A capillary-based centrifugal indicator equipped with in situ pathogenic bacteria culture for fast antimicrobial susceptibility testing.

Antimicrobial resistance has become a major global health threat due to the misuse and overuse of antibiotics. Rapid, affordable, and high-efficiency antimicrobial susceptibility testing (AST) is among the effective means to solve this problem. Herein, we developed a capillary-based centrifugal indicator (CBCI) equipped with an in situ culture of pathogenic bacteria for fast AST. The bacterial incubation and growth were performed by macro-incubation, which seamlessly integrated the capillary indicator. Through simple centrifugation, all the bacterial cells were confined at the nanoliter-level capillary column. The packed capillary column height could linearly reflect the bacterial count, and the minimum inhibitory concentration (MIC) was determined based on the difference in the column height between the drug-added groups and the control group. The AST results could easily be determined by the naked eye or smartphone imaging. Thus, the CBCI realized the combination of macro-bacterial incubation and early micro assessment, which accelerated the phenotypic AST without complex microscopic counting or fluorescent labelling. The whole operation process was simple and easy to use. AST results could be determined for E. coli ATCC strains within 3.5 h, and the output results for clinical samples were consistent with the hospital reports. We expect this AST platform to become a useful tool in limiting antimicrobial resistance, especially in remote/resource-limited areas or in underdeveloped countries.

HBB contributes to individualized aconitine-induced cardiotoxicity in mice via interfering with ABHD5/AMPK/HDAC4 axis.

The root of Aconitum carmichaelii Debx. (Fuzi) is an herbal medicine used in China that exerts significant efficacy in rescuing patients from severe diseases. A key toxic compound in Fuzi, aconitine (AC), could trigger unpredictable cardiotoxicities with high-individualization, thus hinders safe application of Fuzi. In this study we investigated the individual differences of AC-induced cardiotoxicities, the biomarkers and underlying mechanisms. Diversity Outbred (DO) mice were used as a genetically heterogeneous model for mimicking individualization clinically. The mice were orally administered AC (0.3, 0.6, 0.9 mg· kg-1 ·d-1) for 7 d. We found that AC-triggered cardiotoxicities in DO mice shared similar characteristics to those observed in clinic patients. Most importantly, significant individual differences were found in DO mice (variation coefficients: 34.08%-53.17%). RNA-sequencing in AC-tolerant and AC-sensitive mice revealed that hemoglobin subunit beta (HBB), a toxic-responsive protein in blood with 89% homology to human, was specifically enriched in AC-sensitive mice. Moreover, we found that HBB overexpression could significantly exacerbate AC-induced cardiotoxicity while HBB knockdown markedly attenuated cell death of cardiomyocytes. We revealed that AC could trigger hemolysis, and specifically bind to HBB in cell-free hemoglobin (cf-Hb), which could excessively promote NO scavenge and decrease cardioprotective S-nitrosylation. Meanwhile, AC bound to HBB enhanced the binding of HBB to ABHD5 and AMPK, which correspondingly decreased HDAC-NT generation and led to cardiomyocytes death. This study not only demonstrates HBB achievement a novel target of AC in blood, but provides the first clue for HBB as a novel biomarker in determining the individual differences of Fuzi-triggered cardiotoxicity.

Bent-Capillary-Centrifugal-Driven Monodisperse Droplet Generator with Its Application for Digital LAMP Assay.

We developed a bent-capillary-centrifugal-driven (BCCD) monodisperse droplet generator, which could achieve a perfect combination of driving and segmentation for the dispersed phase only using a rotating bent capillary immersed in the continuous phase (mineral oil). The sample could flow continuously to the bent-capillary outlet to form the droplet precursors, which were segmented into homogeneous droplets in the continuous phase. Through the investigation of influence factors on droplet size and stability, we found that the droplet size could be conveniently controlled by the rotational speed of the bent capillary. The droplet volumes could be adjusted with the range from 34 pL to 1 µL, and the coefficient variations (CVs) were less than 3%. Meanwhile, the BCCD droplet generator could realize the controllable droplet output with a high-efficiency sample utilization of 99.75 ± 1.15%, which offered a significant advantage in reducing the waste of precious samples in the droplet generation process. We validated this system with a digital loop-mediated isothermal amplification (dLAMP) assay for the absolute quantification of Mycobacterium tuberculosis complex nucleic acids. The results demonstrated that the BCCD droplet generator was easy to build, was of low cost, and was convenient to operate, as well as avoided sample loss and cross-contamination by coupling with a 96-well plate. Overall, the present platform, as a simple chip-free droplet generator, will provide an especially valuable droplet generation solution for biochemical applications based on droplets.

