysis is the high input material requirement. Here, we present a semi-automated workflow to robustly identify and quantify immunopeptides from reduced amounts of clinical tissue biopsy and peripheral blood mononuclear cell samples. At the core of immunopeptidomics is the enrichment of HLA-associated peptides, followed by identification using mass spectrometry and bioinformatics tools. We optimized the native lysis and a sequential immunoprecipitation workflow for both class I and class II immunopeptides while ensuring scalability and reproducibility. Leveraging the magnetic properties of the beads, 1,000 samples can be processed within a week by a single operator. For both tissue and PBMC samples, we performed a systematic ramping experiment starting with as little as 2.5mg tissue or 5 million PBMCs. In all experiments, The established sample preparation offers high reproducibility and identifications of good quality: 1) Class-I immunopeptides: >60% of the peptides identified are 9-mers, >80% predicted strong binders, and the expected amino acids are enriched at the anchor positions; 2) Class-II immunopeptides, >50% of the peptides identified are 14-to-16-mers, and >50% are predicted strong binders. Furthermore, the pipeline is highly sensitive as we could still identify over 2,800 class-I immunopeptides when processing 2.5 mg fresh frozen tissue and 2,000 - 3,000 class-I immunopeptides when starting from 5 million PBMCs. Furthermore, the developed immunopeptidomics workflow was deployed to profile a cohort of 12 cancerous and matched healthy lung tissue samples. Their Class-I immunopeptidomes clearly displayed a pattern where matched tissues from each subject cluster together, further underlining the fact that the intricate immune-tumor interface is highly personalized. Overall, we established a robust pipeline for for class-I and II immunopeptidome profiling from clinically relevant sample types. Taking advantage of the nature of mass spectrometry-based methods, customized targeted assays can be developed without the need for affinity reagent, allowing specific and absolute quantification of any immunopeptide of interest. Citation Format: Ilja E. Shapiro, Marco Tognetti, Tikira Temu, Oliver M. Bernhardt, Daniel Redfern, Yuehan Feng, Roland Bruderer, Lukas Reiter. Quantitative profiling of HLA class I and class II antigens and neoantigens in tissue biopsy and PBMC samples using an optimized mass spectrometry-based workflow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5376.
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Miskevich, Dmitry, Anastasia Chaban, Maria Dronina, Ifat Abramovich, Eyal Gottlieb e Imad Shams. "Comprehensive Analysis of 13C6 Glucose Fate in the Hypoxia-Tolerant Blind Mole Rat Skin Fibroblasts". Metabolites 11, n.º 11 (27 de outubro de 2021): 734. http://dx.doi.org/10.3390/metabo11110734.
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The bioenergetics of the vast majority of terrestrial mammals evolved to consuming glucose (Glc) for energy production under regular atmosphere (about 21% oxygen). However, some vertebrate species, such as aquatic turtles, seals, naked mole rat, and blind mole rat, Spalax, have adjusted their homeostasis to continuous function under severe hypoxic environment. The exploration of hypoxia-tolerant species metabolic strategies provides a better understanding of the adaptation to hypoxia. In this study, we compared Glc homeostasis in primary Spalax and rat skin cells under normoxic and hypoxic conditions. We used the targeted-metabolomics approach, utilizing liquid chromatography and mass spectrometry (LC-MS) to track the fate of heavy Glc carbons (13C6 Glc), as well as other methodologies to assist the interpretation of the metabolic landscape, such as bioenergetics profiling, Western blotting, and gene expression analysis. The metabolic profile was recorded under steady-state (after 24 h) of the experiment. Glc-originated carbons were unequally distributed between the cytosolic and mitochondrial domains in Spalax cells compared to the rat. The cytosolic domain is dominant apparently due to the hypoxia-inducible factor-1 alpha (HIF-1α) mastering, since its level is higher under normoxia and hypoxia in Spalax cells. Consumed Glc in Spalax cells is utilized for the pentose phosphate pathway maintaining the NADPH pool, and is finally harbored as glutathione (GSH) and UDP-GlcNAc. The cytosolic domain in Spalax cells works in the semi-uncoupled mode that limits the consumed Glc-derived carbons flux to the tricarboxylic acid (TCA) cycle and reduces pyruvate delivery; however, it maintains the NAD+ pool via lactate dehydrogenase upregulation. Both normoxic and hypoxic mitochondrial homeostasis of Glc-originated carbons in Spalax are characterized by their massive cataplerotic flux along with the axis αKG→Glu→Pro→hydroxyproline (HPro). The product of collagen degradation, HPro, as well as free Pro are apparently involved in the bioenergetics of Spalax under both normoxia and hypoxia. The upregulation of 2-hydroxyglutarate production detected in Spalax cells may be involved in modulating the levels of HIF-1α. Collectively, these data suggest that Spalax cells utilize similar metabolic frame for both normoxia and hypoxia, where glucose metabolism is switched from oxidative pathways (conversion of pyruvate to Acetyl-CoA and further TCA cycle processes) to (i) pentose phosphate pathway, (ii) lactate production, and (iii) cataplerotic pathways leading to hexosamine, GSH, and HPro production.
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Thomas, Daniel, Subarna Sinha, Steven M. Chan, Manhong Wu, Damoun Torabi, Gary Peltz, Yang Gao, David L. Dill e Ravi Majeti. "Isocitrate Dehydrogenase 1 Mutant Cancers Are Metabolically Vulnerable to Inhibition of Acetyl CoA Carboxylase Via a 2-Hydroxyglutarate Independent Mechanism". Blood 128, n.º 22 (2 de dezembro de 2016): 1054. http://dx.doi.org/10.1182/blood.v128.22.1054.1054.
