Sequencing Analysis Research Articles

Cannabidiol alleviates LPS-inhibited odonto/osteogenic differentiation in human dental pulp stem cells invitro.

Cannabidiol (CBD), derived from the Cannabis sativa plant, exhibits benefits in potentially alleviating a number of oral and dental pathoses, including pulpitis and periodontal diseases. This study aimed to explore the impact of CBD on several traits of human dental pulp stem cells (hDPSC), such as their proliferation, apoptosis, migration and odonto/osteogenic differentiation. hDPSCs were harvested from human dental pulp tissues. The cells were treated with CBD at concentrations of 1.25, 2.5, 5, 10, 25 and 50 μg/mL. Cell responses in terms of cell proliferation, colony-forming unit, cell cycle progression, cell migration, apoptosis and odonto/osteogenic differentiation of hDPSCs were assessed in the normal culture condition and P. gingivalis lipopolysaccharide (LPS)-induced 'inflammatory' milieus. RNA sequencing and proteomic analysis were performed to predict target pathways impacted by CBD. CBD minimally affects hDPSCs' behaviour under normal culture growth milieu in normal conditions. However, an optimal concentration of 1.25 μg/mL CBD significantly countered the harmful effects of LPS, indicated by the promoting cell proliferation and restoring the odonto/osteogenic differentiation potential of hDPSCs under LPS-treated conditions. The proteomic analysis demonstrated that several proteins involved in cell proliferation and differentiation were upregulated following CBD exposure, including CCL8, CDC42 and KFL5. RNA sequencing data indicated that CBD upregulated the Notch signalling pathway. In an inhibitory experiment, DAPT, a Notch inhibitor, reduced the effect of CBD-rescued LPS-attenuated mineralization in hDPSCs, suggesting that CBD potentially mediates Notch activation to exert its impact on odonto/osteogenic differentiation of hDPSCs. CBD recovers the proliferation and survival of hDPSCs following exposure to LPS. Additionally, we report that CBD-mediated Notch activation effectively restores the odonto/osteogenic differentiation ability of hDPSCs under inflamed conditions. These results underscore the potential role of CBD as a therapeutic option to enhance dentine regeneration.

Genomic insights into drug resistance and virulence determinants in rare pyomelanin-producing clinical isolates of Acinetobacter baumannii.

Clinical isolates of multi-drug resistant Acinetobacter baumannii are a major cause of nosocomial infections, often attributed to the highly adaptable genome that helps it tothrive under environmental selection pressure. Here, we aim to provide genotypic-based surveys and comparative whole genome sequencing (WGS) analysis to explore the genomics of the rare pyomelanin-forming clinical isolates of A. baumannii from India. A total of 54 clinical isolates of A. baumannii obtained from two tertiary care hospitals were genotyped using repetitive sequence-based PCR(REP-PCR) for elucidating their molecular epidemiology, followed by their resistance profiling through the determination of minimum inhibitory concentration using the micro broth dilution method. The isolates' virulence and antibiotic-resistant determinants were detected by PCR screening, followed by biofilm quantification. Pyomelanin pigment produced by A. baumannii isolates was isolated and chemically characterized. Finally, WGS of three pigment-producing and one non-producing A. baumannii strains was performed to explore the factors contributing to their variability. REP-PCR genotyping identified around 8 clusters, with all isolates being multidrug-resistant (MDR). Pyomelanin-producing isolates were strong biofilm formers, characterized by the concurrent presence of 'pgaB, BfmR, BfmS, ompA, and cusE' biofilm-related genes. These pigmented strains belonged to ST2Pas and co-harbored blaOXA-23, blaADC-25, aph (3')-VIa, armA, aph (6)-Id, tet(B) and msr(E) genes. Thirteen common IS elements and biosynthetic gene clusters of arylpolyene, NI-siderophore, and NRP-metallophore were identified. Notably, genomic islands containing aminoglycoside 3'-phosphotransferase, oxidative stress, two-component response regulators, efflux pump-related, toxin-antitoxin protein, and virulence-related genes were also mapped by WGS. The pyomelanin-forming isolates were MDR and virulent. The elucidation of WGS analysis provided critical insights for understanding the epidemiology, virulome, and mobilome of rare pigment-producing A. baumannii strains.

