High-throughput and genome-scale targeted mutagenesis using CRISPR in a nonmodel multicellular organism, Bombyx mori.

Large-scale genetic mutant libraries are powerful approaches to interrogating genotype-phenotype correlations and identifying genes responsible for certain environmental stimuli, both of which are the central goal of life science study. We produced the first large-scale CRISPR-Cas9-induced library in a nonmodel multicellular organism, Bombyx mori We developed a piggyBac-delivered binary genome editing strategy, which can simultaneously meet the requirements of mixed microinjection, efficient multipurpose genetic operation, and preservation of growth-defect lines. We constructed a single-guide RNA (sgRNA) plasmid library containing 92,917 sgRNAs targeting promoters and exons of 14,645 protein-coding genes, established 1726 transgenic sgRNA lines following microinjection of 66,650 embryos, and generated 300 mutant lines with diverse phenotypic changes. Phenomic characterization of mutant lines identified a large set of genes responsible for visual phenotypic or economically valuable trait changes. Next, we performed pooled context-specific positive screens for tolerance to environmental pollutant cadmium exposure, and identified KWMTBOMO12902 as a strong candidate gene for breeding applications in sericulture industry. Collectively, our results provide a novel and versatile approach for functional B. mori genomics, as well as a powerful resource for identifying the potential of key candidate genes for improving various economic traits. This study also shows the effectiveness, practicality, and convenience of large-scale mutant libraries in other nonmodel organisms.

The OPEN STOMATA1-SPIRAL1 module regulates microtubule stability during abscisic acid-induced stomatal closure in Arabidopsis.

Drought stress triggers abscisic acid (ABA) signaling in guard cells and induces stomatal closure to prevent water loss in land plants. Stomatal movement is accompanied by reorganization of the cytoskeleton. Cortical microtubules disassemble in response to ABA, which is required for stomatal closure. However, how ABA signaling regulates microtubule disassembly is unclear, and the microtubule-associated proteins (MAPs) involved in this process remain to be identified. In this study, we show that OPEN STOMATA 1 (OST1), a central component in ABA signaling, mediates microtubule disassembly during ABA-induced stomatal closure in Arabidopsis thaliana. We identified the MAP SPIRAL1 (SPR1) as the substrate of OST1. OST1 interacts with and phosphorylates SPR1 at Ser6, which promotes the disassociation of SPR1 from microtubules and facilitates microtubule disassembly. Compared with the wild type, the spr1 mutant exhibited significantly greater water loss and reduced ABA responses, including stomatal closure and microtubule disassembly in guard cells. These phenotypes were restored by introducing the phosphorylated active form of SPR1. Our findings demonstrate that SPR1 positively regulates microtubule disassembly during ABA-induced stomatal closure, which depends on OST1-mediated phosphorylation. These findings reveal a specific connection between a core component of ABA signaling and MAPs.

mDia formins form hetero-oligomers and cooperatively maintain murine hematopoiesis.

mDia formin proteins regulate the dynamics and organization of the cytoskeleton through their linear actin nucleation and polymerization activities. We previously showed that mDia1 deficiency leads to aberrant innate immune activation and induces myelodysplasia in a mouse model, and mDia2 regulates enucleation and cytokinesis of erythroblasts and the engraftment of hematopoietic stem and progenitor cells (HSPCs). However, whether and how mDia formins interplay and regulate hematopoiesis under physiological and stress conditions remains unknown. Here, we found that both mDia1 and mDia2 are required for HSPC regeneration under stress, such as serial plating, aging, and reconstitution after myeloid ablation. We showed that mDia1 and mDia2 form hetero-oligomers through the interactions between mDia1 GBD-DID and mDia2 DAD domains. Double knockout of mDia1 and mDia2 in hematopoietic cells synergistically impaired the filamentous actin network and serum response factor-involved transcriptional signaling, which led to declined HSPCs, severe anemia, and significant mortality in neonates and newborn mice. Our data demonstrate the potential roles of mDia hetero-oligomerization and their non-rodent functions in the regulation of HSPCs activity and orchestration of hematopoiesis.

Pleckstrin-2 Mediates the Activation of AKT in Prostate Cancer and Is Repressed by Androgen Receptor.

