Bie Jia Jian pill ameliorates BDL-induced cholestatic hepatic fibrosis in rats by regulating intestinal microbial composition and TMAO-mediated PI3K/AKT signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: As a compound of traditional Chinese medicine (TCM), Bie Jia Jian pill (BJJP) is extensively used to treat the clinical chronic liver disease. Nevertheless, the specific mechanism through which BJJP affects hepatic fibrosis (HF) remains unknown. AIM OF THE STUDY: To explore the role and potential mechanism of BJJP involved in treating HF. MATERIALS AND METHODS: HF model of Sprague-Dawley (SD) rats was induced by a bile duct ligation (BDL). The function of BJJP involved in the intestinal microbiota (IM) and its metabolites in BDL-induced HF rats were explored through the 16S rRNA sequencing and untargeted metabolomics technologies. Network pharmacology was used to forecast mechanism underlying BJJP's anti-HF effects, which were validated in BDL-induced rats and trimethylamine N-oxide (TMAO)-induced LX-2 and HSC-T6 cells. RESULTS: BJJP effectively ameliorated pathological liver damage, inflammation, and fibrosis of the BDL-induced HF rats. BJJP regulated IM diversity and composition and interfered with trimethylamine (TMA)-flavin monooxygenase 3 (FMO3)-TMAO process. In vitro, BJJP significantly inhibited the TMAO-induced activation of hepatic stellate cells (HSCs) (rat HSC cell line, HSC-T6; human HSC cell line, LX-2). Network pharmacology results demonstrated that PI3K/AKT signal pathway is crucially involved in BJJP treatment of HF. Further research revealed that BJJP inhibited the PI3K/AKT signal pathway in BDL-induced HF rats. Moreover, TMAO activated the PI3K/AKT pathway, whereas BJJP suppressed TMAO-induced activation. Subsequent intervention with 740Y-P (the PI3K agonist) successfully neutralized the repression effect on PI3K/AKT signal pathway by BJJP. CONCLUSION: These results clearly show that BJJP attenuates HF by regulating the IM, as well as inhibiting PI3K/AKT pathway mediated by TMAO.

DeepPBI-KG: a deep learning method for the prediction of phage-bacteria interactions based on key genes.

Phages, the natural predators of bacteria, were discovered more than 100 years ago. However, increasing antimicrobial resistance rates have revitalized phage research. Methods that are more time-consuming and efficient than wet-laboratory experiments are needed to help screen phages quickly for therapeutic use. Traditional computational methods usually ignore the fact that phage-bacteria interactions are achieved by key genes and proteins. Methods for intraspecific prediction are rare since almost all existing methods consider only interactions at the species and genus levels. Moreover, most strains in existing databases contain only partial genome information because whole-genome information for species is difficult to obtain. Here, we propose a new approach for interaction prediction by constructing new features from key genes and proteins via the application of K-means sampling to select high-quality negative samples for prediction. Finally, we develop DeepPBI-KG, a corresponding prediction tool based on feature selection and a deep neural network. The results show that the average area under the curve for prediction reached 0.93 for each strain, and the overall AUC and area under the precision-recall curve reached 0.89 and 0.92, respectively, on the independent test set; these values are greater than those of other existing prediction tools. The forward and reverse validation results indicate that key genes and key proteins regulate and influence the interaction, which supports the reliability of the model. In addition, intraspecific prediction experiments based on Klebsiella pneumoniae data demonstrate the potential applicability of DeepPBI-KG for intraspecific prediction. In summary, the feature engineering and interaction prediction approaches proposed in this study can effectively improve the robustness and stability of interaction prediction, can achieve high generalizability, and may provide new directions and insights for rapid phage screening for therapy.

Left Pulmonary Vein Trunk Length as a Robust Predictor of Long-Term Success of Atrial Fibrillation Catheter Ablation.