Molecular characterization and safety properties of multi drug-resistant Escherichia coli O157:H7 bacteriophages

The increase in multi drug resistance (MDR) amongst food-borne pathogens such as Escherichia coli O157:H7, coupled with the upsurge of food-borne infections caused by these pathogens is a major public health concern. Lytic phages have been employed as an alternative to antibiotics for use against food-borne pathogens. However, for effective application, phages should be selectively toxic. Therefore, the objective of this study was to characterise lytic E. coli O157:H7 phages isolated from wastewater as possible biocontrol agents and access their genomes for the absence of genes that denotes virulence, resistance, toxins, and lysogeny using whole genome sequencing. E. coli O157:H7 bacteriophages showed clear plaques ranging in size from 1.0 mm to 2.0 mm. Spot test and Efficiency of plating (EOP) analysis demonstrated that isolated phages could infect various environmental E. coli strains. Four phages; vB_EcoM_EP32a, vB_EcoP_EP32b, vB_EcoM_EP57, and vB_EcoM_EP69 demonstrated broad lytic spectra against E. coli O157:H7 strains. Transmission Electron Microscopy (TEM) showed that all phages have tails and were classified as Caudoviricetes. Growth parameters showed an average latent period of 15 ± 3.8 min, with a maximum burst size of 392 PFU/cell. The phages were stable at three distinct temperatures (4 °C, 28 °C, and 37 °C) and at pH values of 3.5, 5.0, 7.0, 9.0, and 11.0. Based on their morphological distinctiveness, three phages were included in the Whole Genome Sequencing (WGS) analysis. WGS results revealed that E. coli O157:H7 phages (vB_EcoM_EP32a, vB_EcoP_EP32b, and vB_EcoM_EP57) were composed of linear double-stranded DNA (dsDNA) with genome sizes 163,906, 156,698, and 130,723 bp and GC contents of 37.61, 37, and 39% respectively. Phages vB_EcoM_EP32a and vB_EcoP_EP32b genomes were classified under the class Caudoviricetes, Straboviridae family, and the new genus “Phapecoctavirus”, while vB_EcoM_EP57 was classified under the class Caudoviricetes, Autographiviridae family. Genome analysis revealed no lysogenic (integrase), virulence, or antimicrobial resistance sequences in all three Escherichia phage genomes. The overall results provided evidence that lytic E. coli O157:H7 bacteriophages in this study, are relatively stable, can infect diverse E. coli strains, and does not contain genes responsible for virulence, resistance, toxins, and lysogeny. Thus, they can be considered as biocontrol candidates against MDR pathogenic E. coli O157:H7 strains in the food industry.

Emergence and clonal dissemination of KPC-2- and NDM-1-coharboring Citrobacter freundii in China with an IncR plasmid.

The rise of carbapenem-resistant Enterobacteriaceae coharboring KPC-2 and NDM-1 poses a significant public health threat. KPC-2-NDM-1-Citrobacter freundii is rarely reported in clinical settings. In this study, we report the largest cohort of eight KPC-2-NDM-1-C. freundii isolated from children with urinary tract infections. Comprehensive characterization, including antimicrobial susceptibility testing, plasmid conjugation, whole-genome sequencing, and comparative genomics, was conducted for these clinical strains. Antimicrobial susceptibility testing indicated that all strains were resistant to meropenem [minimal inhibitory concentration (MICs) ≥ 8 µg/mL] and imipenem (MICs > 8 µg/mL). Genotyping and comparative genomics analyses identified the eight KPC-2-NDM-1- C. freundii belonging to ST523, exhibiting a close relationship characterized by 45 single nucleotide polymorphisms. Whole-genome sequencing and plasmid analysis confirmed the presence of blaKPC-2 and blaNDM-1 on an IncR plasmid named pC275-2 with 46,050 bp. The blaNDM-1 was integrated within the blaNDM-1 related region, which includes ΔISAba125, blaNDM-1, ble-MBL, trpF, dsbD, and ISCR1, while the blaKPC-2 gene was located on a novel non-Tn4401 genetic element. The blaKPC-2 genetic architectures containing blaKPC-2 differed from classical structures, underscoring the ongoing evolution of these genetic elements.IMPORTANCEOur study described the largest cohort to date of eight ST523 KPC-2-NDM-1-C. freundii isolated from children with urinary tract infections. The cooccurrence of KPC-2, a serine β-lactamases, and NDM-1, a metallo-β-lactamase on an IncR plasmid pC275-2 from a clinical carbapenem-resistant C. freundii. The conserved insertion structure mediated the blaNDM-1, and the propagation of blaKPC-2 gene with a new genetic background using IncR plasmid in clinical settings promotes the emergence of superbugs necessitating vigilant monitoring. Our research detected that ST523 KPC-2- NDM-1-C. freundii were disseminated in a children's hospital. The potential spread of an IncR plasmid within the hospital raises concerns about the pandemic potential of this clone that produces two carbapenemases: KPC-2 and NDM-1. Further investigations will be necessary to control and prevent the spread of KPC-2-NDM-1-C. freundiis.