Phosphatidylinositol 3-kinase (PI3K)-AKT and androgen receptor (AR) pathways are commonly activated in prostate cancers. Their reciprocal regulation makes advanced prostate cancers difficult to treat. The current study shows that pleckstrin-2 (PLEK-2, a proto-oncoprotein involved in the activation and stabilization of AKT, connects these two pathways. Genetic evidence is provided that suggests that Plek2 deficiency largely reverted tumorigenesis in Pten prostate-specific knockout mice and that overexpression of PLEK2 promoted the proliferation and colony formation of prostate cancer cells in vitro. In addition, it was found that PLEK2 was negatively regulated by AR, that AR transcriptionally repressed PLEK2 through binding to the PLEK2 promoter region, and that overexpression of AR reduced PLEK2 expression, which inactivated AKT. Conversely, knockdown of AR in prostate cancer cells increased PLEK2 expression and activated the AKT pathway. This reciprocal inhibitory loop can be pharmacologically targeted using the PLEK2 inhibitor. It is shown that the PLEK2 inhibitor dose-dependently inhibited prostate cancer cell proliferation with the inactivation of AKT. Overall, the current study uncovers the crucial role of PLEK2 in prostate cancer proliferation and provides the rationale for targeting PLEK2 to treat prostate cancers.

Alteration of the intestinal microbiota and serum metabolites in a mouse model of Pon1 gene ablation.

Mutations in the Paraoxonase 1 (Pon1) gene underlie aging, cardiovascular disease, and impairments of the nervous and gastrointestinal systems and are linked to the intestinal microbiome. The potential role of Pon1 in modulating the intestinal microbiota and serum metabolites is poorly understood. The present study demonstrated that mice with genomic excision of Pon1 by a multiplexed guide RNA CRISPR/Cas9 approach exhibited disrupted gut microbiota, such as significantly depressed alpha-diversity and distinctly separated beta diversity, accompanied by varied profiles of circulating metabolites. Furthermore, genomic knock in of Pon1 exerted a distinct effect on the intestinal microbiome and serum metabolome, including dramatically enriched Aerococcus, linoleic acid and depleted Bacillus, indolelactic acid. Specifically, a strong correlation was established between bacterial alterations and metabolites in Pon1 knockout mice. In addition, we identified metabolites related to gut bacteria in response to Pon1 knock in. Thus, the deletion of Pon1 affects the gut microbiome and functionally modifies serum metabolism, which can lead to dysbiosis, metabolic dysfunction, and infection risk. Together, these findings put forth a role for Pon1 in microbial alterations that contribute to metabolism variations. The function of Pon1 in diseases might at least partially depend on the microbiome.

Downregulation of miR-503-5p Promotes the Development of Pancreatic Cancer by Targeting Cyclin E2.

This study aimed to elucidate the role of microRNA-503 (miR-503) in pancreatic cancer (PC) progression and the underlying regulatory mechanisms. We acquired miR-503-3p and miR-503-5p expression data along with survival times of PC and normal samples from the UCSC Xena database. Using the t-test, we compared the expression of miR-503-3p and miR-503-5p between PC and normal samples, and evaluated their prognostic significance via Kaplan-Meier survival analysis. The expression of miR-503-5p in PC cells was detected by quantitative PCR. We subsequently overexpressed miR-503-5p in PC cells and examined cell viability, apoptosis, and migration through CCK8 assay, flow cytometry, and Transwell assay, respectively. Potential functional targets were identified using miRTarBase and validated by dual-luciferase reporter assay. Both miR-503-3p and miR-503-5p expression were found to be downregulated in PC; however, only miR-503-5p was linked to cancer prognosis based on public data. In vitro experiments demonstrated that overexpression of miR-503-5p substantially decreased cell viability, induced apoptosis, caused G0/G1 arrest, and inhibited cell migration. miR-503-5p was found to target cyclin E2 (CCNE2), and overexpression of CCNE2 could counteract the effects of miR-503-5p on PC cells. Conclusion: The downregulation of miR-503-5p enhances the progression of PC by targeting CCNE2. The detection of miR-503-5p expression may provide valuable insights for the prevention and prognostic evaluation of PC.