Background: Radiofrequency catheter ablation (RFCA) is a commonly used treatment for atrial fibrillation (AF), but the long-term recurrence rate remains relatively high. Given the inconsistent results regarding the role of left pulmonary vein (PV) ostial anatomy in post-ablative recurrence of RFCA in previous studies, we sought to investigate the role of left PV trunk length using an alternative methodology. Methods: A total of 369 AF patients undergoing catheter ablation were included. The left/right trunk length (LTL/RTL) of the PV was measured from pre-ablative computed tomography (CT) using three-dimensional reconstruction techniques. We constructed three multivariable Cox models, with the inclusion of the LTL, RTL, and no LTL/RTL, and used the Delong test, integrated discrimination index (IDI), and net reclassification index (NRI) to assess model improvement. We identified optimal cut-off values for LTL with the receiver operating characteristic (ROC) curve, and estimated outcomes using the Kaplan-Meier survival curve. We also used subgroup analysis to evaluate interactions. Results: The results of the Delong test, IDI, and NRI indicated that LTL had a favorable impact on the performance of the multivariate model. Subsequently, the multivariate Cox regression analysis identified LTL as a significant risk factor for post-ablative recurrence of AF (adjusted hazard ratio (HR) = 1.08, 95% CI: 1.05-1.12, p < 0.001). According to the ROC curve, the optimal cut-off value for LTL is 11.15 mm, and the Kaplan-Meier estimator revealed different outcomes (p < 0.001). We calculated p for interaction between LTL and other factors, and no significant interaction terms were observed. Conclusions: LTL is a robust prognostic indicator for post-ablative outcome in AF patients receiving RFCA, with a longer LTL indicating a higher risk of recurrence.

Diagnostic and prognostic value of CD44v9 and TIM3 expression in CK­ and CK+ regions in gastric cancer tissues.

The specificity and sensitivity of the current diagnostic and prognostic biomarkers for gastric cancer (GC) are limited. The present study aimed to evaluate the diagnostic and prognostic significance of cluster-of-differentiation gene 44 variant isoform 9 (CD44v9) and T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) expression levels alone or combined in the tumor tissues of patients with GC and reveal the roles of CD44v9 and TIM3 in the cytokeratin (CK)+ and CK- regions. Multiplex immunofluorescence staining was performed for CD44v9, TIM3 and CK using a tissue microarray. The tissues were divided into three regions based on CK expression: Total, CK+, and CK- regions. The diagnostic and prognostic value was evaluated using receiver operating characteristic curves, Kaplan-Meier and Cox regression analyses. The results demonstrated that the density of cells expressing CD44v9, TIM3 and co-expressing CD44v9 and TIM3 (CD44v9/TIM3) in both the CK+ and CK- regions of tumor tissues was significantly higher than those in normal tissues (P<0.001). Moreover, the expression of CD44v9 in the CK- region was significantly positively correlated with age and tumor grade (P<0.05), and the expression of CD44v9/TIM3 in the CK- region of tumor tissues was significantly positively correlated with age, tumor grade and metastasis (P<0.05). Furthermore, the area under the curve for TIM3 expression in the CK+ region was 0.709, with a sensitivity of 45.83% and a specificity of 85.54% (P<0.001). High expression of CD44v9 in the CK- region was also significantly associated with poor survival and independently predicted a poor prognosis in patients with GC (hazard ratio, 2.387; 95% confidence interval, 1.384-4.118; P<0.01). In conclusion, dividing tissue regions based on CK expression is important for the diagnosis of GC. The expression of TIM3 in the CK+ region demonstrated diagnostic potential for GC, and high expression of CD44v9 in the CK- region was an independent prognostic risk factor for patients with GC.

Actin-bundling protein fimbrin serves as a new auxin biosynthesis orchestrator in Arabidopsis root tips.

Plants delicately regulate endogenous auxin levels through the coordination of transport, biosynthesis, and inactivation, which is crucial for growth and development. While it is well-established that the actin cytoskeleton can regulate auxin levels by affecting polar transport, its potential role in auxin biosynthesis has remained largely unexplored. Using LC-MS/MS-based methods combined with fluorescent auxin marker detection, we observed a significant increase in root auxin levels upon deletion of the actin bundling proteins AtFIM4 and AtFIM5. Fluorescent observation, immunoblotting analysis, and biochemical approaches revealed that AtFIM4 and AtFIM5 affect the protein abundance of the key auxin synthesis enzyme YUC8 in roots. AtFIM4 and AtFIM5 regulate the auxin synthesis enzyme YUC8 at the protein level, with its degradation mediated by the 26S proteasome. This regulation modulates auxin synthesis and endogenous auxin levels in roots, consequently impacting root development. Based on these findings, we propose a molecular pathway centered on the 'actin cytoskeleton-26S proteasome-YUC8-auxin' axis that controls auxin levels. Our findings shed light on a new pathway through which plants regulate auxin synthesis. Moreover, this study illuminates a newfound role of the actin cytoskeleton in regulating plant growth and development, particularly through its involvement in maintaining protein homeostasis via the 26S proteasome.