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Abstract Introduction: Mutations substituting arginine 132 of isocitrate dehydrogenase 1 (IDH1) are recurrent in acute myeloid leukemia (AML) and several other cancers, resulting in the aberrant production of the onco-metabolite, R-2-hydroxyglutarate (2-HG), as well as an inability to convert cytoplasmic alpha-ketoglutarate to isocitrate via reductive carboxylation. Currently, small molecules that effectively inhibit the neomorphic enzyme and abrogate the production of 2-HG, such as AG-120, are in clinical trials with promising results. However, these inhibitors have not proven to be curative in most AML cases, indicating a need for additional targeted therapies. We have previously investigated synthetic lethal vulnerabilities in IDH1-mutated AML and identified an interaction with BCL2 leading to increased susceptibility to ABT-199 (Chan et al, 2015). Synthetic lethal approaches targeting 2-HG independent metabolic vulnerabilities conferred by mutant IDH1 may complement IDH1 mutant inhibitors. Using a novel computational method (MiSL) based on Boolean implication (if-then rules) mining of pan-cancer data, we identified acetyl CoA carboxylase (ACACA) as a potential druggable target in IDH1-mutated AML. ACACA is the rate-limiting step in the de novo synthesis of fatty acids, and mutant IDH1 leads to a reduction in malonyl-CoA, a key building block for fatty acids, in a 2-HG independent manner. This finding led us to investigate a potential synthetic lethal interaction between mutant IDH1 and ACACA based on the hypothesis that the combination causes marked inhibition of fatty acid synthesis required for cell growth. Methods: Boolean implications (MiSL) were used to identify candidate synthetic lethal interactions with mutant IDH1 by isolating genes deleted only in the absence of the mutation and with differential gene expression within pan-cancer TCGA data. Validation was performed using THP-1 cells transduced with doxycycline-inducible wildtype and R132H mutant IDH1 lentiviral vectors, and primary patient IDH1-mutant and wildtype AML samples, using both shRNAs and a targeted pharmacologic inhibitor of ACACA. Metabolomics was performed using semi-targeted mass spectrometry and liquid chromatography. Finally, primary AML samples and IDH1-mutant and wildtype cancer cell lines (HT-1080, U118, U87) were transduced with validated shRNA and engrafted into NSG mice. Results: Our computational method found that IDH1 mutation and ACACA deletions were mutually exclusive in pan-cancer TCGA data, ACACA deletions resulted in lowered expression of ACACA, and ACACA was differentially over-expressed in IDH1-mutant AML compared to IDH1-wildtypeAML. Pharmacologic inhibition of ACACA with 2 uM TOFA caused a marked reduction in cell growth in the presence of IDH1 R132H (+ dox), but not in its absence (- dox; p = 0.0001). Similarly, knockdown of ACACA with independent shRNAs caused a defect in viable cell growth in the presence of IDH1 R132H (+ dox), but not in its absence (- dox) or with scrambled shRNA (p=0.009, shRNA #1 vs. scrambled; p=0.01, shRNA #2 vs. scrambled). Primary IDH1 R132 mutated purified AML blasts were selectively sensitive to TOFA treatment compared to IDH1 wildtype normal karyotype blasts (IC50 0.6 uM vs 6 uM, p=0.009) in viable growth assays. Furthermore, when transduced with lentivirus encoding shRNA to ACACA, primary IDH1-mutant AML cells exhibited markedly reduced engraftment of RFP-positive human CD45+CD33+ leukemic cells compared to scrambled non-targeting shRNA (p < 0.05, Mann-Whitney U). As predicted, IDH1-mutant AML blasts pre-treated with 10uM AG-120 (sufficient to inhibit production of detectable 2-HG) remained susceptible to ACACA inhibition in vitro. Strikingly, in vivo models of IDH1 R132C mutated, but not wildtype, sarcoma cell lines exhibited a dramatic decrease in cell growth after ACACA inhibition that was not reversible by treatment with AG-120. Finally, metabolomic profiling revealed a major perturbation in multiple phospholipid fatty acid species and decreased malonyl-CoA conferred by IDH1 R132H, consistent with our proposed mechanism. Conclusion: We have identified de novo lipogenesis through ACACA as a critical metabolic vulnerability linked to IDH1 mutation in AML and provide evidence that therapeutic inhibition of ACACA with small molecules may be beneficial in AML, as well as in other cancers with IDH1 mutations. Disclosures Majeti: Forty Seven Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.
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Verstockt, S., J. Dehairs, F. Vanderhoydonc, B. J. Ke, I. De Greef, J. Sabino, M. Ferrante et al. "P049 The lipidome of creeping fat in Crohn’s disease points towards a harmful microenvironment". Journal of Crohn's and Colitis 18, Supplement_1 (1 de janeiro de 2024): i313—i314. http://dx.doi.org/10.1093/ecco-jcc/jjad212.0179.
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Abstract Background A pathognomonic observation in Crohn’s disease (CD) is the existence of mesenteric adipose tissue (MAT) wrapping around the inflamed and fibrotic intestine, so-called creeping fat. The etiology of this phenomenon is unclear, and both a protective and harmful role in disease pathogenesis and progression have been proposed. Given this conflicting evidence, we aimed to further unravel the role of creeping fat by extensive profiling of its lipidome. Methods We prospectively included 22 CD patients who underwent an ileocecal resection with ileocolonic anastomosis (Table 1). Perioperatively paired adipose tissue samples were collected from 3 locations: (1) creeping fat, (2) MAT close to the proximal unaffected ileum, and (3) MAT far from the diseased ileum, defined as near the base of the ileocecal artery (Fig. 1A). The lipidome of all 66 samples was profiled by quantitative mass spectrometry (targeted C18 LC-MS/MS and HILIC LC-MS/MS; Lipometrix core KU Leuven). This approach enables the detection of mediator lipids, and membrane and storage lipids, of which the latter group represents more complex chemical structures and thus a higher diversity in classification and number of species. Data analysis and paired Wilcoxon rank- sum tests were performed with Python and R 4.3.2. Results Across all CD adipose tissue samples, we detected 38 mediator lipid species, and 456 membrane and storage lipid species . When paired-wise comparing creeping fat with MAT far from the diseased ileum, we observed a significant decrease in 16 lipid mediators (p<0.05), namely arachidonic acid (AA), docosahexaenoic acid (DHA), 4 epoxy and 10 hydroxy mediator lipids; with hydroxy mediator X being the top downregulated in creeping fat (fold change (FC)=-2.11, p=0.0003) (Fig. 1B). While AA and DHA are (semi)-essential fatty acids and function as precursors, the other 8 decreased lipids in creeping fat are known to be anti-inflammatory, and 3 (incl. mediator X) are also anti-fibrotic. The lipid mediator profile of MAT close to the proximal unaffected ileum was similar to that of creeping fat, except for a pro-inflammatory lipid being increased in creeping fat (FC=2.30, p=0.004) (Fig. 1B). Lastly, when evaluating the membrane and storage lipid abundances both at species level (sum notation; fatty acyl composition) and at class level, no paired-wise differences among the three adipose tissue locations were found (p>0.05). Conclusion In this pilot lipidomics study, we did not observe any changes in storage or membrane lipids in creeping fat as compared to paired unaffected MAT. In contrast, a decrease in anti-inflammatory and anti-fibrotic lipid species along with an increase in a pro-inflammatory species points towards a harmful microenvironment within creeping fat.
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Koshy, Nebu V., Ellen Friday e Francesco Turturro. "Thioredoxin Interacting Protein (TXNIP) Correlates with Production of Reactive Oxygen Species (ROS) and Underlines Differences in Stress Response Gene Profile in Patients with Chronic Lymphocytic Leukemia (CLL)." Blood 114, n.º 22 (20 de novembro de 2009): 4375. http://dx.doi.org/10.1182/blood.v114.22.4375.4375.
