Metabolized Research Articles

The conversion of monolignans to sesquilignans and dilignans is closely correlated to the regulation of Arctium lappa seed germination.

The secondary metabolic conversion of monolignans to sesquilignans/dilignans was closely related to seed germination and seedling establishment in Arctium lappa. Arctium lappa plants are used as a kind of traditional Chinese medicines for nearly 1500years, and so far, only a few studies have put focus on the key secondary metabolic changes during seed germination and seedling establishment. In the current study, a combined approach was used to investigate the correlation among secondary metabolites, plant hormone signaling, and transcriptional profiles at the early critical stages of A. lappa seed germination and seedling establishment. Of 50 metabolites in methonolic extracts of A. lappa samples, 35 metabolites were identified with LC-MS/MS and 15 metabolites were identified with GC-MS. Their qualitative properties were examined according to the predicted chemical structures. The quantitative analysis was performed for deciphering their metabolic profiles, discovering that the secondary metabolic conversion from monolignans to sesquilignans/dilignans was closely correlated to the initiation of A. lappa seed germination and seedling establishment. Furthermore, the critical transcriptional changes in primary metabolisms, translational regulation at different cellular compartments, and multiple plant hormone signaling pathways were revealed. In addition, the combined approach provides unprecedented insights into key regulatory mechanisms in both gene transcription and secondary metabolites besides many known primary metabolites during seed germination of an important traditional Chinese medicinal plant species. The results not only provide new insights to understand the regulation of key medicinal components of 'ARCTII FRUCTUS', arctiin and arctigenin at the stages of seed germination and seedling establishment, but also potentially spur the development of seed-based cultivation in A. lappa plants.

Protective Mechanisms of SGLTi in Ischemic Heart Disease.

Ischemic heart disease (IHD) is a common clinical cardiovascular disease with high morbidity and mortality. Sodium glucose cotransporter protein inhibitor (SGLTi) is a novel hypoglycemic drug. To date, both clinical trials and animal experiments have shown that SGLTi play a protective role in IHD, including myocardial infarction (MI) and ischemia/reperfusion (I/R). The protective effects may be involved in mechanisms of energy metabolic conversion, anti-inflammation, anti-fibrosis, ionic homeostasis improvement, immune cell development, angiogenesis and functional regulation, gut microbiota regulation, and epicardial lipids. Thus, this review summarizes the above mechanisms and aims to provide theoretical evidence for therapeutic strategies for IHD.

Effects of age on lacrimal gland bioactive lipids

PurposePolyunsaturated fatty acids (PUFA) are a source of bioactive lipids regulating inflammation and its resolution. MethodsChanges in PUFA metabolism were compared between lacrimal glands (LGs) from young and aged C57BL/6 J mice using a targeted lipidomics assay, as was the gene expression of enzymes involved in the metabolism of these lipids. ResultsGlobal reduction in PUFAs and their metabolites was observed in aged LGs compared to young controls, averaging between 25 and 66 % across all analytes. ꞷ-6 arachidonic acid (AA) metabolites were all reduced in aged LGs, where the changes in prostaglandin E2 (PGE2) and lipoxin A4 (LXA4) were statistically significant. Several other 5-lipoxygenase (5-LOX) mediated metabolites were significantly reduced in the aged LGs, including D-series resolvins (e.g., RvD4, RvD5, and RvD6). Along with the RvDs, several ꞷ-3 docosahexaenoic acid (DHA) metabolites such as 14-HDHA, neuroprotectin D1 (NPD1), Maresin 2 (MaR2), and MaR 1 metabolite (22-COOH-MaR1) were significantly reduced in aged LGs. Similarly, ꞷ-3 eicosapentaenoic acid (EPA) and its metabolites were significantly reduced in aged LGs, where the most significantly reduced was 18-HEPE. Using metabolite ratios (product:precursor) for specific metabolic conversions as surrogate enzymatic measures, reduced 12-LOX activity was identified in aged LGs. ConclusionIn this study, global reduction of PUFAs and their metabolites was found in the LGs of aged female C57BL/6 J compared to young controls. A consistent reduction was observed across all detected lipid analytes except for ꞷ-3 docosapentaenoic acid (DPA) and its special pro-resolving mediator (SPM) metabolites in aged mice, suggesting an increased risk for LG inflammation.