Multi-omics analysis revealed that the protein kinase MoKin1 affected the cellular response to endoplasmic reticulum stress in the rice blast fungus, Magnaporthe oryzae.

BACKGROUND: Previous studies have shown that protein kinase MoKin1 played an important role in the growth, conidiation, germination and pathogenicity in rice blast fungus, Magnaporthe oryzae. ΔMokin1 mutant showed significant phenotypic defects and significantly reduced pathogenicity. However, the internal mechanism of how MoKin1 affected the development of physiology and biochemistry remained unclear in M. oryzae. RESULT: This study adopted a multi-omics approach to comprehensively analyze MoKin1 function, and the results showed that MoKin1 affected the cellular response to endoplasmic reticulum stress (ER stress). Proteomic analysis revealed that the downregulated proteins in ΔMokin1 mutant were enriched mainly in the response to ER stress triggered by the unfolded protein. Loss of MoKin1 prevented the ER stress signal from reaching the nucleus. Therefore, the phosphorylation of various proteins regulating the transcription of ER stress-related genes and mRNA translation was significantly downregulated. The insensitivity to ER stress led to metabolic disorders, resulting in a significant shortage of carbohydrates and a low energy supply, which also resulted in severe phenotypic defects in ΔMokin1 mutant. Analysis of MoKin1-interacting proteins indicated that MoKin1 really took participate in the response to ER stress. CONCLUSION: Our results showed the important role of protein kinase MoKin1 in regulating cellular response to ER stress, providing a new research direction to reveal the mechanism of MoKin1 affecting pathogenic formation, and to provide theoretical support for the new biological target sites searching and bio-pesticides developing.

Role of scientific and technological innovations on industrial upgradation in China: A spatial econometric analysis.

China is in a phase of high-quality development, where scientific and technological innovations are serving as the primary driving force for its development strategy. This emphasis on innovations is expected to fuel the upgrading of the industrial structure. This study investigates the role of scientific and technological innovations in industrial upgradation in China using spatial econometric analysis. Leveraging the data of 31 provinces of China from 2005 to 2022, we employed a spatial Durbin model to determine the spatial spillover effects of scientific and technological innovations on industrial upgradation. Our findings reveal the significant positive spatial spillover effects, indicating that provinces with higher levels of scientific and technological innovations tend to experience greater industrial upgradation, which in turn contributes to regional economic development. Furthermore, the findings suggest a strong spatial correlation between innovation and the upgrading of industrial structures, indicating that regional innovations have the potential to drive China's industrial upgradation. These results underscore the critical role of scientific and technological innovations in promoting industrial upgradation and regional development in China.

Systemic Effects of a Phage Cocktail on Healthy Weaned Piglets.

Numerous studies have demonstrated that bacteriophages (phages) can effectively treat intestinal bacterial infections. However, research on the impact of phages on overall body health once they enter the intestine is limited. This study utilized weaned piglets as subjects to evaluate the systemic effects of an orally administered phage cocktail on their health. Twelve 21-day-old weaned piglets were divided into control (CON) and phage gavage (Phages) groups. The phage cocktail consisted of five lytic phages, targeting Salmonella enterica serovar Choleraesuis (S. choleraesuis), Enteropathogenic Escherichia coli (EPEC), and Shiga tox-in-producing Escherichia coli (STEC). The phages group received 10 mL of phage cocktail orally for 20 consecutive days. The results show that the phage gavage did not affect the piglets' growth performance, serum biochemical indices, or most organ indices, except for the pancreas. However, the impact on the intestine was complex. Firstly, although the pancreatic index decreased, it did not affect the secretion of digestive enzymes in the intestine. Secondly, phages increased the pH of jejunum chyme and relative weight of the ileum, and enhanced intestinal barrier function without affecting the morphology of the intestine. Thirdly, phages did not proliferate in the intestine, but altered the intestinal microbiota structure and increased concentrations of microbial metabolites isobutyric acid and isovaleric acid in the colonic chyme. In addition, phages impacted the immune status, significantly increasing serum IgA, IgG, and IgM, as well as serum and intestinal mucosal IFN-γ, IL-1ß, IL-17, and TGF-ß, and decreasing IL-4 and IL-10. They also activated toll-like receptors TLR-4 and TLR-9. Apart from an increase in basophil numbers, the counts of other immune cells in the blood did not change. This study indicates that the impact of phages on body health is complex, especially regarding immune status, warranting further attention. Short-term phage gavage did not have significant negative effects on health but could enhance intestinal barrier function.