Costunolide Protects Myocardium From Ischemia Reperfusion Injury by Inhibiting Oxidative Stress Through Nrf2/Keap1 Pathway Activation.

ABSTRACT: Costunolide (Cos) is a naturally occurring sesquiterpene lactone that exhibits antioxidative properties. In this study, we demonstrate the protective mechanism of Cos against ischemia/reperfusion (I/R)-induced myocardial injury. Cos significantly decreased levels of reactive oxygen species and ameliorated apoptosis of I/R cardiomyocytes both in vitro and in vivo. Further investigation revealed that Cos increased expression of the antioxidant proteins HO-1 and NQO-1 and decreased the Bax/Bcl-2 ratio, thus protecting cardiac cells. NF-E2-related factor 2 (Nrf2) silencing significantly attenuated the protective effects of Cos in tert-butyl hydroperoxide (TBHP)-treated H9C2 cells. Additionally, Cos significantly intensified the I/R- or TBHP-induced dissociation of the Kelch-like ECH-associated protein 1 (Keap1)/Nrf2 complex both in vitro and in vivo. These results suggest that activation of Nrf2/Keap1 using Cos may be a therapeutic strategy for myocardial I/R injury.

Structure-activity relationship between gluten and dough quality of sprouted wheat flour based on air classification-induced component recombination.

BACKGROUND: Air classification can separate sprouted wheat flour (SWF) into three types: coarse wheat flour (F1), medium wheat flour (F2) and fine wheat flour (F3). The gluten quality of SWF can be indirectly improved by removing inferior parts (F3). In order to reveal the underlying mechanism of this phenomenon, the composition and structural changes of gluten, as well as the rheological properties and fermentation characteristics of gluten in recombinant dough in the process of air classification of all three SWF types, were analyzed in this study. RESULTS: Overall, sprouting significantly reduced the content of high-molecular-weight subunits, such as glutenin subunit and ω-gliadin. It also destroyed the structural content, such as disulfide bonds, α-helix and ß-turn contents, which maintained the stability of gluten gel. Air classification made the above changes in F3 more severe but reversed them in F1. Moreover, rheological properties were more affected by gluten composition, whereas fermentation characteristics were more affected by gluten structure. CONCLUSION: After air classification, particles rich in high molecular weight subunits from SWF are enriched in F1, and the gluten of F1 has more secondary structure that maintain gel stability, which ultimately lead to improved rheology properties and fermentation characteristics. F3 relatively exhibits the opppsite phenomenon. These results further reveal the potential mechanism of improvement of SWF gluten by air classification. Moreover, Thus, this study provides new perspectives for the utilization of SWF. © 2023 Society of Chemical Industry.

Development of high quality T1-weighted and diffusion tensor templates of the older adult brain in a common space.