Antimicrobial resistance and virulence factors analysis of a multidrug-resistant Acinetobacter baumannii isolated from chickens using whole-genome sequencing

Multidrug-resistant (MDR) Acinetobacter baumannii (A. baumannii) is currently recognized not only as a significant nosocomial pathogen but also is an emerging bacterial infection in food-producing animals, posing a critical threat to global health. However, this is a hindrance to detailed bioinformatic studies of MDR A. baumannii of chicken origin due to the lack of its complete genome sequence. Here, we report whole-genome sequencing analysis of MDR A. baumannii Y03 isolated from chickens. The Y03 genome consists of 1 circular chromosome and 4 circular plasmids, The Y03 chromosome harbors 41 antimicrobial resistance genes conferring resistance to major classes of antibiotics, including β-lactams, phenicols, macrolides, lincosamides, aminoglycosides, and nitrofurans, as well as 135 virulence factors involved in effector delivery system, immune modulation, adherence, stress survival, biofilm, exotoxin, and nutritional/metabolic factor. The in vivo infection experiments certificated that Y03 was virulent to chickens. Meanwhile, we used PCR amplification method to detect 10 antimicrobial resistance genes including abeM, adeB, adeH, adeK, blaapmC, blaOXA−90, catB9, macB, folP, and parE, as well as 14 virulence genes including lpxC, pilO, fimT, ompA, basA, bauA, gspL, csu, pgaC, plc2, tssA, tviB, bap, and vgrG. Whole-genome sequencing analysis revealed that Y03 contained 46 horizontal gene transfer elements, including 11 genomic islands, 30 transposons, and 5 prophages, as well as 518 mutations associated with reduced virulence and 44 mutations resulting in loss of pathogenicity. Furthermore, there were 22 antibiotic targets and 28 lethal mutations on the Y03 chromosome that could be used as potential targets to prevent, control, and treat infections caused by MDR A. baumannii Y03. Therefore, this study contributes to the development of strategies for the prevention, control, and treatment of A. baumannii infections and their spread in chickens.

Aging and head and neck cancer insights from single cell and spatial transcriptomic analyses

BackgroundHead and neck squamous cell carcinoma(HNSCC) is the sixth most common malignancy worldwide, with more than 890,000 new cases and 450,000 deaths annually. Its major risk factors include smoking, alcohol abuse, aging, and poor oral hygiene. Due to the lack of early and effective detection and screening methods, many patients are diagnosed at advanced stages with a five-year survival rate of less than 50%. In this study, we deeply explored the expression of Aging-related genes(ARGs) in HNSCC and analyzed their prognostic significance using single-cell sequencing and spatial transcriptomics analysis. This research aims to provide new theoretical support and directions for personalized treatment.Annually, more than 890,000 new cases of head and neck squamous cell carcinoma (HNSCC) are diagnosed globally, leading to 450,000 deaths, making it the sixth most common malignancy worldwide. The primary risk factors for HNSCC include smoking, alcohol abuse, aging, and poor oral hygiene. Many patients are diagnosed at advanced stages due to the absence of early and effective detection and screening methods, resulting in a five-year survival rate of less than 50%. In this research, single cell sequencing and spatial transcriptome analysis were used to investigate the expression of Aging-related genes (ARGs) in HNSCC and to analyse their prognostic significance. This research aims to provide new theoretical support and directions for personalized treatment.MethodsIn this study, we investigated the association between HNSCC and AGRs by utilizing the GSE139324 series in the GEO database alongside the TCGA database, combined with single-cell sequencing and spatial transcriptomics analysis. The data were analyzed using Seurat and tSNE tools to reveal intercellular communication networks. For the spatial transcriptome data, SCTransform and RunPCA were applied to examine the metabolic activities of the cells. Gene expression differences were determined through spacerxr and RCTD tools, while the limma package was employed to identify differentially expressed genes and to predict recurrence rates using Cox regression analysis and column line plots. These findings underscore the potential importance of molecular classification, prognostic assessment, and personalized treatment of HNSCC.ResultsThis study utilized HNSCC single-cell sequencing data to highlight the significance of ARGs in the onset and prognosis of HNSCC. It revealed that the proportion of monocytes and macrophages increased, while the proportion of B cells decreased. Notably, high expression of the APOE gene in monocytes was closely associated with patient prognosis. Additionally, a Cox regression model was developed based on GSTP1 and age to provide personalized prediction tools for clinical use in predicting patient survival.ConclusionsWe utilized single-cell sequencing and spatial transcriptomics to explore the cellular characteristics of HNSCC and its interaction with the tumor microenvironment. Our findings reveal that HNSCC tissues show increased mononuclear cells and demonstrate enhanced activity in ARGs, thereby advancing our understanding of HNSCC development mechanisms.

