Ceftazidime-avibactam (CAZ/AVI, CZA) combination has shown promising efficacy against carbapenem-resistant Klebsiella pneumoniae (CRKP); however, several recent studies have reported resistance to CZA in CRKP. Hence, the present study aimed to elucidate the potential mechanisms of CZA resistance and heteroresistance in clinical carbapenemase (KPC)-producing K. pneumoniae. A total of 25 KPC-producing K. pneumoniae were collected and identified 19 CZA-resistant and 3 CZA-heteroresistant isolates using broth microdilution and population analysis profile methods, respectively. Whole-genome sequencing and comparative genomic analysis of the heteroresistant parental strain and its resistant subpopulation showed mutations in blaKPC-2 (KPC-71 variant) and tandem amplification of the lipoprotein diacylglyceryl transferase umpA allele in the resistant subpopulation. Reverse transcription-quantitative PCR (qRT-PCR) results demonstrated that the copy number of umpA gene is associated with resistance to CZA. umpA overexpression was induced in K. pneumoniae strains involved in CZA heteroresistance and failed CZA therapy in infected mice. Flow cytometry and parallel reaction monitoring (PRM) assays were performed to investigate the heteroresistance mechanisms in umpA overexpression strains. Flow cytometry analysis showed that overexpression of the umpA gene decreased propidium iodide incorporation in the cells of this strain. PRM assays showed that the peptidoglycan-associated lipoprotein Pal and the outer membrane (OM) protein OmpA were significantly upregulated in the umpA-overexpressing K. pneumoniae strain. In conclusion, we demonstrated a new mechanism for CZA heteroresistance wherein tandem umpA amplification generated population heterogeneity and failed CZA therapy in vivo experimentally infected mice. Lipoprotein biosynthetic pathways are considered a potential target for CZA resistance.IMPORTANCEThe novel synthetic β-lactamase inhibitor combination ceftazidime-avibactam has been available clinically for a few years. However, evidence that resistance and heteroresistance to ceftazidime-avibactam are responsible for treatment failure in clinical settings is increasing, and the underlying molecular mechanisms involved in developing heteroresistance have not been elucidated in detail. This study highlights that the lipoprotein diacylglyceryl transferase umpA gene amplification conferred heteroresistance to K. pneumoniae strains. Moreover, we found that umpA gene amplification-driven ceftazidime-avibactam heteroresistance might have resulted in vivo treatment failure during K. pneumoniae infection in mice. We also observed that the umpA gene amplification upregulated the expression levels of lipoproteins and outer membrane proteins and decreased outer membrane permeability in K. pneumoniae strains. These findings suggest that ceftazidime-avibactam heteroresistance is a major clinical concern in eradicating bacterial infections. Furthermore, lipoprotein biosynthetic pathways are considered a promising target for overcoming ceftazidime-avibactam (CZA) resistance.

Fish diseases are significant constraints to sustainable aquaculture. With the rise of uncrewed operations in smart farms, efficient automated fish disease detection is increasingly critical. Existing deep learning models often suffer from excessive parameters, high computational requirements, and lengthy inference times, which limit their deployment on edge development platforms. To address this, this study develops AQU-YOLO, a lightweight detection model based on YOLOv11n. AQU-YOLO integrates three improvements: depthwise separable convolution (DepSepConv) for efficient feature extraction, the Shuffle Attention mechanism for enhanced disease feature perception and discrimination, and a lightweight detection head that reduces parameters and inference latency. Experiments on four fish disease categories- and Argulus-show that AQU-YOLO achieves 83.5% mAP@0.5, 74.2% recall, and 78.8% F1-score, outperforming YOLOv11n while reducing parameters by 25.2%, FLOPs by 30.2%, and inference time by 18.1%. Deployment on RK3568 hardware achieves 28.6 FPS, exceeding YOLOv11n by 43% and meeting real-time requirements. These results demonstrate the potential of AQU-YOLO as an accurate and efficient solution for real-time fish disease detection in innovative aquaculture.

This file contains the legends of supplementary figures.