Altered white matter functional pathways in Alzheimer's disease.

Alzheimer's disease (AD) is associated with functional disruption in gray matter (GM) and structural damage to white matter (WM), but the relationship to functional signal in WM is unknown. We performed the functional connectivity (FC) and graph theory analysis to investigate abnormalities of WM and GM functional networks and corpus callosum among different stages of AD from a publicly available dataset. Compared to the controls, AD group showed significantly decreased FC between the deep WM functional network (WM-FN) and the splenium of corpus callosum, between the sensorimotor/occipital WM-FN and GM visual network, but increased FC between the deep WM-FN and the GM sensorimotor network. In the clinical groups, the global assortativity, modular interaction between occipital WM-FN and visual network, nodal betweenness centrality, degree centrality, and nodal clustering coefficient in WM- and GM-FNs were reduced. However, modular interaction between deep WM-FN and sensorimotor network, and participation coefficients of deep WM-FN and splenium of corpus callosum were increased. These findings revealed the abnormal integration of functional networks in different stages of AD from a novel WM-FNs perspective. The abnormalities of WM functional pathways connect downward to the corpus callosum and upward to the GM are correlated with AD.

The novel GATA1-interacting protein HES6 is an essential transcriptional cofactor for human erythropoiesis.

Normal erythropoiesis requires the precise regulation of gene expression patterns, and transcription cofactors play a vital role in this process. Deregulation of cofactors has emerged as a key mechanism contributing to erythroid disorders. Through gene expression profiling, we found HES6 as an abundant cofactor expressed at gene level during human erythropoiesis. HES6 physically interacted with GATA1 and influenced the interaction of GATA1 with FOG1. Knockdown of HES6 impaired human erythropoiesis by decreasing GATA1 expression. Chromatin immunoprecipitation and RNA sequencing revealed a rich set of HES6- and GATA1-co-regulated genes involved in erythroid-related pathways. We also discovered a positive feedback loop composed of HES6, GATA1 and STAT1 in the regulation of erythropoiesis. Notably, erythropoietin (EPO) stimulation led to up-regulation of these loop components. Increased expression levels of loop components were observed in CD34+ cells of polycythemia vera patients. Interference by either HES6 knockdown or inhibition of STAT1 activity suppressed proliferation of erythroid cells with the JAK2V617F mutation. We further explored the impact of HES6 on polycythemia vera phenotypes in mice. The identification of the HES6-GATA1 regulatory loop and its regulation by EPO provides novel insights into human erythropoiesis regulated by EPO/EPOR and a potential therapeutic target for the management of polycythemia vera.

Strong Protection by 4-Hydroxyestrone against Erastin-Induced Ferroptotic Cell Death in Estrogen Receptor-Negative Human Breast Cancer Cells: Evidence for Protein Disulfide Isomerase as a Mechanistic Target for Protection.

Ferroptosis is a recently identified form of regulated cell death, characterized by excessive iron-dependent lipid peroxidation. Recent studies have demonstrated that protein disulfide isomerase (PDI) is an important mediator of chemically induced ferroptosis and also a new target for protection against ferroptosis-associated cell death. In the present study, we identified that 4-hydroxyestrone (4-OH-E1), a metabolic derivative of endogenous estrogen, is a potent small-molecule inhibitor of PDI, and can strongly protect against chemically induced ferroptotic cell death in the estrogen receptor-negative MDA-MB-231 human breast cancer cells. Pull-down and CETSA assays demonstrated that 4-OH-E1 can directly bind to PDI both in vitro and in intact cells. Computational modeling analysis revealed that 4-OH-E1 forms two hydrogen bonds with PDI His256, which is essential for its binding interaction and thus inhibition of PDI's catalytic activity. Additionally, PDI knockdown attenuates the protective effect of 4-OH-E1 as well as cystamine (a known PDI inhibitor) against chemically induced ferroptosis in human breast cancer cells. Importantly, inhibition of PDI by 4-OH-E1 and cystamine or PDI knockdown by siRNAs each markedly reduces iNOS activity and NO accumulation, which has recently been demonstrated to play an important role in erastin-induced ferroptosis. In conclusion, this study demonstrates that 4-OH-E1 is a novel inhibitor of PDI and can strongly inhibit ferroptosis in human breast cancer cells in an estrogen receptor-independent manner. The mechanistic understanding gained from the present study may also aid in understanding the estrogen receptor-independent cytoprotective actions of endogenous estrogen metabolites in many noncancer cell types.