A biomathematical model of SARS-CoV-2 in Syrian hamsters

When infected with SARS-CoV-2, Syrian hamsters (Mesocricetus auratus) develop moderate disease severity presenting key features of human COVID-19. We here develop a biomathematical model of the disease course by translating known biological mechanisms of virus-host interactions and immune responses into ordinary differential equations. We explicitly describe the dynamics of virus population, affected alveolar epithelial cells, and involved relevant immune cells comprising for example CD4+ T cells, CD8+ T cells, macrophages, natural killer cells and B cells. We also describe the humoral response dynamics of neutralising antibodies and major regulatory cytokines including CCL8 and CXCL10. The model is developed and parametrized based on experimental data collected at days 2, 3, 5, and 14 post infection. Pulmonary cell composition and their transcriptional profiles were obtained by lung single-cell RNA (scRNA) sequencing analysis. Parametrization of the model resulted in a good agreement of model and data. The model can be used to predict, for example, the time course of the virus population, immune cell dynamics, antibody production and regeneration of alveolar cells for different therapy scenarios or after multiple-infection events. We aim to translate this model to the human situation in the future.

PtrA, Piz-t, and a novel minor-effect QTL (qBR12_3.3–4.4) collectively contribute to the durable blast-resistance of rice cultivar Tainung 84

BackgroundRice blast caused by Pyricularia oryzae is a major threat to rice production worldwide. Tainung 84 (TNG84) is an elite japonica rice cultivar developed through the traditional pedigree method. It has maintained superior blast resistance since its release in 2010. This study aimed to investigate the genetic factors underlying the durable resistance of TNG84 in Taiwan.ResultsQuantitative trait locus (QTL) mapping was conducted using 122 F2 individuals and F2:3 families derived from the cross of TNG84 and a susceptible japonica cultivar Tainan 11 (TN11). Using 733 single nucleotide polymorphisms (SNPs) obtained through genotyping-by-sequencing and three P. oryzae isolates (D41-2, 12CY-MS1-2, and 12YL-TT4-1) belonging to different physiological races and Pot2 lineages, a major QTL was identified in the region of 52–54 cM (9.54–15.16 Mb) on chromosome 12. Fine-mapping using 21 F5:6 recombinants delimited the QTL to a 140.4-kb region (10.78 to 10.93 Mb) containing the known resistance gene Ptr. Sequencing analysis indicated that TNG84 carries the resistant PtrA allele and TN11 carries the susceptible PtrD allele. Investigation of the Ptr haplotypes in 41 local japonica rice cultivars revealed that eight PtrA-containing cultivars (19.5%) consistently exhibited good field resistance in Taiwan from 2008 to 2024. Subsequently, a few F5:6 lines (P026, P044, P092, and P167) lacking the resistant Ptr allele were observed to exhibit a resistant phenotype against P. oryzae 12YL-TT4-1-lab. Trait-marker association analyses using eight F6:7 homozygous recombinants, 378 BC1F2 from P044 backcrossed to TN11, and 180 BC1F2 from P092 backcrossed to TN11, identified Piz-t on chromosome 6 and a new QTL located between 3.3 Mb and 4.4 Mb on chromosome 12 (designated as qBR12_3.3–4.4). Analysis of 12 selected BC1F2:3 lines derived from P044 demonstrated that in the absence of Ptr and Piz-t, qBR12_3.3–4.4 alone reduced the disease severity index from approximately 6.3 to 3.9.ConclusionsPtrA is likely the primary gene responsible for the broad-spectrum and durable resistance of TNG84. Piz-t confers narrow-spectrum resistance, while qBR12_3.3–4.4 contributes partial resistance. The discovery of qBR12_3.3–4.4 has provided a new source of blast resistance, and the markers developed in this study can be utilized in future breeding programs.