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Abstract Abstract 4375 Introduction Our previous studies have focused on the role of TXNIP/ROS/TRX axis and hyperglycemia in breast cancer (Clin Cancer Res.13:3724-30, 2007). A main function of thioredoxin-interacting protein (TXNIP) is to bind and inactivate thioredoxin (TRX) leading to increased reactive oxygen species (ROS) production and apoptosis. While ROS and TRX have been examined in CLL, no studies have been performed on TXNIP levels. Based on the fact that CLL is known to have high levels of ROS compared to normal individuals and that TRX has been shown to have a protective function in CLL, we wanted to first determine if there was any correlation between TXNIP RNA and ROS production, and whether there was any relationship with TRX levels in patient plasma. Methods Venous blood samples were obtained from the first ten consecutive patients with definite diagnosis of CLL during their routine clinic visits. Patients were selected without regard to demographics or stage or grade of disease. Prior treatment for CLL was not an exclusion criterion. Cells were purified using a Ficoll density gradient. ROS activity was measured using DCFDA by flow cytometry and reported as mean fluorescence intensity. TXNIP and TRX RNA levels were assessed by semi-quantitative PCR. Thioredoxin levels in plasma were measured using a thioredoxn-1 Elisa. Gene profiling of our patient extremes was accomplished using the Human Stress Response 96 StellARray plate and analyzed using Global Pattern Recognition software Results Over a four week period, 10 CLL patients were seen in our clinic and participated in this study. Six out of the 10 patients were either actively being treated or had finished treatment for CLL. The rest were under observation. Three out of the 10 patients had trisomy 12 on FISH and the remaining had normal cytogenetics. We examined the levels of TXNIP RNA in our samples. The median level of TXNIP was 0.77 relative ratio (range: 0.45-1.01). The median ROS levels in our patient samples was 7108 (range 2340-10861). We found a statistically significant correlation between the level of TXNIP RNA and ROS (Fig. 1). TRX RNA levels did not significantly differ among the patients. We also measured TRX levels in the patient plasma and found a median level of 33 ng/ml (range: 18-46 ng/ml) not significantly correlating with TXNIP, TRX RNA levels or ROS levels (R2 = 0.48). Based on the correlation between TXNIP and ROS, 2 patients at either end of our spectrum (see Fig. 1, circled squares) were chosen for further analysis by targeted microarray. Patient (PT009) with the low TXNIP/ROS (currently undergoing treatment) was set as our baseline and the patient (PT005) with high TXNIP/ROS (observation only) was used as test comparison. The microarray has 96 genes involved in cellular stress response pathways. Comparing PT005 to PT009 we found of the 96 genes, 15 genes were up-regulated greater than 2-fold and 21 genes were down-regulated. Among the up-regulated genes were superoxide dismutase (SOD1), FOXO1 transcription factor, catalase (CAT) and members of the catenin family of proteins. Down-regulated genes included BAX and BCL2L1, GSK3-B, and thioredoxin reductase. In conclusion, we measured the expression of TXNIP in CLL patient samples. We found a range of TXNIP expression that robustly correlates with the level of ROS production, and is associated with differences in the stress response signature the clinical or biological relevance of which deserves further investigation. Disclosures: Turturro: Celgene: Speakers Bureau; Genentech: Speakers Bureau; Merck & Co: Research Funding.
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Hall, Stephanie Jane. "'Quick Reads' May Promote Literacy without Stigma: Findings from Eight UK Public Libraries". Evidence Based Library and Information Practice 1, n.º 2 (5 de junho de 2006): 36. http://dx.doi.org/10.18438/b8d59m.
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A review of:
McLoughlin, Carla, and Anne Morris. "UK Public Libraries: Roles in Adult Literacy Provision." Journal of Librarianship and Information Science 36.1 (March 2004): 37-46.
Objective – To examine the role of public libraries in the provision of adult literacy services, with a detailed look at both the successes and concerns of the libraries under study; to provide recommendations for best practice in establishing or reviewing adult literacy services.
Design – A series of case studies using written reports and semi-structured interviews.
Setting – Eight public libraries in the UK involved in literacy service provision or reader development services.
Subjects – Eight senior staff members in charge of library literacy programming.
Method – A written report of literacy service initiatives was solicited from each participating library. A single interview was conducted with a staff member in charge of literacy service at each of the eight participating libraries. Fact-checking telephone interviews were conducted at three locations where adult literacy programs were in early stages. More in-depth, face-to-face interviews were conducted at the five libraries with better established programs. Each type of interview consisted of a set of scripted questions supplemented by individualized questions based on the written reports.
Main results – There are four key areas of results to be summarized from this study:
Adult Literacy Collections – The authors observed three main approaches to branding literacy collections:
?Emphasis on reading for pleasure (with collections entitled ‘Quick Reads’ or ‘First Choice’);
?Emphasis on reading for skills development;
?Discreet labelling enabling stock recognition without advertising that the reader is borrowing literacy materials.
The authors conclude that the ‘Quick Reads’ approach was the most successful in highlighting the collection without stigmatizing it and in promoting the pleasure of reading. The importance of maintaining relevant, attractive books was highlighted, with collections targeting both entry level readers and emergent readers.
Approaches for Supporting Adult Literacy – The libraries used reader development extensively as a strategy to support adult literacy efforts. Staff tied literacy offerings to other programs or services of interest (for example, promoting adult literacy services alongside audio-visual collections and Internet access). Adult learners were also targeted for library tours, reading groups, and assistance with book selection for the literacy collection. Some of the libraries hired new staff from outside the library profession, choosing candidates with prior experience in basic skills development or community work.
Methods of Attracting Adults with Poor Literacy -- Partnership was identified as a key strategy for the libraries studied. Partnerships were formed with numerous agencies, including the probationary service, a community centre (where the library’s ‘reader in residence’ was installed), a college, and a Peugeot factory. Networking with other literacy service providers and coalitions was also an important strategy, particularly as a way to increase the library’s profile as a literacy service provider. Perhaps the simplest strategy for attracting adults with poor literacy was to identify areas of the library districts where literacy skills were lowest and then to target literacy service to those regions.
Sustainability and Mainstreaming -- Early planning for sustainability was crucial. Incorporating funding for literacy staffing and collections into the core budget and annual library plan was also an important step. While some libraries hired new staff, and one library staffed the literacy project with volunteers, using existing staff for adult literacy work proved to be more efficient and sustainable. Instilling a sense of ownership in the project for both staff and users of the literacy services by involving them in the development and promotion of literacy service and collections was another strategy employed to ensure longevity of the service.
Conclusions – The most successful form of library literacy service provision was found to be the reader development approach (promoting reading for enjoyment and building reading activities around existing interests). The libraries studied showed an understanding of the wide range of reading levels and interests among adult learners. Potential barriers for libraries in the provision of adult literacy service “include restrictive funding criteria, limited staff capacity, and a bidding culture that remains unsympathetic to public library circumstances” (44). The authors make five recommendations for best practices in adult literacy service provision:
Eclectic adult literacy collections: Collections should be fresh and appealing and should incorporate engaging non-fiction.
Standardized criteria for adult literacy stock: Standardized criteria should be developed by a basic skills agency, preferably at a national level.