High-quality T1-weighted (T1w) and diffusion tensor imaging (DTI) brain templates that are representative of the individuals under study enhance the accuracy of template-based neuroimaging investigations, and when they are also located in a common space they facilitate optimal integration of information on brain morphometry and diffusion characteristics. However, such multimodal templates have not been constructed for the brain of older adults. The purpose of this work was threefold: (A) to introduce an iterative method for construction of multimodal T1w and DTI templates that aims at maximizing the quality of each template separately as well as the spatial matching between templates, (B) to use this method to develop T1w and DTI templates of the older adult brain in a common space, and (C) to evaluate the performance of the method across iterations and compare it to the performance of state-of-the-art approaches based on multichannel registration. It was demonstrated that more iterations of the proposed method enhanced the characteristics and spatial matching of the resulting T1w and DTI templates. The templates of the older adult brain generated by the final iteration of the proposed method provided better delineation of brain structures, higher discriminability between tissues, and higher image sharpness near the cortex compared to templates generated with approaches employing multichannel registration. In addition, the spatial matching between the T1w and DTI templates constructed by the proposed method approximated the template alignment achieved with methods employing multichannel registration. Finally, when using the templates generated by the proposed method as references for spatial normalization of older adult T1w and DTI data, both the intra-modality inter-subject normalization precision and the inter-modality spatial matching were higher in most metrics than those achieved with templates constructed with other methods. Overall, the present work brought new insights into multimodal template construction, generated much-needed high quality T1w and DTI templates of the older adult brain in a common space, and conducted a thorough, quantitative evaluation of available multimodal template construction methods.

Single-dose rAAV5-based vaccine provides long-term protective immunity against SARS-CoV-2 and its variants.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus and its variants, has posed unprecedented challenges worldwide. Existing vaccines have limited effectiveness against SARS-CoV-2 variants. Therefore, novel vaccines to match mutated viral lineages by providing long-term protective immunity are urgently needed. We designed a recombinant adeno-associated virus 5 (rAAV5)-based vaccine (rAAV-COVID-19) by using the SARS-CoV-2 spike protein receptor binding domain (RBD-plus) sequence with both single-stranded (ssAAV5) and self-complementary (scAAV5) delivery vectors and found that it provides excellent protection from SARS-CoV-2 infection. A single-dose vaccination in mice induced a robust immune response; induced neutralizing antibody (NA) titers were maintained at a peak level of over 1:1024 more than a year post-injection and were accompanied by functional T-cell responses. Importantly, both ssAAV- and scAAV-based RBD-plus vaccines produced high levels of serum NAs against the circulating SARS-CoV-2 variants, including Alpha, Beta, Gamma and Delta. A SARS-CoV-2 virus challenge showed that the ssAAV5-RBD-plus vaccine protected both young and old mice from SARS-CoV-2 infection in the upper and lower respiratory tracts. Whole genome sequencing demonstrated that AAV vector DNA sequences were not found in the genomes of vaccinated mice one year after vaccination, demonstrating vaccine safety. These results suggest that the rAAV5-based vaccine is safe and effective against SARS-CoV-2 and several variants as it provides long-term protective immunity. This novel vaccine has a significant potential for development into a human prophylactic vaccination to help end the global pandemic.

Ras inhibitor farnesylthiosalicylic acid conjugated with IR783 dye exhibits improved tumor-targeting and altered anti-breast cancer mechanisms in mice.

Ras has long been viewed as a promising target for cancer therapy. Farnesylthiosalicylic acid (FTS), as the only Ras inhibitor has ever entered phase II clinical trials, has yielded disappointing results due to its strong hydrophobicity, poor tumor-targeting capacity, and low therapeutic efficiency. Thus, enhancing hydrophilicity and tumor-targeting capacity of FTS for improving its therapeutic efficacy is of great significance. In this study we conjugated FTS with a cancer-targeting small molecule dye IR783 and characterized the anticancer properties of the conjugate FTS-IR783. We showed that IR783 conjugation greatly improved the hydrophilicity, tumor-targeting and therapeutic potential of FTS. After a single oral administration in Balb/c mice, the relative bioavailability of FTS-IR783 was increased by 90.7% compared with FTS. We demonstrated that organic anion transporting polypeptide (OATP) and endocytosis synergistically drove the uptake of the FTS-IR783 conjugate in breast cancer MDA-MB-231 cells, resulting in superior tumor-targeting ability of the conjugate both in vitro and in vivo. We further revealed that FTS-IR783 conjugate could bind with and directly activate AMPK rather than affecting Ras, and subsequently regulate the TSC2/mTOR signaling pathway, thus achieving 2-10-fold increased anti-cancer therapeutic efficacy against 6 human breast cancer cell lines compared to FTS both in vivo and in vitro. Overall, our data highlights a promising approach for the modification of the anti-tumor drug FTS using IR783 and makes it possible to return FTS back to the clinic with a better efficacy.