Metabolism of 11'-α- and 11'-γ-Tocomonoenols in HepG2 Cells Favors the γ-Congener and Results Predominantly in Carboxymethylbutyl-Hydroxychromans.

Tocomonoenols (T1) are little-known vitamin E derivatives naturally occurring in foods. Limited knowledge exists regarding the cellular uptake and metabolism of α-tocomonoenol (αT1) and none about that of γ-tocomonoenol (γT1). The study investigates the cytotoxicity, uptake, and metabolism of αT1 and γT1 in HepG2 cells compared to the α- and γ-tocopherols (T) and -tocotrienols (T3). None of the studied tocochromanols are cytotoxic up to 100µmolL-1. The uptake of the γ-congeners is significantly higher than that of the corresponding α-forms, whereas no significant differences are observed based on the degree of saturation of the sidechain. Carboxymethylbutyl-hydroxychromans (CMBHC) are the predominant short-chain metabolites of all tocochromanols and conversion is higher for γT1 than αT1 as well as for the γ-congeners of T and T3. The rate of metabolism increases with the number of double bonds in the sidechain. The rate of metabolic conversion of the T1 is more similar to tocopherols than to that of the tocotrienols. This is the first evidence that both αT1 and γT1 follow the same sidechain degradation pathway and exert similar rates of metabolism than tocopherols. Therefore, investigation into the biological activities of tocomonoenols is warranted.

MK591 (Quiflapon), a 5-lipoxygenase inhibitor, kills pancreatic cancer cells via downregulation of protein kinase C-epsilon.

Pancreatic tumors and cell lines derived from them exhibit elevated expression of 5-lipoxygenase (5-Lox), whereas non-tumor glands or normal cells do not exhibit this overexpression. Arachidonic acid stimulates pancreatic cancer cell growth via metabolic conversion through the 5-Lox pathway, and inhibition of 5-Lox activity decreases the viability of pancreatic cancer cells. However, the downstream signaling mechanisms through which 5-Lox exerts its effects on the survival of pancreatic cancer cells remain to be elucidated. The effects of 5-Lox inhibition on cell proliferation, apoptosis, and invasive potential were investigated in pancreatic cancer cells. The protein expression was analyzed by Western blot. Apoptosis was analyzed by Annexin-V binding assay and by detecting the degradation of chromatin-DNA to nucleosomal fragments. The protein kinase C-epsilon (PKCε) activity was measured by an immunoprecipitation-kinase assay. The in vivo effects of MK591 were evaluated in pancreatic tumor xenograft model. MK591, a specific inhibitor of 5-Lox activity, killed pancreatic cancer cells via induction of apoptosis, involving externalization of phosphatidylserine, cleavage of PARP (poly-ADP ribose polymerase) and degradation of chromatin DNA to nucleosomes. MK591 effectively blocked in vitro invasion and soft-agar colony formation by pancreatic cancer cells and decreased pancreatic tumor growth in nude mice xenografts. Furthermore, inhibition of 5-Lox downregulated K-Ras and inhibited phosphorylation of c-Raf and ERKs. Interestingly, 5-Lox inhibition induced apoptosis in pancreatic cancer cells without the inhibition of Akt but the protein level of PKCε was dramatically downregulated. Furthermore, inhibition of 5-Lox decreased the phosphorylation of Stat3 at Serine-727. Pre-treatment of pancreatic cancer cells with peptide activators of PKCε prevented apoptosis induced by 5-Lox inhibition, suggesting that the mechanism by which 5-Lox inhibition causes cell death in pancreatic cancer involves downregulation of PKCε. The combination of low doses of MK591 and gemcitabine synergistically reduced the oncogenic phenotype and killed pancreatic cancer cells by inducing apoptosis. These findings indicate that inhibition of 5-Lox interrupts an Akt-independent, PKCε-dependent survival mechanism in pancreatic cancer cells and suggest that metabolism of arachidonic acid through the 5-Lox pathway plays an integral part in the survival of pancreatic cancer cells via signaling through PKCε, an oncogenic, pro-survival serine/threonine kinase.