Equality for adult readers: Approach adult readers as people who read for enjoyment or who are ‘getting back to reading’, rather than as those needing to ‘improve’ their reading.
Maximum access: Ensure a diverse and well-stocked collection of books that is easy for adult learners to locate.
Community profiling: Optimize service delivery by profiling your community’s literacy levels.
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Pakulska, Urszula, Marta Obacz, Kristina Goller, Michal Combik, Kinga Keska, Jan Zaucha, Ewa Zarzycka et al. "RVU120 (SEL120) CDK8/19 Inhibitor - a Drug Candidate for the Treatment of MDS Can Induce Erythroid Differentiation". Blood 138, Supplement 1 (5 de novembro de 2021): 1518. http://dx.doi.org/10.1182/blood-2021-150662.
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Abstract Background: Myelodysplastic syndromes (MDS) are a group of malignant blood disorders characterized by ineffective hematopoiesis and cytopenias and frequent evolution to acute myeloid leukemia (AML). MDS results from the expansion of genetically and epigenetically changed clones with impaired differentiation and maturation. Primary clinical goals in MDS are to achieve remissions, alleviate symptoms associated with cytopenias, and minimize the transfusion burden. While supportive red blood cell transfusions, erythropoiesis-stimulating agents and novel targeted agents may lead to clinical improvement, an allogeneic bone marrow transplant (BMT) remains the only potential curative option for patients with MDS. RVU120 (SEL120) is a specific, selective inhibitor of CDK8 and its paralog CDK19. A first-in-human Phase Ib clinical trial with RVU120 in patients with AML or high risk (HR)-MDS is currently ongoing. Preclinical studies indicated the strong antileukemic potential of RVU120 that was often associated with the multilineage commitment of CD34+ AML cells. Moreover, RVU120 could improve proliferation and induce erythroid differentiation of CD34+ cells derived from Diamond-Blackfan anemia (DBA) patients. Aims: Primary aim was to evaluate the erythroid differentiation potential of RVU120 in primary MDS and transformed cord blood CD34+ blood cells characterized with an early block in erythroid differentiation. Methods: Efficacy and mechanism of action of RVU120 and other CDK8 inhibitors were investigated in cord blood (CB) cells transduced with TLS-ERG, a fusion gene generated from t(16;21)(p11;q22). Transformed cells displayed an increased capacity for self-renewal, proliferation, and altered erythroid differentiation. Efficacy was further tested in CD34+ cells isolated from BM of MDS patients and established MDS cell lines. Cells were treated with RVU120 and global transcriptional changes and chromatin status were analyzed by RNA-seq, ATAC-seq, and ChIP-seq. Cell cycle, proliferation, and lineage-specific markers were studied by flow cytometry in liquid and semi-solid methylcellulose-based media. Results: RVU120 treatment leads to transcriptional reprogramming of transformed CD34+ CB cells. The most profound changes included decreased CDK8 occupancy followed by increased STAT5 and RNA Polymerase II loading at transcription start site and gene bodies. RVU120 treatment transcriptionally represses multiple genes associated with hematopoietic and leukemia stem cells such as CD34, FLI1, ENG and RGS18 and importantly induce the expression of genes involved in erythroid commitment, including regulators of erythroid/megakaryocytic fate, such as RGS18, KLF1, FLI1, INHBA, GATA1/2 and hemoglobin genes. Detailed analysis of transformed CB and MDS CD34+ cells by flow cytometry at early and late time points reflected sequential changes in the expression of lineage-specific surface markers, leading to erythroid differentiation. Conclusions: Presented results indicate strong erythroid differentiation potential of RVU120 in CD34+ cells that acquired genetic abnormalities leading to arrested erythroid commitment, characteristics of many MDS and AML subtypes. Observed differentiation phenotype strikingly resembles effects of RVU120 in DBA cells caused by disruption of genes encoding ribosomal proteins. Detailed transcriptomic profiling strongly links the differentiation with enrichment of genes representing regulators of erythroid commitment and hemoglobin metabolism. Further studies are warranted to investigate the efficacy of RVU120 in chronic anemias associated with bone marrow failures in AML and MDS patients. Figure 1 Figure 1. Disclosures Pakulska: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Obacz: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Goller: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Combik: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Keska: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mazan: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Juszczynski: Ryvu Therapeutics: Current equity holder in publicly-traded company. Brzozka: Ardigen: Current Employment, Membership on an entity's Board of Directors or advisory committees; Selvita SA: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Dziedzic: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Angelosanto: Ryvu Therapeutics: Current Employment. Shamsili: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Rzymski: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company.
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Kerzeli, Iliana K., Martin Lord, Milena Doroszko, Ramy Elgendy, Aikaterini Chourlia, Ivan Stepanek, Elinor Larsson et al. "Single-cell RNAseq and longitudinal proteomic analysis of a novel semi-spontaneous urothelial cancer model reveals tumor cell heterogeneity and pretumoral urine protein alterations". PLOS ONE 16, n.º 7 (7 de julho de 2021): e0253178. http://dx.doi.org/10.1371/journal.pone.0253178.
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Bladder cancer, one of the most prevalent malignancies worldwide, remains hard to classify due to a staggering molecular complexity. Despite a plethora of diagnostic tools and therapies, it is hard to outline the key steps leading up to the transition from high-risk non–muscle-invasive bladder cancer (NMIBC) to muscle-invasive bladder cancer (MIBC). Carcinogen-induced murine models can recapitulate urothelial carcinogenesis and natural anti-tumor immunity. Herein, we have developed and profiled a novel model of progressive NMIBC based on 10 weeks of OH-BBN exposure in hepatocyte growth factor/cyclin dependent kinase 4 (R24C) (Hgf-Cdk4R24C) mice. The profiling of the model was performed by histology grading, single cell transcriptomic and proteomic analysis, while the derivation of a tumorigenic cell line was validated and used to assess in vivo anti-tumor effects in response to immunotherapy. Established NMIBC was present in females at 10 weeks post OH-BBN exposure while neoplasia was not as advanced in male mice, however all mice progressed to MIBC. Single cell RNA sequencing analysis revealed an intratumoral heterogeneity also described in the human disease trajectory. Moreover, although immune activation biomarkers were elevated in urine during carcinogen exposure, anti-programmed cell death protein 1 (anti-PD1) monotherapy did not prevent tumor progression. Furthermore, anti-PD1 immunotherapy did not control the growth of subcutaneous tumors formed by the newly derived urothelial cancer cell line. However, treatment with CpG-oligodeoxynucleotides (ODN) significantly decreased tumor volume, but only in females. In conclusion, the molecular map of this novel preclinical model of bladder cancer provides an opportunity to further investigate pharmacological therapies ahead with regards to both targeted drugs and immunotherapies to improve the strategies of how we should tackle the heterogeneous tumor microenvironment in urothelial bladder cancer to improve responses rates in the clinic.