HSC-derived exosomal miR-199a-5p promotes HSC activation and hepatocyte EMT via targeting SIRT1 in hepatic fibrosis.

Exosomes have been implicated in inflammation-related diseases, such as hepatic fibrosis (HF) and renal fibrosis, via transferring bioactive cargoes to recipient cells. This study aimed to investigate the possible effect of hepatic stellate cell (HSC)-derived exosomes on the initiation and development of HF by delivering microRNA (miR)-199a-5p. In HF rats with cholestasis induced by ligating the common bile duct, miR-199a-5p was upregulated while SIRT1 was downregulated in liver tissues from bile duct ligation (BDL) rats compared with that of sham rats. Furthermore, miR-199a-5p expression was upregulated, but the mRNA and protein expression levels of SIRT1 were downregulated in TGF-ß1-activated LX-2. miR-199a-5p promoted the proliferation and further activation of LX-2 and enhanced the expression levels of the HF markers COL1A1 and α-SMA. Subsequently, the binding of miR-199a-5p to the 3'UTR of SIRT1 mRNA was predicted by bioinformatics websites and evidenced by fluorescent reporter assay. Knocking down SIRT1 enhanced the abilities of LX-2 cell proliferation, migration, and colony formation and increased the expression levels of the HF markers α-SMA and COL1A1. LX-2-derived exosomal miR-199a-5p transferred to LX-2 and THLE-2, inhibited the proliferation of THLE-2, and promoted the epithelial mesenchymal transition (EMT) and senescence of THLE-2. Furthermore, in vivo results suggested that miR-199a-5p overexpression aggravated HF in BDL rats; increased miR-199a-5p, α-SMA, and COL1A1 expression levels; and significantly upregulated the serum ALT, AST, TBA, and TBIL levels. However, reverse results were obtained with inhibited miR-199a-5p expression. In conclusion, HSC-derived exosomal miR-199a-5p may promote HF by accelerating HSC activation and hepatocyte EMT by targeting SIRT1, suggesting that miR-199a-5p and SIRT1 may serve as potential therapeutic targets for HF.

p21-activated kinase is involved in the sporulation, pathogenicity, and stress response of Arthrobotrys oligospora under the indirect regulation of Rho GTPase-activating protein.

The p21-GTPase-activated protein kinases (PAKs) participate in signal transduction downstream of Rho GTPases, which are regulated by Rho GTPase-activating proteins (Rho-GAP). Herein, we characterized two orthologous Rho-GAPs (AoRga1 and AoRga2) and two PAKs (AoPak1 and AoPak2) through bioinformatics analysis and reverse genetics in Arthrobotrys oligospora, a typical nematode-trapping (NT) fungus. The transcription analyses performed at different development stages suggested that Aopaks and Aorga1 play a crucial role during sporulation and trap formation, respectively. In addition, we successfully deleted Aopak1 and Aorga1 via the homologous recombination method. The disruption of Aopak1 and Aorga1 caused a remarkable reduction in spore yield and the number of nuclei per cell, but did not affect mycelial growth. In ∆Aopak1 mutants, the trap number was decreased at 48 h after the introduction of nematodes, but nematode predatory efficiency was not affected because the extracellular proteolytic activity was increased. On the contrary, the number of traps in ∆Aorga1 mutants was significantly increased at 36 h and 48 h. In addition, Aopak1 and Aorga1 had different effects on the sensitivity to cell-wall-disturbing reagent and oxidant. A yeast two-hybrid assay revealed that AoPak1 and AoRga1 both interacted with AoRac, and AoPak1 also interacted with AoCdc42. Furthermore, the Aopaks were up-regulated in ∆Aorga1 mutants, and Aorga1 was down-regulated in ∆Aopak1 mutants. These results reveal that AoRga1 indirectly regulated AoPAKs by regulating small GTPases.