Probiotic fermentation can promote the release of more effective components from traditional Chinese medicines (TCMs). Astragalus membranaceus (Fisch.) Bunge (A. membranaceus) and Raphani Semen are TCMs that have gained attention for their immunoenhancing activities. This study aimed to investigate the effects and underlying mechanisms of probiotic-fermented A. membranaceus and Raphani Semen (PROAS) in cyclophosphamide (CTX)-induced immunocompromised mice. Changes in the composition of A. membranaceus and Raphani Semen after fermentation by probiotic strains, including Bifidobacterium longum SD5219, Lactobacillus fermentum NCIMB5221, and Lactobacillus paracasei SD5219, were identified using high-performance liquid chromatography. The immunostimulatory effects and mechanisms of PROAS were evaluated in immunosuppressed mice 3 and 7 days after CTX treatment. Probiotic fermentation of TCMs resulted in changes in major bioactive components. PROAS supplementation effectively restored intestinal integrity in CTX-treated mice by upregulating the mRNA expression of the tight junction proteins. PROAS significantly ameliorated the reduction in the spleen index and number of B lymphocytes caused by CTX treatment and regulated the secretion of cytokines in serum and colon tissues. PROAS administration modulated gut microbial dysbiosis and short-chain fatty acid (SCFA) content in CTX-treated mice. These results suggest that PROAS enhances B lymphocyte function by increasing the regulation of intestinal microbiota to produce high levels of SCFA, repairs the intestinal barrier damage induced by CTX, and promotes intestinal mucosal immunity.

Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide, with its pathogenesis intricately linked to metabolic and immune dysregulation. This study aims to elucidate the molecular mechanisms underpinning HCC by analyzing metabolic and immune-related pathways and constructing a prognostic risk model.Methods: We utilized data from The Cancer Genome Atlas (TCGA) to analyze genomic and clinical characteristics of HCC. Techniques such as single-sample gene set enrichment analysis (ssGSEA), weighted gene coexpression network analysis (WGCNA), and gene set variation analysis (GSVA) were employed to explore the interplay between metabolic pathways, immune responses, and HCC progression. In addition, a prognostic risk model was developed using univariate Cox regression and LASSO regression analysis based on PPAR signaling and immune-related genes.Results: Our ssGSEA results indicated a significant involvement of metabolism-related pathways in HCC. The WGCNA identified key immune-related genes, with particular modules correlating with macrophage activity. The prognostic model, comprising five key genes, effectively stratified patients into low- and high-risk groups, with implications for overall survival (OS). Further analyses revealed the model's correlation with clinical characteristics and immune-related indexes, suggesting its utility in predicting HCC progression.Conclusion: This study provides a comprehensive molecular portrait of HCC, emphasizing the role of metabolic reprogramming and immune responses. The prognostic model offers potential for personalized therapeutic strategies and improved clinical outcomes. Future research should focus on validating these findings and exploring the therapeutic potential of targeting metabolic and immune pathways in HCC.

Figure S3 describes that lack of tryptophan impairs the function of CD8+T cells.

BackgroundThe intricate interplay between dietary habits and the development of Parkinson’s Disease (PD) has long been a subject of scientific inquiry. Mendelian Randomization (MR) emerges as a potent tool, harnessing genetic variants to infer causality in observational data. While evidence links diet to Parkinson’s Disease (PD) etiology, a thorough MR exploration of dietary impacts on PD, particularly involving gut microbiota, is still emerging.MethodsThis research leverages the IEU Open GWAS project’s vast GWAS database1 to address the knowledge gap in understanding diet’s influence on PD, employing a diverse range of dietary variables. Our holistic dataset includes various foods like processed fava beans, bap, red wine, to cheese, reflecting a commitment to untangling dietary complexities in PD etiology. Advancing from initial dietary-PD associations, we innovatively explore the gut microbiota, focusing on Parabacteroides goldsteinii, in relation to bap intake and PD, employing MR. Utilizing weighted median, MR-Egger, and inverse variance weighting methods, we ensure rigorous causality assessments, meticulously mitigating pleiotropy and heterogeneity biases to uphold finding validity.ResultsOur findings indicate red wine (OR: 1.031; 95% CI 1.001–1.062; p = 0.044) and dried fruit consumption (OR: 2.019; 95% CI 1.052–3.875; p = 0.035) correlate with increased PD risk, whereas broad beans (OR: 0.967; 95% CI 0.939–0.996; p = 0.024) and bap intake (OR: 0.922; 95% CI 0.860–0.989; p = 0.023) show protective effects against PD. Employing MR, specifically the IVW method, revealed a significant inverse association between bap intake and gut microbiota, marked by an 8.010-fold decrease in Parabacteroides goldsteinii per standard deviation increase in bap intake (95% CI 1.005–63.818, p = 0.049). Furthermore, a connection between PD and Parabacteroides goldsteinii was observed (OR: 0.810; 95% CI 0.768–0.999; p = 0.049), suggesting a potential microbiota-mediated pathway in PD etiology.ConclusionOur study links dietary habits to PD risk, showing higher PD risk with red wine and dried fruit consumption, and a protective effect from broad beans and bap. Using MR, we found bap intake inversely correlates with Parabacteroides goldsteinii in the gut, suggesting bap influences microbiota. Further, higher Parabacteroides goldsteinii levels correlate with lower PD risk, highlighting a complex interplay of diet, gut microbiome, and neurological health. These insights shed light on potential dietary interventions for PD.