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Astone, Giuseppina, Luca Vincenzo Cappelli, William Chiu, Clarisse Kayembe, Rui Wang, Bin Yang, Kirti Sharma et al. "Selective STAT3 Degraders Dissect Peripheral T-Cell Lymphomas Vulnerabilities Empowering Personalized Regimens". Blood 138, Supplement 1 (5 de novembro de 2021): 865. http://dx.doi.org/10.1182/blood-2021-153995.
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Abstract Introduction: Peripheral T-cell lymphomas (PTCLs) include heterogeneous entities of rare and aggressive neoplasms. The improved understanding of the biological/molecular mechanisms driving T-cell transformation and tumor maintenance has powerfully propelled new therapeutic programs. However, despite this progress, PTCLs remain an unmet medical need. Recurrent aberrations and the deregulated activation of distinct signaling pathways have been mapped and linked to selective subtypes. The JAK/STAT signaling pathway's deregulated activation plays a pathogenetic role in PTCL, including ALCL subtypes. STATs regulate the differentiation/phenotype, survival and cell-growth, metabolism, and drug resistance of T-cell lymphomas as well as host immunosuppressive microenvironments. Although many drugs' discovery programs were launched, a plethora of compounds has failed. Methods: We have discovered heterobifunctional molecules by an iterative medicinal chemistry SAR campaign that potently and selectively degrade STAT3 in a proteasome-dependent manner. Conventional PTCL cell lines and Patient Derived Tumor Xenograft (PDTX) and/or derived cell lines (PDTX-CL), carrying either WT- or mutated-STAT3, were exposed to increasing amounts (50nM⇒5µM) of STAT3-degraders. Proteins and mRNA transcripts (2⇒144hrs) were assessed by deep-proteomics and paired-end RNA sequencing, combined with WB/flow cytometry and qRT-PCR. Cell-titer-glo, cell titer blue, Annexin-V and S-cell cycle analyses were used as readouts. Chromatin accessibility, STAT3 DNA binding, 3D chromosomal architecture reorganization and 5-hmC profiling were assessed by ATACseq, CHIPseq and Hi-C and H3K27ac Hi-CHIP and mass-spectrometry. Drug testing/discovery combinations (96-well-plate) were performed using a semi-automated flow-cytometry. A battery of PTCL PDTX models were tested in pre-clinical trials. Results: Treatment of ALK+ ALCL (SU-DHL1) led to the rapid (~6hrs.) and profound down-regulation of STAT3 followed by the loss of canonical STAT3-regulated proteins (SOCS3, MYC, Granzyme B, GAS1, and IL2RA), without appreciable changes for other STAT family members (STAT1, STAT5b). In vitro, cytoplasmic, nuclear, and mitochondrial STAT3 downregulation was maintained up to 144 hrs. Loss of STAT3 in ALK+/- ALCL and BIA-ALCL cells was associated with major transcriptional changes (7116-10615 and 15114 DEGs in ALK- and ALK+ ALCL, respectively), underscoring public/shared as well as private time-dependent signatures. Main down-regulated pathways included JAK-STAT, MAPK, NF-kB, PI3K, TGFb, and TNFa. Comparison of STAT3 shRNA (ALK+ ALCL) and STAT3 degrader (ALK-/ALK+ ALCL) signatures demonstrated a substantial and concordant gene modulation (24hrs) among all models with the highest overlaps between ALK+ ALCL (Figure 3). To identify direct STAT3 gene targets, we analyzed CHIPseq peaks and predicted bindings sites, demonstrating that canonical genes, i.e., SOCS3, Granzyme B, GAS1, IL2RA, STAT3, and CD30, were significantly downregulated. Conversely, CD58, CD274, and MCH-I/II were upregulated at late time points. By mapping the STAT3 binding sites in ALK+ and ALK- ALCL, we have identified 1077 and 2763 STAT3 peaks within promoter/5'-/3'- and distant intergenic regions, corresponding to both coding and non-coding genes. Therapeutically, in vitro treatments led to cell cycle arrest and profound growth inhibition, and over time increased cell death of PTCL cells, including ALCL. Accordingly, growth inhibition of ALCL xenograft and PDTX tumors was also achieved (Figure 2). To identify drugs that could synergize withSTAT3-degrader activity, we tested a compound library (40) targeting pro-tumorigenic PTCL pathways as well as FDA-approved compounds. Ongoing studies are in progress. Conclusion: We have discovered selective STAT3 degraders which control PTCL growth. STAT3 degraders are powerful tools to define the STAT3 pathogenetic mechanisms and dissect genes/pathways to be targeted for T-cell lymphoma eradication. These data provide additional rationale for testing STAT3 degraders in the clinic for the treatment of aggressive malignancies including PTCL/ALCL. Figure 1 Figure 1. Disclosures Yang: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sharma: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Dey: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Karnik: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Elemento: Owkin: Consultancy, Other: Current equity holder; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Johnson and Johnson: Research Funding; Eli Lilly: Research Funding; Janssen: Research Funding; Champions Oncology: Consultancy; Freenome: Consultancy, Other: Current equity holder in a privately-held company; One Three Biotech: Consultancy, Other: Current equity holder; AstraZeneca: Research Funding. Horwitz: Affimed: Research Funding; Aileron: Research Funding; ADC Therapeutics, Affimed, Aileron, Celgene, Daiichi Sankyo, Forty Seven, Inc., Kyowa Hakko Kirin, Millennium /Takeda, Seattle Genetics, Trillium Therapeutics, and Verastem/SecuraBio.: Consultancy, Research Funding; Acrotech Biopharma, Affimed, ADC Therapeutics, Astex, Merck, Portola Pharma, C4 Therapeutics, Celgene, Janssen, Kura Oncology, Kyowa Hakko Kirin, Myeloid Therapeutics, ONO Pharmaceuticals, Seattle Genetics, Shoreline Biosciences, Inc, Takeda, Trillium Th: Consultancy; Celgene: Research Funding; C4 Therapeutics: Consultancy; Crispr Therapeutics: Research Funding; Daiichi Sankyo: Research Funding; Forty Seven, Inc.: Research Funding; Kura Oncology: Consultancy; Kyowa Hakko Kirin: Consultancy, Research Funding; Millennium/Takeda: Research Funding; Myeloid Therapeutics: Consultancy; ONO Pharmaceuticals: Consultancy; Seattle Genetics: Consultancy, Research Funding; Secura Bio: Consultancy; Shoreline Biosciences, Inc.: Consultancy; Takeda: Consultancy; Trillium Therapeutics: Consultancy, Research Funding; Tubulis: Consultancy; Verastem/Securabio: Research Funding. DeSavi: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Liu: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company.
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Trusov, Nikita V., Sergey A. Apryatin, Andrej N. Timonin, Vladimir A. Shipelin, Ivan V. Gmoshinski e Dmitriy B. Nikityuk. "Comparative evaluation of the effect of resveratrol and carnitine on the full transcriptomic profile of liver tissue in mice with different sensitivity to the development of alimentary obesity". Vestnik Tomskogo gosudarstvennogo universiteta. Biologiya, n.º 54 (2021): 83–115. http://dx.doi.org/10.17223/19988591/54/5.