Evaluation of an Algorithm for the Segmentation of Lung Nodules in Computerized Tomography Images Based on the Automatic Location of A Threshold.

BACKGROUND: Early detection of pulmonary nodules is critical for the clinical diagnosis and management of pulmonary nodules. Computed tomography imaging is currently the best imaging method for detecting pulmonary nodules. OBJECTIVE: This study proposes and applies a new thresholding-based method for identifying pulmonary nodules in computed tomography images. METHODS: The proposed method involves segmenting the lung volume and identifying candidate nodules based on their intensity levels, which are higher than those of the lung parenchyma. Reference points on the histogram curve are used to determine a threshold value, and filtering by geometric characteristics is applied to reduce false positives. The performance of the proposed method is evaluated on a training set consisting of 35 nodules distributed among 16 cases with ground truth using the SPIE-AAPM Lung CT Challenge Database and ELCAP Public Lung Image Database. RESULTS: The proposed method shows a significant reduction in false positives, filtering from an average of 12,380 candidate nodules to 19 detected nodules. The method also demonstrates a sensitivity of 88.6% for detecting pulmonary nodules with an error of 1 nodule in cases where complete detection is not reached. CONCLUSION: The proposed thresholding-based method improves the sensitivity of identifying pulmonary nodules in computed tomography images while reducing false positives.

Fus3 regulates asexual development and trap morphogenesis in the nematode-trapping fungus Arthrobotrys oligospora.

Mitogen-activated protein kinase (MAPK) Fus3 is an essential regulator of cell differentiation and virulence in fungal pathogens of plants and animals. However, the function and regulatory mechanism of MAPK signaling in nematode-trapping (NT) fungi remain largely unknown. NT fungi can specialize in the formation of "traps", an important indicator of transition from a saprophytic to a predatory lifestyle. Here, we characterized an orthologous Fus3 in a typical NT fungus Arthrobotrys oligospora using multi-phenotypic analysis and multi-omics approaches. Our results showed that Fus3 plays an important role in asexual growth and development, conidiation, stress response, DNA damage, autophagy, and secondary metabolism. Importantly, Fus3 plays an indispensable role in hyphal fusion, trap morphogenesis, and nematode predation. Moreover, we constructed the regulatory networks of Fus3 by means of transcriptomic and yeast two-hybrid techniques. This study provides insights into the mechanism of MAPK signaling in asexual development and pathogenicity of NT fungi.

Comparison of two diagnostic strategies for patients with stable chest pain suggestive of chronic coronary syndrome: rationale and design of the double-blind, pragmatic, randomized and controlled OPERATE Trial.

BACKGROUND: To achieve potential financial savings and avoid exposing the patients to unnecessary risk, an optimal diagnostic strategy to identify low risk individual who may derive minimal benefit from further cardiac imaging testing (CIT) is important for patients with stable chest pain (SCP) suggestive of chronic coronary syndrome (CCS). Although several diagnostic strategies have been recommended by the most recent guidelines, few randomized controlled trials (RCTs) have prospectively investigated the actual effect of applying these strategies in clinical practice. METHODS: OPERATE (OPtimal Evaluation of stable chest pain to Reduce unnecessAry utilization of cardiac imaging TEsting) trial is an investigator-initiated, multicenter, coronary computed tomography angiography (CCTA)-based, 2-arm parallel-group, double-blind, pragmatic and confirmative RCT planning to include 800 subjects with SCP suggestive of CCS. After enrollment, all subjects will be randomized to two arms (2016 U.K. National Institute of Health and Care Excellence guideline-determined and 2019 European Society of Cardiology guideline-determined diagnostic strategy) on a 1:1 basis. According to each strategy, CCTA should be referred and deferred for a subject in high and low risk group, respectively. The primary (effectiveness) endpoint is CCTA without obstructive coronary artery disease. Safety of each strategy will be mainly assessed by 1-year major adverse cardiovascular event rates. DISCUSSION: The OPERATE trial will provide comparative effectiveness and safety evidences for two different diagnostic strategies for patients with SCP suggestive of CCS, with the intension of improving the diagnostic yield of CCTA at no expense of safety. CLINICAL TRIAL REGISTRATION: ClinicalTrial.org Identifier NCT05640752.