The choroid plexus (ChP) helps maintain the homeostasis of the brain by forming the blood-CSF barrier via tight junctions (TJ) at the choroid plexus epithelial cells, and subsequently preventing neuroinflammation by restricting immune cells infiltration into the central nervous system. However, whether chronic cerebral hypoperfusion causes ChP structural damage and blood-CSF barrier impairment remains understudied. The bilateral carotid stenosis (BCAS) model in adult male C57BL/6 J mice was used to induce cerebral hypoperfusion, a model for vascular contributions to cognitive impairment and dementia (VCID). BCAS-mediated changes of the blood-CSF barrier TJ proteins, apical secretory Na+-K+-Cl− cotransporter isoform 1 (NKCC1) protein and regulatory serine-threonine kinases SPAK, and brain infiltration of myeloid-derived immune cells were assessed. BCAS triggered dynamic changes of TJ proteins (claudin 1, claudin 5) accompanied with stimulation of SPAK-NKCC1 complex and NF-κB in the ChP epithelial cells. These changes impacted the integrity of the blood-CSF barrier, as evidenced by ChP infiltration of macrophages/microglia, neutrophils and T cells. Importantly, pharmacological blockade of SPAK with its potent inhibitor ZT1a in BCAS mice attenuated brain immune cell infiltration and improved cognitive neurological function. BCAS causes chronic ChP blood-CSF damage and immune cell infiltration. Our study sheds light on the SPAK-NKCC1 complex as a therapeutic target in neuroinflammation.

The EP300-ZNF384 fusion gene is an oncogenic driver in B-cell acute lymphoblastic leukemia (B-ALL). In the present study, we demonstrated that EP300-ZNF384 substantially induces the transcription of IL3RA and the expression of IL3Rα (CD123) on B-ALL cell membranes. Interleukin 3 (IL-3) supplementation promotes the proliferation of EP300-ZNF348-positive B-ALL cells by activating STAT5. Conditional knockdown of IL3RA in EP300-ZF384-positive cells inhibited the proliferation in vitro, and induced a significant increase in overall survival of mice, which is attributed to impaired propagation ability of leukemia cells. Mechanistically, the EP300-ZNF384 fusion protein transactivates the promoter activity of IL3RA by binding to an A-rich sequence localized at -222/-234 of IL3RA. Furthermore, forced EP300-ZNF384 expression induces the expression of IL3Rα on cell membranes and the secretion of IL-3 in CD19-positive B precursor cells derived from healthy individuals. Doxorubicin displayed a selective killing of EP300-ZNF384-positive B-ALL cells in vitro and in vivo. Collectively, we identify IL3RA as a direct downstream target of EP300-ZNF384, suggesting CD123 is a potent biomarker for EP300-ZNF384-driven B-ALL. Targeting CD123 may be a novel therapeutic approach to EP300-ZNF384-positive patients, alternative or, more likely, complementary to standard chemotherapy regimen in clinical setting.

BackgroundVoriconazole is primarily metabolized by CYP2C19 and CYP3A4. Drug interactions that affect this pathway can alter its plasma exposures, resulting in untargeted voriconazole concentrations.Case summaryIn this case report, we describe the case of a 64-year-old man who was treated for non-Hodgkin’s lymphoma with continuous glucocorticoids co-administrated with voriconazole against invasive pulmonary aspergillosis. A decrease in trough concentration (Cmin) of voriconazole was observed and related with co-administration of dexamethasone in the patient carrying the CYP2C19 *1*2 genotype: voriconazole Cmin/dose ratios of 0.018 (0.1 mg L−1/5.7 mg kg−1 day−1), 0.18 (1 mg L−1/5.7 mg kg−1 day−1), and 0.23 (2 mg L−1/8.6 mg kg−1 day−1) at dexamethasone doses of 20, 12.5, and 2.5 mg, respectively. Sub-therapeutic voriconazole Cmin was associated with high- and moderate-dose dexamethasone (20 and 12.5 mg), leading to failure of antifungal treatment.ConclusionThe extent of voriconazole–dexamethasone interaction was determined by the dose of dexamethasone and associated with the CYP2C19 *1*2 genotype. Therapeutic drug monitoring of voriconazole is necessary to avoid clinically relevant interactions for optimal antifungal therapy.