Integrated chemical profiling, network pharmacology and pharmacological evaluation to explore the potential mechanism of Xinbao pill against myocardial ischaemia-reperfusion injury.

CONTEXT: Xinbao pill (XBW), a traditional Chinese herbal formula, is widely used in clinical treatment for cardiovascular diseases; however, the therapeutic effect of XBW on myocardial ischaemia-reperfusion injury (MI/RI) is unclear. OBJECTIVE: This study evaluates the cardioprotective effect and molecular mechanism of XBW against MI/RI. MATERIALS AND METHODS: A phytochemistry-based network pharmacology analysis was used to uncover the mechanism of XBW against MI/RI. Ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry method was used to identify chemicals. MI/RI-related targets of XBW were predicted using TargetNet database, OMIC database, etc. Sprague-Dawley (SD) rats under anterior descending artery ligation model were divided into Sham, MI/RI and XBW (180 mg/kg, intragastric administration). After 30 min ischaemia and 24 h reperfusion, heart tissues were collected for measurement of myocardial infarct size. After oxygen glucose deprivation for 6 h, H9c2 cells were treated with XBW (60, 240 and 720 µg/mL) and diazoxide (100 µM) for 18 h of reperfusion. RESULTS: Thirty-seven chemicals were identified in XBW; 50 MI/RI-related targets of XBW were predicted using indicated databases. XBW significantly reduced infarct size and creatine kinase MB (CK-MB) level after MI/RI; XBW protected H9c2 cells against OGD/R injury. Gene ontology (GO) and KEGG pathway enrichment analyses by String database showed that the cardioprotective effect of XBW was associated with autophagy and apoptosis signalling pathways. Experimental investigation also verified that XBW suppressed apoptosis, autophagy and endoplasmic reticulum (ER) stress. CONCLUSIONS: XBW showed therapeutic effects against MI/RI mainly via attenuating apoptosis though suppressing excessive autophagy and ER stress.

Regionconnect: Rapidly extracting standardized brain connectivity information in voxel-wise neuroimaging studies.

Reporting white matter findings in voxel-wise neuroimaging studies typically lacks specificity in terms of brain connectivity. Therefore, the purpose of this work was to develop an approach for rapidly extracting standardized brain connectivity information for white matter regions with significant findings in voxel-wise neuroimaging studies. The new approach was named regionconnect and is based on precalculated average healthy adult brain connectivity information stored in standard space in a fashion that allows fast retrieval and integration. Towards this goal, the present work first generated and evaluated the white matter connectome of the IIT Human Brain Atlas v.5.0. It was demonstrated that the edges of the atlas connectome are representative of those of individual participants of the Human Connectome Project in terms of the spatial organization of streamlines and spatial patterns of track-density. Next, the new white matter connectome was used to develop multi-layer, connectivity-based labels for each white matter voxel of the atlas, consistent with the fact that each voxel may contain axons from multiple connections. The regionconnect algorithm was then developed to rapidly integrate information contained in the multi-layer labels across voxels of a white matter region and to generate a list of the most probable connections traversing that region. Usage of regionconnect does not require high angular resolution diffusion MRI or any MRI data. The regionconnect algorithm as well as the white matter tractogram and connectome, multi-layer, connectivity-based labels, and associated resources developed for the IIT Human Brain Atlas v.5.0 in this work are available at www.nitrc.org/projects/iit. An interactive, online version of regionconnect is also available at www.iit.edu/~mri.

Development and evaluation of a high performance T1-weighted brain template for use in studies on older adults.

Τhe accuracy of template-based neuroimaging investigations depends on the template's image quality and representativeness of the individuals under study. Yet a thorough, quantitative investigation of how available standardized and study-specific T1-weighted templates perform in studies on older adults has not been conducted. The purpose of this work was to construct a high-quality standardized T1-weighted template specifically designed for the older adult brain, and systematically compare the new template to several other standardized and study-specific templates in terms of image quality, performance in spatial normalization of older adult data and detection of small inter-group morphometric differences, and representativeness of the older adult brain. The new template was constructed with state-of-the-art spatial normalization of high-quality data from 222 older adults. It was shown that the new template (a) exhibited high image sharpness, (b) provided higher inter-subject spatial normalization accuracy and (c) allowed detection of smaller inter-group morphometric differences compared to other standardized templates, (d) had similar performance to that of study-specific templates constructed with the same methodology, and (e) was highly representative of the older adult brain.