Spatial Distribution of Pinus koraiensis Trees and Community-Level Spatial Associations in Broad-Leaved Korean Pine Mixed Forests in Northeastern China.

Investigating the spatial distributions and associations of tree populations provides better insights into the dynamics and processes that shape the forest community. Korean pine (Pinus koraiensis) is one of the most important tree species in broad-leaved Korean pine mixed forests (BKMFs), and little is known about the spatial point patterns of and associations between Korean pine and community-level woody species groups such as coniferous and deciduous trees in different developmental stages. This study investigated the spatial patterns of Korean pine (KP) trees and then analyzed how the spatial associations between KP trees and other tree species at the community level vary in different BKMFs. Extensive data collected from five relatively large sample plots, covering a substantial area within the natural distribution range of KP in northeastern China, were utilized. Uni- and bivariate pair correlation functions and mark correlation functions were applied to analyze spatial distribution patterns and spatial associations. The DBH (diameter at breast height) histogram of KP trees in northeastern China revealed that the regeneration process was very poor in the Changbai Mountain (CBS) plot, while the other four plots exhibited moderate or expanding population structures. KP trees were significantly aggregated at scales up to 10 m under the HPP null model, and the aggregation scales decreased with the increase in size classes. Positive or negative spatial associations were observed among different life stages of KP trees in different plots. The life history stages of the coniferous tree group showed positive spatial associations with KP saplings and juvenile trees at small scales, and spatial independence or negative correlations with larger KP trees at greater scales. All broad-leaved tree groups (canopy, middle, and understory layers) exhibited only slightly positive associations with KP trees at small scales, and dominant negative associations were observed at most scales. Our results demonstrate that mature KP trees have strong importance in the spatial patterns of KP populations, and site heterogeneity, limited seed dispersal, and interspecific competition characterize the spatial patterns of KP trees and community-level spatial associations with respect to KP trees, which can serve as a theoretical basis for the management and restoration of BKMFs in northeastern China.

Biological and genomic characterization of a polyvalent bacteriophage (S19cd) strongly inhibiting Salmonella enterica serovar Choleraesuis.

Bacteriophages are a promising alternative for the control of pathogenic bacteria. In this study, we isolated a virulent bacteriophage, S19cd, from pig gut that could infect both a non-pathogenic bacteria Escherichia coli 44 (EC44) and two pathogenic bacterial strains (ATCC 13312 (SC13312) and CICC 21493 (SC21493)) of Salmonella enterica serovar Choleraesuis (SC). S19cd exhibited strong lytic ability in both SC13312 and SC21493 with an optimal multiplicity of infection (MOI) of 10-6 and 10-5, respectively, and inhibited their growth at an MOI of 10-7 within 24 h. Mice pre-treated with S19cd exhibited protection against the SC13312 challenge. Moreover, S19cd has good heat resistance (80 â„ƒ) and pH tolerance (pH 3-12). Genome analysis revealed that S19cd belongs to the Felixounavirus genus and does not contain any virulence or drug-resistance-related genes. Additionally, S19cd encodes an adenine-specific methyltransferase that has no similarity to methyltransferases from other Felixounavirus phages and shares limited similarity with other methyltransferases in the NCBI protein database. Metagenomic analysis of S19cd genomes from 500 pigs revealed that S19cd-like phages may be widespread in Chinese pig gut. In conclusion, S19cd can be a potential phage therapy targeting SC infections.

Involvement of AoMdr1 in the Regulation of the Fluconazole Resistance, Mycelial Fusion, Conidiation, and Trap Formation of Arthrobotrys oligospora.

Multidrug resistance (Mdr) proteins are critical proteins for maintenance of drug resistance in fungi. Mdr1 has been extensively studied in Candida albicans; its role in other fungi is largely unknown. In this study, we identified a homologous protein of Mdr (AoMdr1) in the nematode-trapping (NT) fungus Arthrobotrys oligospora. It was found that the deletion of Aomdr1 resulted in a significant reduction in the number of hyphal septa and nuclei as well as increased sensitivity to fluconazole and resistance to hyperosmotic stress and SDS. The deletion of Aomdr1 also led to a remarkable increase in the numbers of traps and mycelial loops in the traps. Notably, AoMdr1 was able to regulate mycelial fusion under low-nutrient conditions, but not under nutrient-rich conditions. AoMdr1 was also involved in secondary metabolism, and its deletion caused an increase in arthrobotrisins (specific compounds produced by NT fungi). These results suggest that AoMdr1 plays a crucial role in the fluconazole resistance, mycelial fusion, conidiation, trap formation, and secondary metabolism of A. oligospora. Our study contributes to the understanding of the critical role of Mdr proteins in mycelial growth and the development of NT fungi.