Study on the regulatory mechanism of vinegar grains metabolism via different vinegar starters

The application of biofortification technology in regulating the microbial community of Sichuan bran vinegar (SBV) to enhance its quality has garnered significant attention recently. Here, we utilized Cuqu fortified (JQ) by synthetic microbiota composed of two different strains to enhance SBV fermentation, while simultaneously revealing community succession and metabolic regulatory mechanisms. Compared to control samples, the fortified vinegar grains (ZJV) community exhibited a 1.60-fold increase in bacterial biomass during major stages. Dominant fungi dynamics in ZJV significantly shifted, with a notable increase in Monascus and Rasamsonia. Upregulation of enzymes involved in starch and cellulose degradation led to increased contents of volatiles, 4-ethylguaiacol (4-EG), and raw starch conversion rate in the fermented ZJV by 1.75 times, 40 times, and 21.92%, respectively. Biofortification boosted interspecies interactions, clustering functional microbes into a module. Functional bacteria served as connectors, enhancing fungal-bacterial communication, optimizing information and energy exchange, reducing primary metabolite accumulation like ethanol, and improving nutrient utilization and metabolic conversion for secondary metabolite synthesis. These findings establish crucial groundwork for the practical application of synthetic microbiota in various engineering applications.

Comprehensive characterization of Epimedium-Rhizoma drynariae herb pair in rat plasma, urine, and feces metabolic profiles by UHPLC-Q-Orbitrap HRMS combined with diagnostic extraction strategy and multicomponent pharmacokinetic study by UHPLC-MS/MS.

Epimedium-Rhizoma drynariae (EP-RD) was a well-known herb commonly used to treat bone diseases in traditional Chinese medicine. Nevertheless, there was incomplete pharmacokinetic behavior, metabolic conversion and chemical characterization of EP-RD in vivo. Therefore, this study aimed to establish metabolic profiles combined with multicomponent pharmacokinetics to reveal the in vivo behavior of EP-RD. Firstly, the diagnostic product ions (DPIs) and neutral losses (NLs) filtering strategy combined with UHPLC-Q-Orbitrap HRMS for the in vitro chemical composition of EP-RD and metabolic profiles of plasma, urine, and feces after oral administration of EP-RD to rats were proposed to comprehensively characterize the 47 chemical compounds and the 97 exogenous in vivo (35 prototypes and 62 metabolites), and possible biotransformation pathways of EP-RD were proposed, which included phase I reactions such as hydrolysis, hydrogenation, dehydrogenation, hydroxylation, dehydroxylation, isomerization, and demethylation and phase II reactions such as glucuronidation, acetylation, methylation, and sulfation. Moreover, a UHPLC-MS/MS quantitative approach was established for the pharmacokinetic analysis of seven active components: magnoflorine, epimedin A, epimedin B, epimedin C, icariin, baohuoside II, and icariin II. Results indicated that the established method was reliably used for the quantitative study of plasma active ingredients after oral administration of EP-RD in rats. Compared to oral EP alone, the increase in area under curves and maximum plasma drug concentration (P < 0.05). This study increased the understanding of the material basis and biotransformation profiles of EP-RD in vivo, which was of great significance in exploring the pharmacological effects of EP-RD.

Ligustilide covalently binds to Cys703 in the pre-S1 helix of TRPA1, blocking the opening of channel and relieving pain in rats with acute soft tissue injury

Ethnopharmacological relevanceThe natural anodyne Ligustilide (Lig), derived from Angelica sinensis (Oliv.) Diels and Ligusticum chuanxiong Hort., has been traditionally employed for its analgesic properties in the treatment of dysmenorrhea and migraine, and rheumatoid arthritis pain. Despite the existing reports on the correlation between TRP channels and the analgesic effects of Lig, a comprehensive understanding of their underlying mechanisms of action remains elusive. Aim of the studyThe objective of this study is to elucidate the mechanism of action of Lig on the analgesic target TRPA1 channel. MethodsThe therapeutic effect of Lig was evaluated in a rat acute soft tissue injury model. The analgesic target was identified through competitive inhibition of TRP channel agonists at the animal level, followed by Fluo-4/Ca2+ imaging on live cells overexpressing TRP proteins. The potential target was verified through in-gel imaging, colocalization using a Lig-derived molecular probe, and a drug affinity response target stability assay. The binding site of Lig was identified through protein spectrometry and further analyzed using molecular docking, site-specific mutation, and multidisciplinary approaches. ResultsThe administration of Lig effectively ameliorated pain and attenuated oxidative stress and inflammatory responses in rats with soft tissue injuries. Moreover, the analgesic effects of Lig were specifically attributed to TRPA1. Mechanistic studies have revealed that Lig directly activates TRPA1 by interacting with the linker domain in the pre-S1 region of TRPA1. Through metabolic transformation, 6,7-epoxyligustilide (EM-Lig) forms a covalent bond with Cys703 of TRPA1 at high concentrations and prolonged exposure time. This irreversible binding prevents endogenous electrophilic products from entering the cysteine active center of ligand-binding pocket of TRPA1, thereby inhibiting Ca2+ influx through the channel opening and ultimately relieving pain. ConclusionsLig selectively modulates the TRPA1 channel in a bimodal manner via non-electrophilic/electrophilic metabolic conversion. The epoxidized metabolic intermediate EM-Lig exerts analgesic effects by irreversibly inhibiting the activation of TRPA1 on sensory neurons. These findings not only highlight the analgesic mechanism of Lig but also offer a novel nucleophilic attack site for the development of TRPA1 antagonists in the pre-S1 region.