The novel FAT4 activator jujuboside A suppresses NSCLC tumorigenesis by activating HIPPO signaling and inhibiting YAP nuclear translocation.

FAT atypical cadherin 4 (FAT4) has been identified as a tumor suppressor in lung cancers. However, no agent for lung cancer treatment targeting FAT4 has been used in the clinic. Jujuboside A (JUA) is a major active compound in Semen Ziziphi Spinosae. Semen Ziziphi Spinosae is a traditional Chinese herbal medicine used clinically for tumor treatment to improve patients' quality of life. However, the anti-lung cancer activity and the underlying mechanisms of JUA are not yet fully understood. Here, we demonstrated the anti-lung cancer activity of JUA in two lung cancer mice models and three non-small cell lung cancer (NSCLC) cell lines, and further illustrated its underlying mechanisms. JUA suppressed the occurrence and development of lung cancer and extended mice survival in vivo, and suppressed NSCLC cell activities through cell cycle arrest, proliferation suppression, stemness inhibition and senescence promotion. Moreover, JUA directly bound with and activated FAT4, subsequently activating FAT4-HIPPO signaling and inhibiting YAP nuclear translocation. Knockdown of FAT4 diminished JUA's effects on HIPPO signaling, YAP nuclear translocation, cell proliferation and cellular senescence. In conclusion, JUA significantly suppressed NSCLC tumorigenesis by regulating FAT4-HIPPO-YAP signaling. Our findings suggest that JUA is a novel FAT4 activator that can be developed as a promising NSCLC therapeutic agent targeting the FAT4-HIPPO-YAP pathway.

Irinotecan-mediated diarrhea is mainly correlated with intestinal exposure to SN-38: Critical role of gut Ugt.

BACKGROUND AND PURPOSE: Irinotecan-induced diarrhea (IID) results from intestinal damages by its active metabolite SN-38. Alleviation of these damages has focused on lowering luminal SN-38 concentrations. However, it is unclear if the enteric bioavailability of SN-38 is mostly dependent on luminal SN-38 concentrations. EXPERIMENTAL APPROACH: Irinotecan (50 mg/kg, i.p. once daily for 6 days) was administered to female wildtype FVB, Mdr1a (-/-), Mrp2 (-/-) and Bcrp1 (-/-) mice for pharmacokinetic (PK), toxicokinetic (TK) and biodistribution studies. Plasma PK/TK profiles and tissues drug distribution were determined after first or sixth daily doses, along with activities of blood and gut esterases and intestinal Ugts. Caco-2 cells and bile-cannulate mice were used to further investigate intestinal and biliary disposition of irinotecan and its metabolites. KEY RESULTS: Significant differences in IID severity were observed with the susceptible rank of Bcrp1(-/-) > wildtype FVB > Mdr1a(-/-) > Mrp2(-/-). This rank order did not correlate with biliary excretion rates of SN-38/SN-38G. Rather, the severity was best correlated (R = 0.805) with the intestinal ratio of Css SN-38/SN-38G, a measure of gut Ugt activity. On the contrary, IID was poorly correlated with plasma AUC ratio of SN-38/SN-38G (R = 0.227). Increased intestinal esterase activities due to repeated dosing and gut efflux transporter functionality are the other key factors that determine SN-38 enteric exposures. CONCLUSION AND IMPLICATIONS: Intestinal SN-38 exposure is mainly affected by intestinal Ugt activities and blood esterase activities, and strongly correlated with severity of IID. Modulating intestinal SN-38 concentration and gut Ugt expression should be the focus of future studies to alleviate IID.

Mdr1a, Bcrp and Mrp2 regulate the efficacy and toxicity of mesaconitine and hypaconitine by altering their tissue accumulation and in vivo residence.