[ZEB2 Regulates the Migration and Invasion of PANC-1 Pancreatic Cancer Cells: An Experimental Study].

Objective: To investigate the effects and mechanisms of zinc finger E-box binding homeobox transcription factor-2 ( ZEB2) on the proliferation, colony formation, migration, and invasion abilities and the epithelial-mesenchymal transition (EMT) of PANC-1 cells, a human pancreatic cancer cell line. Methods: Data on the expression of ZEB2 in pancreatic cancer tissues and paracancerous tissues from The Cancer Genome Atlas (TCGA) database were analyzed. PANC-1 pancreatic cancer cells were divided into si-NC group, si- ZEB2 group, pcDNA3.1 group, and pcDNA3.1- ZEB2 group. qRT-PCR and Western blot were conducted to confirm the effectiveness of ZEB2 knockdown or overexpression. CCK-8, colony formation, wound healing, and Transwell assays were conducted to examine the effects of ZEB2 on the proliferation, colony formation, migration, and invasion of PANC-1 cells. qRT-PCR and immunofluorescence assays were performed to examine the expression of E-cadherin and vimentin, the EMT markers, in the cells. Prediction of proteins interacting with ZEB2 was made through the STRING database. Results: TCGA database analysis showed that the expression level of ZEB2 in pancreatic cancer tissues was significantly higher than that in adjacent tissues ( P<0.05). Compared with those of cells in the control group, the proliferation, colony formation, migration, and invasion of cells in the si- ZEB2 group were decreased ( P<0.05). Compared with those of cells in the pcDNA3.1 group, the proliferation, colony formation, migration and invasion of cells in the pcDNA3.1- ZEB2 group were increased (all P<0.05). According to the results of qRT-PCR and immunofluorescence assays, compared with those of the si-NC group, the expression of E-cadherin mRNA, an epithelial marker, in the si- ZEB2 group increased, while the expression of vimentin mRNA, an mesenchymal marker, and the protein decreased. Compared with those of the pcDNA3.1 group, the expression of E-cadherin mRNA in the PANC-1 cells of the pcDNA3.1- ZEB2 group decreased, while the expression of vimentin mRNA and the protein increased (all P<0.05). Analysis with the STRING database predicted that 10 proteins had close interaction with ZEB2. Conclusion: Overexpression of ZEB2 promotes the migration, invasion, and the EMT process of PANC-1 pancreatic cancer cells.

AoMae1 Regulates Hyphal Fusion, Lipid Droplet Accumulation, Conidiation, and Trap Formation in Arthrobotrys oligospora.

Malate dehydrogenase (MDH) is a key enzyme in the tricarboxylic acid (TCA) cycle and is essential for energy balance, growth, and tolerance to cold and salt stresses in plants. However, the role of MDH in filamentous fungi is still largely unknown. In this study, we characterized an ortholog of MDH (AoMae1) in a representative nematode-trapping (NT) fungus Arthrobotrys oligospora via gene disruption, phenotypic analysis, and nontargeted metabolomics. We found that the loss of Aomae1 led to a weakening of MDH activity and ATP content, a remarkable decrease in conidia yield, and a considerable increase in the number of traps and mycelial loops. In addition, the absence of Aomae1 also caused an obvious reduction in the number of septa and nuclei. In particular, AoMae1 regulates hyphal fusion under low nutrient conditions but not in nutrient-rich conditions, and the volumes and sizes of the lipid droplets dynamically changed during trap formation and nematode predation. AoMae1 is also involved in the regulation of secondary metabolites such as arthrobotrisins. These results suggest that Aomae1 has an important role in hyphal fusion, sporulation, energy production, trap formation, and pathogenicity in A. oligospora. Our results enhance the understanding of the crucial role that enzymes involved in the TCA cycle play in the growth, development, and pathogenicity of NT fungi.

Integrated transcriptome and metabolome analysis revealed that flavonoids enhanced the resistance of Oryza sativa against Meloidogyne graminicola.