Laser Precise Synthesis of Oxidation-Free High-Entropy Alloy Nanoparticle Libraries.

High-entropy alloy nanoparticles (HEA-NPs) show exceptional properties and great potential as a new generation of functional materials, yet a universal and facile synthetic strategy in air toward nonoxidized and precisely controlled composition remains a huge challenge. Here we provide a laser scribing method to prepare single-phase solid solution HEA-NPs libraries in air with tunable composition at the atomic level, taking advantage of the laser-induced metastable thermodynamics and substrate-assisted confinement effect. The three-dimensional porous graphene substrate functions as a microreactor during the fast heating/cooling process, which is conductive to the generation of the pure alloy phase by effectively blocking the binding of oxygen and metals, but is also beneficial for realizing accurate composition control via microstructure confinement-endowed favorable vapor pressure. Furthermore, by combining an active learning approach based on an adaptive design strategy, we discover an optimal composition of quinary HEA-NP catalysts with an ultralow overpotential for Li-CO2 batteries. This method provides a simple, fast, and universal in-air route toward the controllable synthesis of HEA-NPs, potentially integrated with machine learning to accelerate the research on HEAs.

Pan-cancer characterization of cell-free immune-related miRNA identified as a robust biomarker for cancer diagnosis.

Minimally invasive testing is essential for early cancer detection, impacting patient survival rates significantly. Our study aimed to establish a pioneering cell-free immune-related miRNAs (cf-IRmiRNAs) signature for early cancer detection. We analyzed circulating miRNA profiles from 15,832 participants, including individuals with 13 types of cancer and control. The data was randomly divided into training, validation, and test sets (7:2:1), with an additional external test set of 684 participants. In the discovery phase, we identified 100 differentially expressed cf-IRmiRNAs between the malignant and non-malignant, retaining 39 using the least absolute shrinkage and selection operator (LASSO) method. Five machine learning algorithms were adopted to construct cf-IRmiRNAs signature, and the diagnostic classifies based on XGBoost algorithm showed the excellent performance for cancer detection in the validation set (AUC: 0.984, CI: 0.980-0.989), determined through 5-fold cross-validation and grid search. Further evaluation in the test and external test sets confirmed the reliability and efficacy of the classifier (AUC: 0.980 to 1.000). The classifier successfully detected early-stage cancers, particularly lung, prostate, and gastric cancers. It also distinguished between benign and malignant tumors. This study represents the largest and most comprehensive pan-cancer analysis on cf-IRmiRNAs, offering a promising non-invasive diagnostic biomarker for early cancer detection and potential impact on clinical practice.

Histone Variant H3.3 Controls Arabidopsis Fertility by Regulating Male Gamete Development.

Reprograming of chromatin structures and changes in gene expression are critical for plant male gamete development, and epigenetic marks play an important role in these processes. Histone variant H3.3 is abundant in euchromatin and is largely associated with transcriptional activation. The precise function of H3.3 in gamete development remains unclear in plants. Here, we report that H3.3 is abundantly expressed in Arabidopsis anthers and its knockout mutant h3.3-1 is sterile due to male sterility. Transcriptome analysis of young inflorescence has identified 2348 genes downregulated in h3.3-1 mutant, among which 1087 target genes are directly bound by H3.3, especially at their 3' ends. As a group, this set of H3.3 targets is enriched in the reproduction-associated processes including male gamete generation, pollen sperm cell differentiation and pollen tube growth. The function of H3.3 in male gamete development is dependent on the Anti-Silencing Factor 1A/1B (ASF1A/1B)-Histone regulator A (HIRA)-mediated pathway. Our results suggest that ASF1A/1B-HIRA-mediated H3.3 deposition at its direct targets for transcription activation forms the regulatory networks responsible for male gamete development.

Compressed cerebro-cerebellar functional gradients in children and adolescents with attention-deficit/hyperactivity disorder.