Mesaconitine (MA) and hypaconitine (HA) are the main bioactive/toxic alkaloids of Aconitum carmichaelii Debx, and MDR1, BCRP and MRP2 are involved in their efflux in vitro. This study aimed to explore the effects of Mdr1a, Bcrp and Mrp2 on the efficacy/toxicity of MA and HA by using efflux transporter gene knockout mouse models. The analgesic and anti-inflammatory effects, neurotoxicity/cardiotoxicity, and pharmacokinetic profiles of MA and HA were studied. Compared to wild-type mice, the analgesic effects of MA or HA were significantly enhanced in Mdr1a--/-, Bcrp1-/- and Mrp2-/- mice, and the anti-inflammatory effects notably increased in Bcrp1-/- and Mrp2-/- mice. Compared to wild-type mice, Mdr1a-/-, Bcrp1-/- and Mrp2-/- mice suffered from severe karyopyknosis and edema in the brain after MA or HA treatment. Meanwhile, significant arrhythmia appeared, and the heart rate and RR-interval were greatly altered in Mdr1a-/-, Bcrp1-/- and Mrp2-/- mice. Additionally, obvious disorder of cardiomyocytes were observed, and the CK and cTnT (indicators of heart injury) levels were greatly enhanced in efflux transporter gene knockout mice. The brain levels of MA and HA were markedly increased in Mdr1a-/-, Bcrp1-/- and Mrp2-/- mice, and the heart levels of MA and HA enhanced greatly in Mdr1a-/- mice. The MRT0-t values of MA and HA were remarkably enhanced in most efflux transporter gene knockout mice. In conclusion, Mdr1a, Bcrp and Mrp2 were all involved in regulating the efficacy/toxicity of MA and HA by altering their tissue accumulation and in vivo residence. Among the three efflux transporters, Mdr1a had a superior regulatory effect.

Protein kinase C is involved in the neuroprotective effect of berberine against intrastriatal injection of quinolinic acid-induced biochemical alteration in mice.

Protein kinase C (PKC) shows a neuronal protection effect in neurodegenerative diseases. In this study, we test whether berberine has a positive effect on the activity of PKC in quinolinic acid (QA)-induced neuronal cell death. We used intrastriatal injections of QA mice model to test the effect of berberine on motor and cognitive deficits, and the PKC signalling pathway. Treatment with 50 mg/kg b.w of berberine for 2 weeks significantly prevented QA-induced motor and cognitive impairment and related pathologic changes in the brain. QA inhibited the phosphorylation of PKC and its downstream molecules, GSK-3ß, ERK and CREB, enhanced the glutamate level and release of neuroinflammatory cytokines; these effects were attenuated by berberine. We used in vivo infusion of Go6983, a PKC inhibitor to disturb PKC activity in mice brain, and found that the effect of berberine to reverse motor and cognitive deficits was significantly reduced. Moreover, inhibition of PKC also blocked the anti-excitotoxicity effect of berberine, which is induced by glutamate in PC12 cells and BV2 cells, as well as anti-neuroinflammatory effect in LPS-stimulated BV2 cells. Above all, berberine showed neuroprotective effect against QA-induced acute neurotoxicity by activating PKC and its downstream molecules.

Breast Cancer Resistance Protein and Multidrug Resistance Protein 2 Determine the Disposition of Esculetin-7-O-Glucuronide and 4-Methylesculetin-7-O-Glucuronide.