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Specialized food products and biologically active food supplements enriched with minor biologically active substances are considered as a useful supplement in the treatment of obesity and other nutrition-dependent diseases. Biologically active substances of food can have a complex effect on the expression of a large number of genes, which can affect the results of a therapy. The aim of the study was to analyze the nutrigenomic mechanisms of the effect of biologically active substances - l-carnitine and resveratrol on the expression of liver genes of DBA/2J mice and DBCB tetrahybrid, differing in genotype and sensitivity to the development of diet-induced obesity, using the method of full transcriptomic profiling of liver tissue. We carried out the experiment on male DBA/2J mice and the hybrid of the 2nd generation DBCB, obtained by crossing 4 lines of mice (DBA/2J, BALB/c, CBA/ lac and С57Black/6J). Mice for the experiment were obtained from Stolbovaya nursery, Federal State Budgetory Scientific Institution Scientific Center of Biomedical Technologies of the Federal Medical-Biological Agency (Moscow region, Russia). We worked with animals in accordance with international recommendations (Directive 2010/63/EU on the protection of animals used for scientific purposes adopted on September 22, 2010; Guide for the care and use of laboratory animals. Eighth Edition / Committee for the Update of the Guide for the Care and Use of Laboratory Animals; Institute for Laboratory Animal Research (ILAR); Division on Earth and Life Studies (DELS); National Research Council of the National Academies. Washington: The National Academies Press. 2011).The mice were divided into four groups with an equal number of 8 individuals. During 65 days, animals of the 1st (control) groups received a balanced semi-synthetic diet and purified drinking water, the 2nd groups received a high-carbohydrate and high-fat diet with a high fat content (30% by of dry matter of the diet) and replacing drinking water by 20% fructose solution, 3rd groups - high-carbohydrate and high-fat diet with the addition of resveratrol at a dose of 25 mg/kg body weight, 4th groups - high-carbohydrate and high-fat diet with the addition of l-carnitine at a dose of 300 mg/kg body weight. Full transcriptome analysis was performed using the Gene Expression Hybridization Kit (Agilent Technologies, USA) on SurePrint G3 Mouse GE 8×60K Microarray Kit microarrays. Differential gene expression was expressed as base 2 logarithm of increasing or decreasing fluorescence (log2 FC) compared to control groups, separately for DBA/2J and DBCB mice. Chip scan data and calculation of differential expression values were exported to the “R” IDE and bioinformatics analysis was performed with quantile normalization and further analysis in the limma package. The packages AnnotationDbi, org.Rn.eg.db, pathview, gage, gageData were used to identify metabolic pathways among the genes, metabolic pathways and functions of biological systems presented in the international database Kyoto Encyclopedia of Genes and Genomes and to visualize them. To visualize the results at all stages, the standard “R” graphics and additional packages ggplot2, ggrepel, and gplots were used. Liver morphology was studied by light microscopy after staining with hematoxyline-eosine (See Fig. 1). We revealed differential expression for at least one of the intergroup comparisons in the amount of │log2FC│≥0.5 (towards both enhancement and attenuation) and at a p-value ≤ 0.05 for 415 transcripts, of which 311 were identified with proteins or RNA with a known function (See Tables 1-3). Consumption of a high-carbohydrate and high-fat diet was reflected in differential expression of 62 genes in DBA/2J mice and 97 in DBCB mice. In DBA/2J mice fed on a high-carbohydrate and high-fat diet, supplementation with resveratrol and l-carnitine caused a differential expression of 26 genes each. At the same time, only 2 genes (Pklr, Tkfc) responded to resveratrol and l-carnitine in mice of this strain. In DBCB tetrahybrid mice, resveratrol consumption corresponded to differential expression of 147 genes, and l-carnitine consumption corresponded to 221 genes. 61 genes from DBCB mice responded to both supplements, and the number of genes simultaneously targeted by high-carbohydrate and high-fat diets, resveratrol and l-carnitine was 10 (See Fig. 2). The gene expression profiles in DBA/2J and DBCB mice formed two separate clusters, the differences within which, determined by the composition of the diets, were less significant than the interstrain differences (See Fig. 3). Differential expression values in DBCB and DBA mice responding to HFCD and both supplements correlated negatively (See Fig. 4). The consumption of a high-carbohydrate and high-fat diet in DBA/2J mice resulted in significant changes in 4 metabolic pathways, and in DBCB mice, in addition, in 5 more metabolic pathways. Resveratrol consumption did not cause significant changes in DBA/2J mice, and in tetrahybrid mice it affected mmu04512 ECM-receptor interaction. L-carnitine supplementation caused significant changes in mmu00830 Retinol metabolism only in DBCB mice (See Table 4). Consumption of a high-carbohydrate and high-fat diet produced similar changes in the mmu00830 Retinol metabolism pathway in both mice (See Fig. 5). In metabolic pathway mmu03320 PPAR signaling pathway DBA/2J and DBCB mice showed positive differential expression of the PPARγ gene and negative Scd1. At the same time, only DBCB mice in this metabolic pathway are characterized by activation of the RXR gene expression and suppression of FABP, and the direction in changing Cyp4a1 in both mice is opposite (See Fig. 6). Changes in the metabolic pathway mmu00590 Arachidonic acid metabolism characterized by the imbalance in the expression of Cyp4a and Cyp2 isoforms, which are responsible for the synthesis of various hydroxy and epoxy derivatives of arachidonic acid, is characteristic only of DBCB mice (See Fig. 7). Thus, the experiments performed revealed both a certain similarity and differences in the response of the transcriptome of DBA/2J and DBCB mice to the consumption of a high-carbohydrate and high-fat diet, resveratrol and l-carnitine. The mechanisms that determine the direction of changes induced in the transcriptome of mice (and in coupled phenotypic changes) are, apparently, in the intervention of the studied dietary factors in key metabolic pathways, such as the PPAR signaling pathway, the metabolism of retinoids and eicosanoids. The data obtained indicate the importance of an adequate choice of a in vivo model of obesity and metabolic syndrome in preclinical studies of biologically active substances, in diet therapy and the enrichment of specialized food products with them.
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Wang, Jingtao, Gregory J. Fonseca e Jun Ding. "scSemiProfiler: Advancing large-scale single-cell studies through semi-profiling with deep generative models and active learning". Nature Communications 15, n.º 1 (16 de julho de 2024). http://dx.doi.org/10.1038/s41467-024-50150-1.