Neuroinflammation is a pathological event associated with many neurological disorders, including dementia and stroke. The choroid plexus (ChP) is a key structure in the ventricles of the brain that secretes cerebrospinal fluid (CSF), forms a blood-CSF barrier, and responds to disease conditions by recruiting immune cells and maintaining an immune microenvironment in the brain. Despite these critical roles, the exact structural and functional changes to the ChP over post-stroke time remain to be elucidated. We induced ischemic stroke in C57BL/6J mice via transient middle cerebral artery occlusion which led to reduction of cerebral blood flow and infarct stroke. At 1-7 days post-stroke, we detected time-dependent increase in the ChP blood-CSF barrier permeability to albumin, tight-junction damage, and dynamic changes of SPAK-NKCC1 protein complex, a key ion transport regulatory system for CSF production and clearance. A transient loss of SPAK protein complex but increased phosphorylation of the SPAK-NKCC1 complex was observed in both lateral ventricle ChPs. Most interestingly, stroke also triggered elevation of proinflammatory Lcn2 mRNA and its protein as well as infiltration of anti-inflammatory myeloid cells in ChP at day 5 post-stroke. These findings demonstrate that ischemic strokes cause significant damage to the ChP blood-CSF barrier, contributing to neuroinflammation in the subacute stage.

Intratumoral hypoxia is a microenvironmental feature that promotes breast cancer progression and is associated with cancer mortality. Plexin B3 (PLXNB3) is highly expressed in estrogen receptor-negative breast cancer, but the underlying mechanisms and consequences have not been thoroughly investigated. Here, we report that PLXNB3 expression is increased in response to hypoxia and that PLXNB3 is a direct target gene of hypoxia-inducible factor 1 (HIF-1) in human breast cancer cells. PLXNB3 expression is correlated with HIF-1α immunohistochemistry, breast cancer grade and stage, and patient mortality. Mechanistically, PLXNB3 is required for hypoxia-induced MET/SRC/focal adhesion kinase (FAK) and MET/SRC/STAT3/NANOG signaling as well as hypoxia-induced breast cancer cell migration, invasion, and cancer stem cell specification. PLXNB3 knockdown impairs tumor formation and lung metastasis in orthotopic breast cancer mouse models.

Background & Aims:Exploiting key regulators responsible for hepatocarcinogenesis is of great importance for the prevention and treatment of hepatocellular carcinoma (HCC). However, the key players contributing to hepatocarcinogenesis remain poorly understood. We explored the molecular mechanisms underlying the carcinogenesis and progression of HCC for the development of potential new therapeutic targets. Methods:The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) and Genotype-Tissue Expression (GTEx) databases were used to identify genes with enhanced expression in the liver associated with HCC progression. A murine liver-specific Ftcd knockout (Ftcd-LKO) model was generated to investigate the role of formimidoyltransferase cyclodeaminase (FTCD) in HCC. Multi-omics analysis of transcriptomics, metabolomics, and proteomics data were applied to further analyse the molecular effects of FTCD expression on hepatocarcinogenesis. Functional and biochemical studies were performed to determine the significance of loss of FTCD expression and the therapeutic potential of Akt inhibitors in FTCD-deficient cancer cells. Results:FTCD is highly expressed in the liver but significantly downregulated in HCC. Patients with HCC and low levels of FTCD exhibited worse prognosis, and patients with liver cirrhosis and low FTCD levels exhibited a notable higher probability of developing HCC. Hepatocyte-specific knockout of FTCD promoted both chronic diethylnitrosamine-induced and spontaneous hepatocarcinogenesis in mice. Multi-omics analysis showed that loss of FTCD affected fatty acid and cholesterol metabolism in hepatocarcinogenesis. Mechanistically, loss of FTCD upregulated peroxisome proliferator-activated receptor (PPAR)γ and sterol regulatory element-binding protein 2 (SREBP2) by regulating the PTEN/Akt/mTOR signalling axis, leading to lipid accumulation and hepatocarcinogenesis. Conclusions:Taken together, we identified a FTCD-regulated lipid metabolic mechanism involving PPARγ and SREBP2 signaling in hepatocarcinogenesis and provide a rationale for therapeutically targeting of HCC driven by downregulation of FTCD. Impact and implications:Exploiting key molecules responsible for hepatocarcinogenesis is significant for the prevention and treatment of HCC. Herein, we identified formimidoyltransferase cyclodeaminase (FTCD) as the top enhanced gene, which could serve as a predictive and prognostic marker for patients with HCC. We generated and characterised the first Ftcd liver-specific knockout murine model. We found loss of FTCD expression upregulated peroxisome proliferator-activated receptor (PPAR)γ and sterol regulatory element-binding protein 2 (SREBP2) by regulating the PTEN/Akt/mTOR signalling axis, leading to lipid accumulation and hepatocarcinogenesis, and provided a rationale for therapeutic targeting of HCC driven by downregulation of FTCD.