Metabolic Dysregulation and Its Role in Postoperative Pain among Knee Osteoarthritis Patients.

The inflammation observed in joint tissues is linked to pain caused by the production of proinflammatory cytokines. Since the biosynthesis of cytokines requires energy, their production is supported by extensive metabolic conversions of carbohydrates and fatty acids, which could lead to a disruption in cellular homeostasis. Therefore, this study aimed to investigate the association between POP development and disturbances in energy metabolic conversions, focusing on carbohydrate and fatty acid metabolism. Peripheral blood samples were collected from 26 healthy subjects and 50 patients with end-stage OA before joint replacement surgery. All implants were validated by orthopedic surgeons, and patients with OA demonstrated no inherent abnormalities to cause pain from other reasons than OA disease, such as malalignment, aseptic loosening, or excessive bleeding. Pain levels were assessed before surgery using the visual analogue scale (VAS) and neuropathic pain questionnaires, DN4 and PainDETECT. Functional activity was evaluated using the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). Three and six months after surgery, pain indices according to a VAS of 30 mm or higher were considered. Total RNA isolated from whole blood was analyzed using quantitative real-time RT-PCR (qRT-PCR) for the expression of genes related to carbohydrate and fatty acid metabolism. Protein levels of the examined genes were measured using an ELISA in the peripheral blood mononuclear cells (PBMCs). We used qRT-PCR because it is the most sensitive and reliable method for gene expression analysis, while an ELISA was used to confirm our qRT-PCR results. Among the study cohort, 17 patients who reported POP demonstrated significantly higher (p < 0.05) expressions of the genes PKM2, LDH, SDH, UCP2, CPT1A, and ACLY compared to pain-free patients with KOA. Receiver-operating characteristic (ROC) curve analyses confirmed the association between these gene expressions and pain development post-arthroplasty. A principle component analysis identified the prognostic values of ACLY, CPT1A, AMPK, SDHB, Caspase 3, and IL-1β gene expressions for POP development in the examined subjects. These findings suggest that the disturbances in energy metabolism, as observed in the PBMCs of patients with end-stage KOA before arthroplasty, may contribute to POP development. An understanding of these metabolic processes could provide insights into the pathogenesis of KOA. Additionally, our findings can be used in a clinical setting to predict POP development in end-stage patients with KOA before arthroplasty.

Denitrification enhanced by composite carbon sources in AAO-biofilter: Efficiency and metagenomics research

Nitrogen removal from domestic sewage is usually limited by insufficient carbon source and electron donor. An economical solid carbon source was developed by composition of polyvinyl alcohol, sodium alginate, and corncob, which was utilized as external carbon source in the anaerobic anoxic oxic (AAO)-biofilter for the treatment of low carbon-to-nitrogen ratio domestic sewage, and the nitrogen removal was remarkably improved from 63.2% to 96.5%. Furthermore, the effluent chemical oxygen demand maintained at 35 mg/L or even lower, and the total nitrogen was reduced to less than 2 mg/L. Metagenomic analysis demonstrated that the microbial communities responsible for potential denitrification and organic matter degradation in both AAO and the biofilter reactors were mainly composed of Proteobacteria and Bacteroides, respectively. The solid carbon source addition resulted in relatively high abundance of functional enzymes responsible for NO3−-N to NO2−-N conversion in both AAO and the biofilter reactors, thus enabled stable reaction. The carbon source addition during glycolysis primarily led to the increase of genes associated with the metabolic conversion of fructose 1.6P2 to glycerol-3P The reactor maintained high abundance of genes related to the tricarboxylic acid cycle, and then guaranteed efficient carbon metabolism. The results indicate that the composite carbon source is feasible for denitrification enhancement of AAO-biofilter, which contribute to the theoretical foundation for practical nitrogen removal application.