Both cortical and cerebellar developmental differences have been implicated in attention-deficit/hyperactivity disorder (ADHD). Recently accumulating neuroimaging studies have highlighted hierarchies as a fundamental principle of brain organization, suggesting the importance of assessing hierarchy abnormalities in ADHD. A novel gradient-based resting-state functional connectivity analysis was applied to investigate the cerebro-cerebellar disturbed hierarchy in children and adolescents with ADHD. We found that the interaction of functional gradient between diagnosis and age was concentrated in default mode network (DMN) and visual network (VN). At the same time, we also found that the opposite gradient changes of DMN and VN caused the compression of the cortical main gradient in ADHD patients, implicating the co-occurrence of both low- (visual processing) and high-order (self-related thought) cognitive dysfunction manifesting in abnormal cerebro-cerebellar organizational hierarchy in ADHD. Our study provides a neurobiological framework to better understand the co-occurrence and interaction of both low-level and high-level functional abnormalities in the cortex and cerebellum in ADHD.

Salicylic acid inducing the expression of maize anti-insect gene SPI: a potential control strategy for Ostrinia furnacalis.

BACKGROUND: Due to being rooted in the ground, maize (Zea mays L.) is unable to actively escape the attacks of herbivorous insects such as the Asian corn borer (Ostrinia furnacalis). In contrast to the passive damage, plants have evolved defense mechanisms to protect themselves from herbivores. Salicylic acid, a widely present endogenous hormone in plants, has been found to play an important role in inducing plant resistance to insects. In this study, we screened and identified the insect resistance gene SPI, which is simultaneously induced by SA and O. furnacalis feeding, through preliminary transcriptome data analysis. The functional validation of SPI was carried out using bioinformatics, RT-qPCR, and heterologous expression protein feeding assays. RESULTS: Both SA and O. furnacalis treatment increased the expression abundance of SA-synthesis pathway genes and SPI in three maize strains, and the upregulation of SPI was observed strongly at 6 hours post-treatment. The expression of SPI showed a temporal relationship with SA pathway genes, indicating that SPI is a downstream defense gene regulated by SA. Protein feeding assays using two different expression vectors demonstrated that the variation in SPI protein activity among different strains is mainly due to protein modifications. CONCLUSIONS: Our research results indicate that SPI, as a downstream defense gene regulated by SA, is induced by SA and participates in maize's insect resistance. The differential expression levels of SPI gene and protein modifications among different maize strains are one of the reasons for the variation in insect resistance. This study provides new insights into ecological pest control in maize and valuable insights into plant responses to SA-induced insect resistance.

SiC@NiO Core-Shell Nanowire Networks-Based Optoelectronic Synapses for Neuromorphic Computing and Visual Systems at High Temperature.

1D nanowire networks, sharing similarities of structure, information transfer, and computation with biological neural networks, have emerged as a promising platform for neuromorphic systems. Based on brain-like structures of 1D nanowire networks, neuromorphic synaptic devices can overcome the von Neumann bottleneck, achieving intelligent high-efficient sensing and computing function with high information processing rates and low power consumption. Here, high-temperature neuromorphic synaptic devices based on SiC@NiO core-shell nanowire networks optoelectronic memristors (NNOMs) are developed. Experimental results demonstrate that NNOMs attain synaptic short/long-term plasticity and modulation plasticity under both electrical and optical stimulation, and exhibit advanced functions such as short/long-term memory and "learning-forgetting-relearning" under optical stimulation at both room temperature and 200 °C. Based on the advanced functions under light stimulus, the constructed 5 × 3 optoelectronic synaptic array devices exhibit a stable visual memory function up to 200 °C, which can be utilized to develop artificial visual systems. Additionally, when exposed to multiple electronic or optical stimuli, the NNOMs effectively replicate the principles of Pavlovian classical conditioning, achieving visual heterologous synaptic functionality and refining neural networks. Overall, with abundant synaptic characteristics and high-temperature thermal stability, these neuromorphic synaptic devices offer a promising route for advancing neuromorphic computing and visual systems.

Electroactive Nanomaterials for the Prevention and Treatment of Heart Failure: From Materials and Mechanisms to Applications.