Esculetin (ET)-7-O-glucuronide (ET-G) and 4-methylesculetin (4-ME)-7-O-glucuronide (4-ME-G) are the main glucuronide of ET and 4-ME, respectively. The disposition mediated by efflux transporters for glucuronide has significant influence on the pharmacokinetic profile and efficacy of bioactive compounds. In the current study, transporter gene knockout mice and Caco-2 cells were used to explore the effects of breast cancer resistance protein (BCRP) and multidrug resistance-associated protein 2 (MRP2) on the disposition of ET-G and 4-ME-G. After oral or i.v. administration of ET and 4-ME, the area under the plasma concentration-time curve from time 0 to the last data point or infinity values of ET, 4-ME, and their glucuronides (ET-G and 4-ME-G) were remarkably and significantly increased in most Bcrp1-/- and Mrp2-/- mice compared with those in wild-type FVB mice (P < 0.05). These results were accompanied with a significant increase of maximum plasma concentration values (P < 0.05). In Caco-2 monolayers, the efflux and clearance rates of ET-G and 4-ME-G were markedly reduced by the BCRP inhibitor Ko143 and MRP2 inhibitor MK571 on the apical side (P < 0.05). In an intestinal perfusion study, the excretion of ET-G was significantly decreased in perfusate and increased in plasma in Bcrp1-/- mice compared with those in wild-type FVB mice (P < 0.05). The 4-ME-G concentration was also decreased in the bile in transporter gene knockout mice. ET and 4-ME showed good permeability in both Caco-2 monolayers [apparent permeability (Papp ) ≥ 0.59 × 10-5 cm/s] and duodenum (Papp ≥ 1.81). In conclusion, BCRP and MRP2 are involved in excreting ET-G and 4-ME-G. ET and 4-ME are most likely absorbed via passive diffusion in the intestines.

Interplay of Efflux Transporters with Glucuronidation and Its Impact on Subcellular Aglycone and Glucuronide Disposition: A Case Study with Kaempferol.

Glucuronidation is a major process of drug metabolism and elimination that generally governs drug efficacy and toxicity. Publications have demonstrated that efflux transporters control intracellular glucuronidation metabolism. However, it is still unclear whether and how efflux transporters interact with UDP-glucuronosyltransferases (UGTs) in subcellular organelles. In this study, kaempferol, a model fluorescent flavonoid, was used to investigate the interplay of glucuronidation with transport at the subcellular level. Human recombinant UGTs and microsomes were utilized to characterize the in vitro glucuronidation kinetics of kaempferol. The inhibition of UGTs and efflux transporters on the subcellular disposition of kaempferol were determined visually and quantitatively in Caco-2/TC7 cells. The knockout of transporters on the subcellular accumulation of kaempferol in liver and intestine were evaluated visually. ROS and Nrf2 were assayed to evaluate the pharmacological activities of kaempferol. The results showed that UGT1A9 is the primary enzyme responsible for kaempferol glucuronidation. Visual and quantitative data showed that the UGT1A9 inhibitor carvacrol caused a significant rise in subcellular aglycone and reduction in subcellular glucuronides of kaempferol. The inhibition and knockout of transporters, such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated proteins (MRPs), exhibited a marked increase in subcellular kaempferol and decrease in its subcellular glucuronides. Correspondingly, inhibition of UGT1A9 and transporters led to increased kaempferol and, consequently, a significantly enhanced ROS scavenging efficiency and nuclear translocation of Nrf2. In conclusion, the interplay of efflux transporters (P-gp, BCRP, and MRPs) and UGTs govern the subcellular exposure and corresponding pharmacological activity of kaempferol.

DACT2 Epigenetic Stimulator Exerts Dual Efficacy for Colorectal Cancer Prevention and Treatment.

DACT2, a tumor suppressor gene in various tumors, is frequently down-regulated via hypermethylation. We found DACT2 gene expressions were dramatically silenced (P = 0.002, n = 8) in our clinical colorectal cancer (CRC) tissues, and TCGA data revealed DACT2 hypermethylation correlated to CRC poor prognosis (P = 0.0129, HR = 0.2153, n = 248). Thus, by screening twelve nutritional compounds, we aimed to find out an effective DACT2 epigenetic stimulator to determine whether DACT2 epigenetic restoration could reverse CRC tumorigenesis. We found that kaempferol significantly increased DACT2 expressions up to 3.47-fold in three CRC cells (HCT116, HT29, and YB5). Furthermore, kaempferol remarkably decreased DACT2 methylation (range: 19.58%-67.00%, P < 0.01), while increased unmethylated DACT2 by 13.72-fold (P < 0.01) via directly binding to DNA methyltransferases DNMT1. By epigenetic reactivating DACT2 transcription, kaempferol notably inhibited nuclear ß-catenin expression to inactivate Wnt/ß-catenin pathway, which consequently restricted CRC cells proliferation and migration. Moreover, in AOM/DSS-induced CRC tumorigenesis, kaempferol-demethylated DACT2 effectively decreased tumor load (range: 50.00%-73.52%, P < 0.05). By determining the chemopreventive and chemotherapeutic efficacy of a novel DACT2 demethylating stimulator, we demonstrated that DACT2 epigenetic restoration could successfully slow down and reverse CRC tumorigenesis.