Search for molecular descriptors and computer prediction of biological activity in a series of S-derivatives (1,2,4-triazole-3(2H)-yl)methyl)thiopyrimidines

Most pharmaceutical compounds interact with various molecular compositions in the body, leading to complex biological effects. Furthermore, during biotransformation, they may generate one or more metabolites with potent bioactive properties. Therefore, the development and careful use of new medications require thorough examination of their biological impact profiles, including considerations of human metabolic processes. In silico techniques are currently widely used to evaluate the interactions of emerging drug candidates with pharmacological conditions and predict their metabolic conversions. To investigate the bioactivity of compounds based on the structure of 1,2,4-triazole-3(2H)-thione with pyrimidine-2-thiol, we generated a combinatorial library of bioregulators using the computer programs SuperPred and SwissADME. The relevance of this research is underscored by the ongoing quest for novel biomolecular compounds that are highly effective yet low in toxicity, spanning both natural and synthetic sources. Much attention is directed towards nitrogen-containing heterocycles, particularly derivatives of 1,2,4-triazole, due to their substantial medical and biological potential. The aim of the work is to create a combinatorial library of bioregulators, which contains the structures of 1,2,4-triazole and pyrimidine, and to carry out in silico screening of heterocycle derivatives using the SuperPred web server to determine promising directions for the study of their bioactivity. Materials and methods. The subject of our research is S-derivatives of (1,2,4-triazole-3(2H)-yl)methyl)thiopyrimidines. Virtual screening of compounds was carried out using the SuperPred computer program. The ADME study was performed using the free service SwissADME, which is used to evaluate the pharmacokinetics, bioavailability, and interaction of small molecules with enzymes in medicinal chemistry. Results. Predicting the likelihood of a substance demonstrating a particular biological activity enables the selection of the most appropriate tests for studying the activity of a given chemical compound, as well as identifying which substances available to the researcher are most likely to enhance the effectiveness of the effects. This principle is grounded in the concept that “the biological activity of a substance is a function of its chemical structure”. Such predictions are generated based on the structural formula of a chemical compound and can be made during the planning stage of synthesis. Conclusions. A combinatorial library of bioregulators was created by combining the structure of 1,2,4-triazole-3(2H)-thione with pyrimidine-2-thiol. The conducted virtual screening of the 1,2,4-triazole-3(2H)-thione heterocycle with pyrimidine-2-thiol shows promise for obtaining non-toxic compounds with antineoplastic biological activity. Screening of derivatives, particularly S-derivatives of (1,2,4-triazole-3(2H)-yl)methyl)thiopyrimidines, indicates the potential for discovering biologically active substances with antineoplastic, antibacterial, analgesic, antidiabetic, antihypertensive, and other types of biological activities within this compound series.

Effects of Itraconazole on Pharmacokinetics of Mitragynine and 7-Hydroxymitragynine in Healthy Volunteers.

CYP3A4-mediated metabolic conversion of mitragynine to 7-hydroxymitragynine (7OH) has been demonstrated in human liver microsomes, and in rodents. Pharmacokinetics (PK) of mitragynine and 7OH in humans is still limited. We aimed to examine the pharmacokinetics of mitragynine and the formation of 7OH in healthy volunteers. To elucidate involvement of CYP3A4 in 7OH formation, inhibition by itraconazole was implemented. Two study periods with PK study of mitragynine alone in period 1, followed by period 2 including itraconazole pretreatment was conducted. Freshly prepared kratom tea consisting of 23.6 mg of mitragynine was given to participants in both study periods. Serial blood samplings were performed for 72 hours, and analyzed using a validated LCMS in multiple reaction monitoring mode. The median Cmax for mitragynine of 159.12 ± 8.68 ng/mL was attained in 0.84 h. While median Cmax for 7OH of 12.81 ± 3.39 ng/mL was observed at 1.77 h. In period 1, Cmax and AUC 0-inf of 7OH accounted for 9% and 20 %, respectively, of those parameters for mitragynine. The geometric mean ratio of AUC0-72 for 7OH/mitragynine (metabolic ratio, MR) was 13.25 ± 1.07. Co-administration of itraconazole 200 mg per day orally for 4 days (period 2) decreased 7OH exposure by 56% for Cmax and 43% for AUC0-72 after a single oral dose of kratom tea. While the Cmax of mitragynine increased by 1.5 folds without a significant change in Tmax. The geometric mean metabolic ratio was 3.30 ± 1.23 (period 2), indicating the attenuation for the formation of 7OH by the pretreatment with itraconazole. This suggested the CYP3A4-mediated formation of 7OH from mitragynine in healthy volunteers. This study provides the first evidence of metabolic conversion of mitragynine to 7OH in humans.