Heart failure (HF) represents a cardiovascular disease that significantly threatens global well-being and quality of life. Electroactive nanomaterials, characterized by their distinctive physical and chemical properties, emerge as promising candidates for HF prevention and management. This review comprehensively examines electroactive nanomaterials and their applications in HF intervention. It presents the definition, classification, and intrinsic characteristics of conductive, piezoelectric, and triboelectric nanomaterials, emphasizing their mechanical robustness, electrical conductivity, and piezoelectric coefficients. The review elucidates their applications and mechanisms: 1) early detection and diagnosis, employing nanomaterial-based sensors for real-time cardiac health monitoring; 2) cardiac tissue repair and regeneration, providing mechanical, chemical, and electrical stimuli for tissue restoration; 3) localized administration of bioactive biomolecules, genes, or pharmacotherapeutic agents, using nanomaterials as advanced drug delivery systems; and 4) electrical stimulation therapies, leveraging their properties for innovative pacemaker and neurostimulation technologies. Challenges in clinical translation, such as biocompatibility, stability, and scalability, are discussed, along with future prospects and potential innovations, including multifunctional and stimuli-responsive nanomaterials for precise HF therapies. This review encapsulates current research and future directions concerning the use of electroactive nanomaterials in HF prevention and management, highlighting their potential to innovating in cardiovascular medicine.

Coffee consumption during pregnancy increases the risk of preeclampsia in rats by inhibiting 2-methoxyestradiol production.

Preeclampsia (PE) is a pregnancy-specific disease that causes maternal symptoms such as high blood pressure and adverse pregnancy outcomes. 2-methoxyestradiol (2-MeO-E2), an endogenous metabolite of 17ß-estradiol (E2) formed by Catechol-O-Methyltransferase (COMT), plays an important role in pregnancy. Our earlier studies have shown that polyphenols present in coffee can inhibit COMT activity, which may inhibit the formation of 2-MeO-E2 and contribute to PE. Therefore, the current study aims to investigate the possible effect and mechanism of coffee intake during pregnancy on PE in SD rats. Coffee is administered with or without cotreatment of 2-MeO-E2 to pregnant rats from the10th to the18th day of pregnancy. The results show that pregnant rats with coffee intake had prominent fetal growth restriction, hypertension and proteinuria, which can be ameliorated by co-treatment of 2-MeO-E2. In addition, coffee treatment leads to significantly decreased serum 2-MeO-E2. Therefore, the PE symptoms induced by coffee treatment is probably mediated by decreased 2-MeO-E2. Our findings provide new mechanistic insight into how coffee intake could lead to increased risk of PE, and demonstrate the effectiveness of 2-MeO-E2 supplementation as a potential therapeutic agent for PE.

The N-terminal coiled-coil domain of Arabidopsis CROWDED NUCLEI 1 is required for nuclear morphology maintenance.

The Arabidopsis CROWDED NUCLEI (CRWN) family proteins form a lamina-like meshwork beneath the nuclear envelope with multiple functions, including maintenance of nuclear morphology, genome organization, DNA damage repair and transcriptional regulation. CRWNs can form homodimers/heterodimers through protein‒protein interactions; however, the exact molecular mechanism of CRWN dimer formation and the diverse functions of different CRWN domains are not clear. In this report, we show that the N-terminal coiled-coil domain of CRWN1 facilitates its homodimerization and heterodimerization with the coiled-coil domains of CRWN2-CRWN4. We further demonstrated that the N-terminus but not the C-terminus of CRWN1 is sufficient to rescue the defect in nuclear morphology of the crwn1 crwn2 mutant to the WT phenotype. Moreover, both the N- and C-terminal fragments of CRWN1 are necessary for its normal function in the regulation of plant development. Collectively, our data shed light on the mechanism of plant lamina network formation and the functions of different domains in plant lamin-like proteins.

The compositional and functional imbalance of the gut microbiota in CKD linked to disease patterns.