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AbstractSingle-cell sequencing is a crucial tool for dissecting the cellular intricacies of complex diseases. Its prohibitive cost, however, hampers its application in expansive biomedical studies. Traditional cellular deconvolution approaches can infer cell type proportions from more affordable bulk sequencing data, yet they fall short in providing the detailed resolution required for single-cell-level analyses. To overcome this challenge, we introduce “scSemiProfiler”, an innovative computational framework that marries deep generative models with active learning strategies. This method adeptly infers single-cell profiles across large cohorts by fusing bulk sequencing data with targeted single-cell sequencing from a few rigorously chosen representatives. Extensive validation across heterogeneous datasets verifies the precision of our semi-profiling approach, aligning closely with true single-cell profiling data and empowering refined cellular analyses. Originally developed for extensive disease cohorts, “scSemiProfiler” is adaptable for broad applications. It provides a scalable, cost-effective solution for single-cell profiling, facilitating in-depth cellular investigation in various biological domains.
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Tais, Leslie, Hartwig Schulz e Christoph Böttcher. "Comprehensive profiling of semi‐polar phytochemicals in whole wheat grains (Triticum aestivum) using liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight mass spectrometry". Metabolomics 17, n.º 2 (27 de janeiro de 2021). http://dx.doi.org/10.1007/s11306-020-01761-4.
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Abstract Introduction Wheat (Triticum aestivum) it is one of the most important staple food crops worldwide and represents an important resource for human nutrition. Besides starch, proteins and micronutrients wheat grains accumulate a highly diverse set of phytochemicals. Objectives This work aimed at the development and validation of an analytical workflow for comprehensive profiling of semi-polar phytochemicals in whole wheat grains. Method Reversed-phase ultra-high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC/ESI-QTOFMS) was used as analytical platform. For annotation of metabolites accurate mass collision-induced dissociation mass spectra were acquired and interpreted in conjunction with literature data, database queries and analyses of reference compounds. Results Based on reversed-phase UHPLC/ESI-QTOFMS an analytical workflow for comprehensive profiling of semi-polar phytochemicals in whole wheat grains was developed. For method development the extraction procedure and the chromatographic separation were optimized. Using whole grains of eight wheat cultivars a total of 248 metabolites were annotated and characterized by chromatographic and tandem mass spectral data. Annotated metabolites comprise hydroquinones, hydroxycinnamic acid amides, flavonoids, benzoxazinoids, lignans and other phenolics as well as numerous primary metabolites such as nucleosides, amino acids and derivatives, organic acids, saccharides and B vitamin derivatives. For method validation, recovery rates and matrix effects were determined for ten exogenous model compounds. Repeatability and linearity were assessed for 39 representative endogenous metabolites. In addition, the accuracy of relative quantification was evaluated for six exogenous model compounds. Conclusions In conjunction with non-targeted and targeted data analysis strategies the developed analytical workflow was successfully applied to discern differences in the profiles of semi-polar phytochemicals accumulating in whole grains of eight wheat cultivars.
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Medana, Claudio, Umile Gianfranco Spizzirri, Valentina Schiavo, Fabio Fusi, Alice Panti, Simona Saponara, Paola Marcolongo et al. "Screening of the antioxidant and vasorelaxant activity of wine waste ultrasonic extracts, and HRMS targeted and semi-targeted profiling of glycosylated polyphenols vs free polyphenols". LWT, agosto de 2024, 116666. http://dx.doi.org/10.1016/j.lwt.2024.116666.
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Suriyanarayanan, Tanujaa, Lye Siang Lee, Sharon Hong Yu Han, Jianhong Ching e Chaminda J. Seneviratne. "Targeted metabolomics analysis approach to unravel the biofilm formation pathways of Enterococcus faecalis clinical isolates". International Endodontic Journal, 18 de junho de 2024. http://dx.doi.org/10.1111/iej.14110.
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AbstractAim(i) To characterize Enterococcus faecalis biofilm formation pathways by semi‐targeted metabolomics and targeted nitrogen panel analysis of strong (Ef63) and weak (Ef 64) biofilm forming E. faecalis clinical isolates and (ii) to validate the identified metabolic markers using targeted inhibitors.MethodologyPrevious proteomics profiling of E. faecalis clinical isolates with strong and weak biofilm formation revealed that differences in metabolic activity levels of small molecule, nucleotide and nitrogen compound metabolic processes and biosynthetic pathways, cofactor metabolic process, cellular amino acid and derivative metabolic process and lyase activity were associated with differences in biofilm formation. Hence, semi‐targeted analysis of Ef 63, Ef 64 and ATC control strain Ef 29212 was performed by selecting metabolites that were part of both the previously identified pathways and a curated library with confirmed physical and chemical identity, followed by confirmatory targeted nitrogen panel analysis. Significantly regulated metabolites (p < .05) were selected based on fold change cut‐offs of 1.2 and 0.8 for upregulation and downregulation, respectively, and subjected to pathway enrichment analysis. The identified metabolites and pathways were validated by minimum biofilm inhibitory concentration (MBIC) and colony forming unit (CFU) assays with targeted inhibitors.ResultsMetabolomics analysis showed upregulation of betaine, hypoxanthine, glycerophosphorylcholine, tyrosine, inosine, allantoin and citrulline in Ef 63 w.r.t Ef 64 and Ef 29212, and thesemetabolites mapped to purinemetabolism, urea cycle and aspartate metabolism pathways. MBIC and CFU assays using compounds against selected metabolites and metabolic pathways, namely glutathione against hypoxanthine and hydroxylamine against aspartate metabolism showed inhibitory effects against E. faecalis biofilm formation.ConclusionsThe study demonstrated the importance of oxidative stress inducers such as hypoxanthine and aspartate metabolism pathway in E. faecalis biofilm formation. Targeted therapeutics against these metabolic markers can reduce the healthcare burden associated with E. faecalis infections.
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Zamani, Arief Izzairy, Susann Barig, Sarah Ibrahim, Hirzun Mohd. Yusof, Julia Ibrahim, Jaime Yoke Sum Low, Shwu Fun Kua, Syarul Nataqain Baharum, Klaus-Peter Stahmann e Chyan Leong Ng. "Comparative metabolomics of Phialemonium curvatum as an omnipotent fungus cultivated on crude palm oil versus glucose". Microbial Cell Factories 19, n.º 1 (9 de setembro de 2020). http://dx.doi.org/10.1186/s12934-020-01434-w.