Effects of topical application of resveratrol on tight junction barrier and antimicrobial compound production in lactating goat mammary glands

In mammary glands, the formation of less-permeable tight junctions (TJs) and the production of antimicrobial compounds like lactoferrin and defensins are important for preventing mastitis. Resveratrol, a polyphenol contained in red grapes, is known to protect mammary epithelial cells (MECs) from oxidative stress; however, oral administration of resveratrol causes a decrease in certain biological processes through conjugation and metabolic conversion. In this study, we determined the beneficial effects of resveratrol on TJs and antimicrobial compounds in cultured goat MECs by adding it to the medium, and in lactating goat mammary glands by topical application for percutaneous absorption. TJ barrier function was evaluated by transepithelial resistance and expression or localization pattern of claudins for culture model in vitro and by somatic cell count, Na+, albumin, and IgG in milk for topical application in vivo. Concentrations of antimicrobial compounds and cytokines were measured using ELISA. Activation of STAT3 was evaluated by Western blotting. Resveratrol strengthened TJ barrier function by upregulating claudin-3 in cultured MECs and topical application to udders reduced somatic cell count, Na+, albumin, and IgG in milk. Resveratrol increased β-defensin and S100A7 levels in cultured MECs and milk. In addition, resveratrol down-regulated cytokine production and STAT3 pathway. These findings suggest that the topical application of resveratrol to udders may be effective in preventing mastitis.

Microbial Pathway Thermodynamics: Stoichiometric Models Unveil Anabolic and Catabolic Processes.

The biotechnological exploitation of microorganisms enables the use of metabolism for the production of economically valuable substances, such as drugs or food. It is, thus, unsurprising that the investigation of microbial metabolism and its regulation has been an active research field for many decades. As a result, several theories and techniques were developed that allow for the prediction of metabolic fluxes and yields as biotechnologically relevant output parameters. One important approach is to derive macrochemical equations that describe the overall metabolic conversion of an organism and basically treat microbial metabolism as a black box. The opposite approach is to include all known metabolic reactions of an organism to assemble a genome-scale metabolic model. Interestingly, both approaches are rather successful at characterizing and predicting the expected product yield. Over the years, macrochemical equations especially have been extensively characterized in terms of their thermodynamic properties. However, a common challenge when characterizing microbial metabolism by a single equation is to split this equation into two, describing the two modes of metabolism, anabolism and catabolism. Here, we present strategies to systematically identify separate equations for anabolism and catabolism. Based on metabolic models, we systematically identify all theoretically possible catabolic routes and determine their thermodynamic efficiency. We then show how anabolic routes can be derived, and we use these to approximate biomass yield. Finally, we challenge the view of metabolism as a linear energy converter, in which the free energy gradient of catabolism drives the anabolic reactions.

Abstract A013: Cell-extrinsic mechanisms of accelerated therapy resistance mediated by altered pharmacokinetics