BACKGROUND: The prevalence of chronic kidney disease (CKD) is on the rise, posing a significant public health challenge. Although gut microbiome dysbiosis has been implicated in the impairment of kidney functions, the existence of pathological subtypes-linked differences remains largely unknown. We aimed to characterize the intestinal microbiota in patients with membranous nephropathy (MN), IgA nephropathy (IgAN), minimal change disease (MCD), and ischemic renal injury (IRI) in order to investigate the intricate relationship between intestinal microbiota and CKD across different subtypes. METHODS: We conducted a cross-sectional study involving 94 patients with various pathological patterns of CKD and 54 healthy controls (HCs). The clinical parameters were collected, and stool samples were obtained from each participant. Gut microbial features were analyzed using 16S rRNA sequencing and taxon annotation to compare the HC, CKD, MN, IgAN, MCD, and IRI groups. RESULTS: The CKD subjects exhibited significantly reduced alpha diversity, modified community structures, and disrupted microbial composition and potential functions compared to the control group. The opportunistic pathogen Klebsiella exhibited a significant enrichment in patients with CKD, whereas Akkermansia showed higher abundance in HCs. The study further revealed the presence of heterogeneity in intestinal microbial signatures across diverse CKD pathological types, including MN, IgAN, MCD, and IRI. The depression of the family Lachnospiraceae and the genus Bilophila was prominently observed exclusively in patients with MN, while suppressed Streptococcus was detected only in individuals with MCD, and a remarkable expansion of the genus Escherichia was uniquely found in cases of IRI. The study also encompassed the development of classifiers employing gut microbial diagnostic markers to accurately discriminate between distinct subtypes of CKD. CONCLUSIONS: The dysregulation of gut microbiome was strongly correlated with CKD, exhibiting further specificity towards distinct pathological patterns. Our study emphasizes the significance of considering disease subtypes when assessing the impact of intestinal microbiota on the development, diagnosis, and treatment of CKD.

Natural Products Inhibition of Cytochrome P450 2B6 Activity and Methadone Metabolism.

Methadone is cleared predominately by hepatic cytochrome P450 (CYP) 2B6-catalyzed metabolism to inactive metabolites. CYP2B6 also catalyzes the metabolism of several other drugs. Methadone and CYP2B6 are susceptible to pharmacokinetic drug-drug interactions. Use of natural products such as herbals and other botanicals is substantial and growing, and concomitant use of prescription medicines and non-prescription herbals is common and may result in interactions, often precipitated by CYP inhibition. Little is known about herbal product effects on CYP2B6 activity, and CYP2B6-catalyzed methadone metabolism. We screened a family of natural product compounds used in traditional medicines, herbal teas, and synthetic analogs of compounds found in plants, including kavalactones, flavokavains, chalcones and gambogic acid, for inhibition of expressed CYP2B6 activity and specifically inhibition of CYP2B6-mediated methadone metabolism. An initial screen evaluated inhibition of CYP2B6-catalyzed 7-ethoxy-4-(trifluoromethyl) coumarin O-deethylation. Hits were further evaluated for inhibition of racemic methadone metabolism, including mechanism of inhibition and kinetic constants. In order of decreasing potency, the most effective inhibitors of methadone metabolism were dihydromethysticin (competitive, K i 0.074 µM), gambogic acid (noncompetitive, K i 6 µM), and 2,2'-dihydroxychalcone (noncompetitive, K i 16 µM). Molecular modeling of CYP2B6-methadone and inhibitor binding showed substrate and inhibitor binding position and orientation and their interactions with CYP2B6 residues. These results show that CYP2B6 and CYP2B6-catalyzed methadone metabolism are inhibited by certain natural products, at concentrations which may be clinically relevant. SIGNIFICANCE STATEMENT: This investigation identified several natural product constituents which inhibit in vitro human recombinant CYP2B6 and CYP2B6-catalyzed N-demethylation of the opioid methadone. The most potent inhibitors (K i) were dihydromethysticin (0.074 µM), gambogic acid (6 µM) and 2,2'-dihydroxychalcone (16 µM). Molecular modeling of ligand interactions with CYP2B6 found that dihydromethysticin and 2,2'-dihydroxychalcone bound at the active site, while gambogic acid interacted with an allosteric site on the CYP2B6 surface. Natural product constituents may inhibit CYP2B6 and methadone metabolism at clinically relevant concentrations.