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Abstract Background Sugars and triglycerides are common carbon sources for microorganisms. Nonetheless, a systematic comparative interpretation of metabolic changes upon vegetable oil or glucose as sole carbon source is still lacking. Selected fungi that can grow in acidic mineral salt media (MSM) with vegetable oil had been identified recently. Hence, this study aimed to investigate the overall metabolite changes of an omnipotent fungus and to reveal changes at central carbon metabolism corresponding to both carbon sources. Results Targeted and non-targeted metabolomics for both polar and semi-polar metabolites of Phialemonium curvatum AWO2 (DSM 23903) cultivated in MSM with palm oil (MSM-P) or glucose (MSM-G) as carbon sources were obtained. Targeted metabolomics on central carbon metabolism of tricarboxylic acid (TCA) cycle and glyoxylate cycle were analysed using LC–MS/MS-TripleQ and GC–MS, while untargeted metabolite profiling was performed using LC–MS/MS-QTOF followed by multivariate analysis. Targeted metabolomics analysis showed that glyoxylate pathway and TCA cycle were recruited at central carbon metabolism for triglyceride and glucose catabolism, respectively. Significant differences in organic acids concentration of about 4- to 8-fold were observed for citric acid, succinic acid, malic acid, and oxaloacetic acid. Correlation of organic acids concentration and key enzymes involved in the central carbon metabolism was further determined by enzymatic assays. On the other hand, the untargeted profiling revealed seven metabolites undergoing significant changes between MSM-P and MSM-G cultures. Conclusions Overall, this study has provided insights on the understanding on the effect of triglycerides and sugar as carbon source in fungi global metabolic pathway, which might become important for future optimization of carbon flux engineering in fungi to improve organic acids production when vegetable oil is applied as the sole carbon source.
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Villette, Claire, Julie Zumsteg, Hubert Schaller e Dimitri Heintz. "Non-targeted metabolic profiling of BW312 Hordeum vulgare semi dwarf mutant using UHPLC coupled to QTOF high resolution mass spectrometry". Scientific Reports 8, n.º 1 (4 de setembro de 2018). http://dx.doi.org/10.1038/s41598-018-31593-1.
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Chang, Jing Kai, Guoshou Teo, Yael Pewzner-Jung, Daniel J. Cuthbertson, Anthony H. Futerman, Markus R. Wenk, Hyungwon Choi e Federico Torta. "Q-RAI data-independent acquisition for lipidomic quantitative profiling". Scientific Reports 13, n.º 1 (7 de novembro de 2023). http://dx.doi.org/10.1038/s41598-023-46312-8.
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AbstractUntargeted lipidomics has been increasingly adopted for hypothesis generation in a biological context or discovery of disease biomarkers. Most of the current liquid chromatography mass spectrometry (LC–MS) based untargeted methodologies utilize a data dependent acquisition (DDA) approach in pooled samples for identification and MS-only acquisition for semi-quantification in individual samples. In this study, we present for the first time an untargeted lipidomic workflow that makes use of the newly implemented Quadrupole Resolved All-Ions (Q-RAI) acquisition function on the Agilent 6546 quadrupole time-of-flight (Q-TOF) mass spectrometer to acquire MS2 spectra in data independent acquisition (DIA) mode. This is followed by data processing and analysis on MetaboKit, a software enabling DDA-based spectral library construction and extraction of MS1 and MS2 peak areas, for reproducible identification and quantification of lipids in DIA analysis. This workflow was tested on lipid extracts from human plasma and showed quantification at MS1 and MS2 levels comparable to multiple reaction monitoring (MRM) targeted analysis of the same samples. Analysis of serum from Ceramide Synthase 2 (CerS2) null mice using the Q-RAI DIA workflow identified 88 lipid species significantly different between CerS2 null and wild type mice, including well-characterized changes previously associated with this phenotype. Our results show the Q-RAI DIA as a reliable option to perform simultaneous identification and reproducible relative quantification of lipids in exploratory biological studies.
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Andrew-Peter-Leon, M. T., Ramchander Selvaraj, K. K. Kumar, Mehanathan Muthamilarasan, Jeshima Khan Yasin e M. Arumugam Pillai. "Loss of Function of OsFBX267 and OsGA20ox2 in Rice Promotes Early Maturing and Semi-Dwarfism in γ-Irradiated IWP and Genome-Edited Pusa Basmati-1". Frontiers in Plant Science 12 (22 de setembro de 2021). http://dx.doi.org/10.3389/fpls.2021.714066.
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Targeted mutagenesis is now becoming the most favored methodology to improve traits in popular rice cultivars selectively. Understanding the genetic basis of already available mutants could be the first step in designing such experiment. Improved White Ponni (IWP), a popularly grown South Indian rice variety, was subjected to γ irradiation to develop WP-22-2, an M6 line superior in semi-dwarfism, early flowering, and high yield, and it has grain qualities similar to those of IWP. The exogenous application of gibberellic acid (GA3) on WP-22-2 resulted in the elongation of shorter internodes to a level similar to IWP. The expression profiling of six genes regulating plant height showed their differential expression pattern at different time points post GA3 treatment. Furthermore, the sequencing of WP-22-2 and IWP genomes revealed several single nucleotide polymorphisms (SNPs) and large-scale deletions in WP-22-2. The conversion of functional codons to stop codons was observed in OsGA20ox2 and OsFBX267, which have been reported to have roles in regulating semi-dwarfism and early flowering, respectively. The loss of function of OsGA20ox2 and OsFBX267 in WP-22-2 resulted in reduced plant height as well as early flowering, and the same has been confirmed by editing OsGA20ox2 in the rice variety Pusa Basmati1 (PB1) using the CRISPR-Cas9 approach. The targeted editing of OsGA20ox2 in PB1 conferred shorter plant height to the edited lines compared with the wild type. Altogether, the study provides evidence on mutating OsGA20ox2 and OsFBX267 genes to develop early maturing and semi-dwarf varieties that can be released to farmers after functional characterization and field trials.
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Gutiérrez-Martín, Daniel, Esteban Restrepo-Montes, Oksana Golovko, Rebeca López-Serna, Reza Aalizadeh, Nikolaos S. Thomaidis, Montse Marquès, Pablo Gago-Ferrero e Rubén Gil-Solsona. "Comprehensive profiling and semi-quantification of exogenous chemicals in human urine using HRMS-based strategies". Analytical and Bioanalytical Chemistry, 9 de novembro de 2023. http://dx.doi.org/10.1007/s00216-023-04998-9.
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AbstractChemicals infiltrate our daily experiences through multiple exposure pathways. Human biomonitoring (HBM) is routinely used to comprehensively understand these chemical interactions. Historically, HBM depended on targeted screening methods limited to a relatively small set of chemicals with triple quadrupole instruments typically. However, recent advances in high-resolution mass spectrometry (HRMS) have facilitated the use of broad-scope target, suspect, and non-target strategies, enhancing chemical exposome characterization within acceptable detection limits. Despite these advancements, establishing robust and efficient sample treatment protocols is still essential for trustworthy broad-range chemical analysis. This study sought to validate a methodology leveraging HRMS-based strategies for accurate profiling of exogenous chemicals and related metabolites in urine samples. We evaluated five extraction protocols, each encompassing various chemical classes, such as pharmaceuticals, plastic additives, personal care products, and pesticides, in terms of their extraction recoveries, linearity, matrix effect, sensitivity, and reproducibility. The most effective protocol was extensively validated and subsequently applied to 10 real human urine samples using wide-scope target analysis encompassing over 2000 chemicals. We successfully identified and semi-quantified a total of 36 chemicals using an ionization efficiency-based model, affirming the methodology’s robust performance. Notably, our results dismissed the need for a deconjugation step, a typically labor-intensive and time-consuming process. Graphical Abstract