Abstract Background: Targeted therapies directed against oncogenic signaling addictions, including one resulting from EML4-ALK fusions, can induce strong and durable clinical responses. However, some tumors do not respond (intrinsic resistance), while the initially responsive advanced cancers eventually relapse (acquired resistance). Our earlier studies with experimental models of ALK+ lung cancer revealed that acquired resistance to ALK inhibitors represents a gradual, multifactorial adaptation that progresses through multiple intermediate stages. These evolutionary intermediates can display unique collateral sensitivities to pharmacological inhibitors, including high sensitivity to a KDM histone demethylase inhibitor JIB-04. Results: To explore the utility of using JIB04 to suppress the evolution of resistance in vivo, we examined its ability to enhance responses to clinical ALKi alectinib and lorlatinib in mouse xenograft models. Surprisingly, JIB04 blunted the in vivo tumor responses and accelerated their relapse. The discrepancy between the in vitro and in vivo effects of JIB04 implied environmental mediation. Therefore, we examined the potential involvement of several mechanisms of environment-mediated resistance. We found that rather than directly decreasing cell-intrinsic therapy sensitivity, JIB04 altered ALKi pharmacokinetics. Using mass spectrometry measurements, we found a strong reduction in systemic and intratumor levels of the ALKi. This reduction is attributable to an accelerated metabolic conversion and excretion of metabolites. Our ongoing and planned efforts focus on the elucidation of mechanistic details and the exploration of clinical correlation. Significance: Both intrinsic and acquired resistance are assumed to reflect reduced sensitivities of tumor cells due to mutational, plasticity-related, and microenvironmental mechanisms. Pharmacokinetics studies are the essential component of drug development and clinical trial pipelines. However, after clinical approval, drug concentrations are not routinely examined in clinics. Whereas JIB04 is not used in clinics, its effects are mediated by mechanisms that might be triggered by more relevant dietary or pharmacological factors. Our results highlight the potential danger of blind spots in consideration of causes of therapy resistance and warrant the inclusion of pharmacokinetics analyses in diagnosing/treating therapy resistance. Citation Format: Pragya Kumar, Robert Vander Velde, Virginia Turati, Andriy Marusyk. Cell-extrinsic mechanisms of accelerated therapy resistance mediated by altered pharmacokinetics [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A013.

Oligoribonuclease mediates high adaptability of P. aeruginosa through metabolic conversion

BackgroundOligoribonuclease (orn) of P. aeruginosa is a highly conserved exonuclease, which can regulate the global gene expression levels of bacteria through regulation of both the nanoRNA and c-di-GMP. NanoRNA can regulate the expression of the bacterial global genome as a transcription initiator, and c-di-GMP is the most widely second messenger in bacterial cells.ObjectiveThis study seeks to elucidate on the regulation by orn on pathogenicity of P. aeruginosa.MethodsP. aeruginosa with orn deletion was constructed by suicide plasmid homologous recombination method. The possible regulatory process of orn was analyzed by TMT quantitative labeling proteomics. Then experiments were conducted to verify the changes of Δorn on bacterial motility, virulence and biofilm formation. Bacterial pathogenicity was further detected in cell and animal skin trauma models. ELISA detection c-di-GMP concentration and colony aggregation and biofilm formation were observed by scanning electron microscope.Resultsorn deletion changed the global metabolism of P. aeruginosa and reduced intracellular energy metabolism. It leads to the disorder of the quorum sensing system, the reduction of bacterial motility and virulence factors pyocyanin and rhamnolipids. But, orn deletion enhanced pathogenicity in vitro and in vivo, a high level of c-di-GMP and biofilm development of P. aeruginosa.Conclusionorn regulates the ability of P. aeruginosa to adapt to the external environment.

Comprehensive metabolic investigation of dopamine reuptake inhibitor HDMP-28 in equine liver microsomes and Cunninghamella elegans for doping control.

A dopamine reuptake inhibitor is a type of medication or substance that works by blocking the reuptake of dopamine in the brain. Dopamine reuptake inhibitors offer multiple effects, including increased alertness, improved mood, and therapeutic potential for conditions like depression, ADHD, and Parkinson's disease. HDMP-28, or methylnaphthidate, is a potent synthetic stimulant from the phenyltropane class. It surpasses methylphenidate in both dopamine reuptake inhibition and half-life. As a dopamine reuptake inhibitor, it boosts dopamine levels by hindering reuptake into nerve cells, resulting in heightened stimulation and increased energy. In order to comprehensively address both the tangible and potential repercussions of the unauthorized utilization of the aforementioned substance in sports, it is imperative to establish analytical methodologies for the identification of the parent drug and its primary metabolites. Additionally, a comprehensive analysis of the metabolic characteristics of HDMP-28 in both human and animal subjects has yet to be published. This study explores the metabolic conversion of HDMP-28 mediated by equine liver microsomes and Cunninghamella elegans. An extraction and detection method was developed, optimized, and validated for doping assessment in equine urine and plasma. Liquid chromatography-high-resolution mass spectrometry was employed to determine metabolite structures. The study identified 31 (22 phase I and 9 phase II) metabolites of HDMP-28, including hydroxylated, hydrogenated, and hydrolyzed analogs. Glucuronic acid-conjugated metabolites were also detected. This manuscript describes metabolites based on the in vitro studies, which might not be the same in vivo. These findings aid in the detection and understanding of the illicit use of HDMP-28 in equestrian sports.