Dear Editor, Harnessing the power of the immune system via immune checkpoint inhibition has revolutionized the treatment paradigm of many malignancies,including hepatocellular carcinoma(HCC).1 Despite encouraging efficacy seen with the immune checkpoint blockade(ICB)agents in a subset of patients with HCC,however,there remain a large number of HCC patients experience ICB resistance and failed to derive durable benefit from these agents,which underscores the need of patient selection for ICB treatment.1 Previous study showed that HCC tumors could be stratified into two classes:inflamed and non-inflamed classes.2 In contrast to non-inflamed class,inflamed class exhibits higher immune infiltration and cytolytic activity,and is associated with enhanced responsiveness to ICB therapy.2,3 Accordingly,deter-mining the immune status of HCC tumors has important clinical significance,which can be leveraged to identify patients who are most likely to benefit from ICB therapy.Recently,a study by Montironi et al.reports a 20-gene signature that can be used to divide HCC tumors into these two classes.4 This signature was developed based on transcriptomic data from single-region samples,and has been demonstrated to have a favorable performance for immune status prediction.4

PCBP-1, a multifunctional RNA binding protein, is expressed in various human cell/tissue types and involved in post-transcriptional gene regulation. PCBP-1 has important roles in cellular Iron homeostasis, mitochondrial stability, and other cellular activities involved in the pathophysiological process of neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Huntington’s disease (HD). However, it remains enigmatic whether PCPB-1 is associated with the pathogenesis of PD. In this study, we cloned and constitutively overexpressed PCBP-1 in rat PC12 cells (PC12 cell is the common cell line studying neurodegenerative disease include PD). RNA-seq was performed to analyze PCBP-1-regulated differentially expressed genes (DEGs) and alternative splicing events (ASEs) between control and PCBP1-overexpressed cells. GO and KEGG pathway analyses were performed to identify functional DEGs and alternatively spliced genes. Consequently, we validated PCBP-1-regulated genes using RT-qPCR. Finally, we downloaded CLIP-seq data from GEO (GSE84700) to analyze the mechanisms of PCBP-1’s regulation of gene expression and ASEs by revealing the binding profile of PCBP-1 on its target pre-mRNAs. Overexpression of PCBP-1 partially regulated the ASE and expression of genes enriched in neuroinflammation and protein ubiquitination, which were also associated with PD pathogenesis. Moreover, RT-qPCR assay verified the PCBP-1-modulated expression of neuroinflammatory genes, like LCN-2, and alternative splicing (AS) of ubiquitination-related gene WWP-2. Finally, CLIP-seq data analysis indicated that the first UC motif was the critical site for PCBP-1 binding to its targets. In this study, we provided evidence that PCBP-1 could regulate the expression of LCN-2 gene expression associated with neuroinflammation and AS of WWP-2 in relation to protein ubiquitination. These findings thus provided novel insights into the potential application of PCBP-1 as the disease pathophysiological or therapeutic target for neurodegenerative disease.

Hepatocellular carcinoma (HCC) is one of the most common solid malignancies worldwide. A large proportion of patients with HCC are diagnosed at advanced stages and are only amenable to systemic therapies. We have witnessed the evolution of systemic therapies from single-agent targeted therapy (sorafenib and lenvatinib) to the combination of a checkpoint inhibitor plus targeted therapy (atezolizumab plus bevacizumab therapy). Despite remarkable advances, only a small subset of patients can obtain durable clinical benefit, and therefore substantial therapeutic challenges remain. In the past few years, emerging systemic therapies, including new molecular-targeted monotherapies (for example, donafenib), new immuno-oncology monotherapies (for example, durvalumab) and new combination therapies (for example, durvalumab plus tremelimumab), have shown encouraging results in clinical trials. In addition, many novel therapeutic approaches with the potential to offer improved treatment effects in patients with advanced HCC, such as sequential combination targeted therapy and next-generation adoptive cell therapy, have also been proposed and developed. In this Review, we summarize the latest clinical advances in the treatment of advanced HCC and discuss future perspectives that might inform the development of more effective therapeutics for advanced HCC.