Rice is a crucial food crop worldwide, but its yield and quality are significantly affected by Meloidogyne graminicola is a root knot nematode. No rice variety is entirely immune to this nematode disease in agricultural production. Thus, the fundamental strategy to combat this disease is to utilize rice resistance genes. In this study, we conducted transcriptome and metabolome analyses on two rice varieties, ZH11 and IR64. The results indicated that ZH11 showed stronger resistance than IR64. Transcriptome analysis revealed that the change in gene expression in ZH11 was more substantial than that in IR64 after M. graminicola infection. Moreover, GO and KEGG enrichment analysis of the upregulated genes in ZH11 showed that they were primarily associated with rice cell wall construction, carbohydrate metabolism, and secondary metabolism relating to disease resistance, which effectively enhanced the resistance of ZH11. However, in rice IR64, the number of genes enriched in disease resistance pathways was significantly lower than that in ZH11, which further explained susceptibility to IR64. Metabolome analysis revealed that the metabolites detected in ZH11 were enriched in flavonoid metabolism and the pentose phosphate pathway, compared to IR64, after M. graminicola infection. The comprehensive analysis of transcriptome and metabolome data indicated that flavonoid metabolism plays a crucial role in rice resistance to M. graminicola infection. The content of kaempferin, apigenin, and quercetin in ZH11 significantly increased after M. graminicola infection, and the expression of genes involved in the synthetic pathway of flavonoids also significantly increased in ZH11. Our study provides theoretical guidance for the precise analysis of rice resistance and disease resistance breeding in further research.

AoSte12 Is Required for Mycelial Development, Conidiation, Trap Morphogenesis, and Secondary Metabolism by Regulating Hyphal Fusion in Nematode-Trapping Fungus Arthrobotrys oligospora.

Nematode-trapping (NT) fungi are a unique group of carnivorous microorganisms that can capture and digest nematodes by producing ingenious trapping devices (traps). Arthrobotrys oligospora, a representative NT fungus, can develop adhesive three-dimensional networks for nematode predation. Hyphal fusion is indispensable for the trap formation of A. oligospora. Here, we characterized an orthologous Ste12 protein (AoSte12) in A. oligospora via gene disruption, DNA affinity purification sequencing (DAP-Seq), and multi-omics approaches. The disruption of the Aoste12 gene caused an increase in hyphal fusion and resulted in defects in mycelial growth, conidiation, trap morphology, and stress resistance, as well as reducing the number of nuclei and lipid droplet accumulation. Moreover, transcriptome and DAP-Seq analysis revealed that AoSte12 was involved in cellular processes associated with growth, cell fusion, the tricarboxylic acid cycle, vesicles, actin filaments, and lipid metabolism. In addition, combining metabolome with transcriptome and DAP-Seq analysis indicated that AoSte12 was involved in the mitogen-activated protein kinase signaling pathway, lipid metabolism, and secondary metabolites. A yeast two-hybrid assay revealed that AoSte12 can interact with diverse proteins, such as the MAK-2 orthologue protein Fus3, the vacuolar sorting protein Pep3, and UDP-glycosyltransferase. Our results suggest that AoSte12 plays an indispensable role in hyphal fusion and thus regulates sporulation and trap morphogenesis. These results provide deep insights into the connection between hyphal fusion and trap formation in NT fungi. IMPORTANCE Nematode-trapping (NT) fungi are an important natural enemy of nematodes and can capture their prey by producing traps. Hyphal anastomosis and fusion are important for mycelial growth and the colony morphological development of filamentous fungi and are also crucial for the trap morphogenesis of NT fungi. Arthrobotrys oligospora can form complex three-dimensional networks (traps) when sensing the presence of nematodes. This study revealed that AoSte12 is indispensable for hyphal fusion and that it regulates mycelial growth, conidiation, trap morphogenesis, stress resistance, the number of nuclei, and lipid droplet accumulation in A. oligospora. In addition, DNA affinity purification sequencing, transcriptome, and metabolome analyses further revealed that AoSte12 is involved in the mitogen-activated protein kinase pathway, lipid metabolism, and secondary metabolism. Overall, these findings expand the important role of AoSte12 in NT fungus A. oligospora and provide a broad foundation for elucidating the regulatory mechanism of trap development and the lifestyle transitions of pathogenic fungi.