Sugar Transport in Thermophiles: Bridging Lignocellulose Deconstruction and Bioconversion.

Biomass degrading thermophiles play an indispensable role in building lignocellulose-based supply chains. They operate at high temperatures to improve process efficiencies and minimize mesophilic contamination, can overcome lignocellulose recalcitrance through their native carbohydrate-active enzyme (CAZyme) inventory, and can utilize a wide range of sugar substrates. However, sugar transport in thermophiles is poorly understood and investigated, as compared to enzymatic lignocellulose deconstruction and metabolic conversion of sugars to value-added chemicals. Here, we review the general modes of sugar transport in thermophilic bacteria and archaea, covering the structural, molecular, and biophysical basis of their high-affinity sugar uptake. We also review how thermophilic sugar transporters are identified and characterized, including recent genetic studies on their function. With this understanding of sugar transport, we discuss strategies for how sugar transport can be engineered in thermophiles, with the potential to enhance the conversion of lignocellulosic biomass into renewable products.

Hyperpolarized 13 C metabolic imaging of the human abdomen with spatiotemporal denoising.

Improving the quality and maintaining the fidelity of large coverage abdominal hyperpolarized (HP) 13 C MRI studies with a patch based global-local higher-order singular value decomposition (GL-HOVSD) spatiotemporal denoising approach. Denoising performance was first evaluated using the simulated [1-13 C]pyruvate dynamics at different noise levels to determine optimal kglobal and klocal parameters. The GL-HOSVD spatiotemporal denoising method with the optimized parameters was then applied to two HP [1-13 C]pyruvate EPI abdominal human cohorts (n = 7 healthy volunteers and n = 8 pancreatic cancer patients). The parameterization of kglobal = 0.2 and klocal = 0.9 denoises abdominal HP data while retaining image fidelity when evaluated by RMSE. The kPX (conversion rate of pyruvate-to-metabolite, X = lactate or alanine) difference was shown to be <20% with respect to ground-truth metabolic conversion rates when there is adequate SNR (SNRAUC > 5) for downstream metabolites. In both human cohorts, there was a greater than nine-fold gain in peak [1-13 C]pyruvate, [1-13 C]lactate, and [1-13 C]alanine apparent SNRAUC . The improvement in metabolite SNR enabled a more robust quantification of kPL and kPA . After denoising, we observed a 2.1 ± 0.4 and 4.8 ± 2.5-fold increase in the number of voxels reliably fit across abdominal FOVs for kPL and kPA quantification maps. Spatiotemporal denoising greatly improves visualization of low SNR metabolites particularly [1-13 C]alanine and quantification of [1-13 C]pyruvate metabolism in large FOV HP 13 C MRI studies of the human abdomen.

Exploring core functional fungi driving the metabolic conversion in the industrial pile fermentation of Qingzhuan tea

The distinct sensory quality of Qingzhuan tea is mainly formed in pile fermentation by a group of functional microorganisms but the core functional ones was poorly characterized. Therefore, this study investigated the dynamic changes in the fungal community and metabolic profile by integrating microbiomics and metabolomics, and explored the core functional fungi driving the metabolic conversion in the industrial pile fermentation of Qingzhuan tea. Indicated by microbiomics analysis, Aspergillus dominated the entire pile-fermentation process, while Thermoascus, Rasamsonia, and Cylindrium successively abounded in the different stages of the pile fermentation. A total of 50 differentially changed metabolites were identified, with the hydrolysis of galloyl/polymeric catechins, biosynthesis of theabrownins, oxidation of catechins, N-ethyl-2-pyrrolidinone substitution of catechins, and deglycosylation of flavonoid glucosides. Nine fungal genera were identified as core functional fungi, in which Aspergillus linked to the hydrolysis of polymeric catechins and insoluble polysaccharides as well as biosynthesis of theabrownins, while Thermoascus participated in the biosynthesis of theabrownins, deglycosylation of flavonoid glucosides, and N-ethyl-2-pyrrolidinone substitution of catechins. These findings would advance our understanding of the quality formation of Qingzhuan tea and provide a benchmark for precise inoculation for its quality improvement.