Tissue Distribution and Gender-Specific Protein Expression of Cytochrome P450 in five Mouse Genotypes with a Background of FVB.

PURPOSE: To systematically investigate tissue distribution and gender-specific protein expression of Cytochrome P450 (Cyps) in five mouse genotypes with a background of Friend virus B (FVB). METHODS: The Cyps were extracted from the tissue S9 fractions of the main metabolic organs and then absolutely quantified by applying the UHPLC-MS/MS method. RESULTS: The liver had the highest expression of Cyps, followed by the small intestine and kidney. In the liver, Cyp1a2, Cyp2c29, Cyp2c39, Cyp2d22, Cyp2e1, and Cyp3a11 were the main isoforms. Cyp1a2 and Cyp2c29 were male-specific, while Cyp2c39 was female-specific. Compared with the expression in Wild-type (WT) FVB mice, the expression of Cyp1a2, Cyp1b1, Cyp2d22, and Cyp3a25 significantly decreased in female efflux transporter (ET) knockout mice. In the small intestine, Cyp2c29 and Cyp3a11 were the major isoforms. Knockout of ET didn't alter the expression levels of most Cyps. However, female ET knockout mice presented higher Cyp2c29 expression than WT FVB mice. The Cyp7a1 expression was markedly decreased in ET knockout mice except Bcrp1-/- mice. In the kidney, Cyp2e1 was the main isoform and exhibited male specificity. Knockout of ET slightly affected the protein expression of Cyps in the brain, heart, lung, spleen and stomach. CONCLUSIONS: A comprehensive understanding of the distribution characteristics and gender-specific expression of Cyps in major metabolic organs of WT and ET knockout FVB mice should contribute to a better understanding of drug efficacy and toxicity, and drug-drug interactions.

Age-related changes in hepatic expression and activity of drug metabolizing enzymes in male wild-type and breast cancer resistance protein knockout mice.

This study aimed to reveal age-related changes in the expression and activity of seven hepatic drug metabolizing enzymes (DMEs) in male wild-type and breast cancer resistance protein knockout (Bcrp1-/- ) FVB mice. The protein expression of four cytochrome P450 (Cyps) (Cyp3a11, 2d22, 2e1, and 1a2), and three UDP-glucuronosyltransferases (Ugts) (Ugt1a1, 1a6a, and 1a9) in liver microsomes of wild-type and Bcrp1-/- FVB mice at different ages were determined using a validated ultra high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) method. The activities and mRNA levels of these DMEs were measured using the probe substrates method and real-time PCR, respectively. In the liver of wild-type FVB mice, Cyp3a11, 2d22, 2e1, 1a2, Ugt1a1, and 1a6a displayed maximum protein levels at 6-9 weeks of age. Cyp1a2, Ugt1a1, 1a6a, and 1a9 showed maximum activities at 6-9 weeks of age, whereas Cyp3a11, 2d22, and 2e1 showed maximum activities in 1-3-week-old mice. Additionally, most of the DMEs showed maximum mRNA levels in 17-week-old mice liver. Compared with wild-type FVB mice, the protein levels of these DMEs showed no significant changes in Bcrp1-/- FVB mice liver. However, the activity of Cyp2e1 was increased and that of Cyp2d22 was decreased. In conclusion, the seven hepatic DMEs in FVB mice liver showed significant alterations in an isoform-specific manner with increased age. Although the protein levels of these DMEs showed no significant changes, the activities of Cyp2e1 and 2d22 were changed in Bcrp1-/- mice.