ZO-1 boosts the in vitro self-renewal of pre-haematopoietic stem cells from OCT4-reprogrammed human hair follicle mesenchymal stem cells through cytoskeleton remodeling

BackgroundThe challenge of expanding haematopoietic stem/progenitor cells (HSPCs) in vitro has limited their clinical application. Human hair follicle mesenchymal stem cells (hHFMSCs) can be reprogrammed to generate intermediate stem cells by transducing OCT4 (hHFMSCsOCT4) and pre-inducing with FLT3LG/SCF, and differentiated into erythrocytes. These intermediate cells exhibit gene expression patterns similar to pre-HSCs, making them promising for artificial haematopoiesis. However, further investigation is required to elucidate the in vitro proliferation ability and mechanism underlying the self-renewal of pre-HSCs derived from hHFMSCs.MethodshHFMSCsOCT4 were pre-treated with FLT3LG and SCF cytokines, followed by characterization and isolation of the floating cell subsets for erythroid differentiation through stimulation with hematopoietic cytokines and nutritional factors. Cell adhesion was assessed through disassociation and adhesion assays. OCT4 expression levels were measured using immunofluorescence staining, RT-qPCR, and Western blotting. RNA sequencing and Gene Ontology (GO) enrichment analysis were then conducted to identify proliferation-related biological processes. Proliferative capacity was evaluated through CCK-8, colony formation assays, Ki67 index, and cell cycle analysis. Cytoskeleton was observed through Wright‒Giemsa, Coomassie brilliant blue, and phalloidin staining. Expression of adherens junction (AJ) core members was confirmed through RT‒qPCR, Western blotting, and immunofluorescence staining before and after ZO-1 knockdown. A regulatory network was constructed to determine relationships among cytoskeleton, proliferation, and the AJ pathway. Student’s t tests (GraphPad Prism 8.0.2) were used for group comparisons. The results were considered significant at P < 0.05.ResultsPre-treatment of hHFMSCsOCT4 with FLT3LG and SCF leads to the emergence of floating cell subsets exhibiting small, globoid morphology, suspended above adherent cells, forming colonies, and displaying minimal expression of CD45. Excessive OCT4 expression weakens adhesion in floating hHFMSCsOCT4. Floating cells moderately enhanced proliferation and undergo cytoskeleton remodelling, with increased contraction and aggregation of F-actin near the nucleus. The upregulation of ZO-1 could impact the expressions of F-actin, E-cadherin, and β-catenin genes, as well as the nuclear positioning of β-catenin, leading to variations in the cytoskeleton and cell cycle. Finally, a regulatory network revealed that the AJ pathway cored with ZO-1 critically bridges cytoskeletal remodelling and haematopoiesis-related proliferation in a β-catenin-dependent manner.ConclusionsZO-1 improved the self-renewal of pre-HSCs from OCT4-overexpressing hHFMSCs by remodeling the cytoskeleton via the ZO-1-regulated AJ pathway, suggesting floating hHFMSCsOCT4 as the promising seed cells for artificial hematopoiesis.

The role of gut microbiota and metabolomic pathways in modulating the efficacy of SSRIs for major depressive disorder

This study aims to explore the mechanism by which gut microbiota influences the antidepressant effects of serotonin reuptake inhibitors (SSRIs) through metabolic pathways. A total of 126 patients were analyzed for their gut microbiota and metabolomics. Patients received SSRI treatment and were categorized into responder and non-responder groups based on changes in their Hamilton Depression Rating Scale (HAMD-17) scores before and after treatment. The association between gut microbiota composition and the efficacy of SSRIs was investigated through 16S rRNA gene sequencing and metabolomic analysis, and a predictive model was developed. As a result, the study found significant differences in gut microbiota composition between the responder and resistant groups. Specific taxa, such as Ruminococcus, Bifidobacterium, and Faecalibacterium, were more abundant in the responder group. Functional analysis revealed upregulation of acetate degradation and neurotransmitter synthesis pathways in the responder group. The machine learning model indicated that gut microbiota and metabolites are potential biomarkers for predicting SSRIs efficacy. In conclusion, gut microbiota influences the antidepressant effects of SSRIs through metabolic pathways. The diversity and function of gut microbiota can serve as biomarkers for predicting the treatment response, providing new insights for personalized treatment.

MACC1 ablation suppresses the dedifferentiation process of non-CSCs in lung cancer through stabilizing KLF4

Metastasis-associated in colon cancer-1 (MACC1) was identified as a new player in lung cancer development, and some stemness-related genes can be novel transcriptional targets of MACC1. Cancer stem cells (CSCs) are responsible for sustaining tumorigenesis and plasticity. Both CSCs and non-CSCs are plastic and capable of undergoing phenotypic transition, especially the dedifferentiation of non-CSCs switch to CSC-like cells. However, the precise role of MACC1 during this process is largely unknown. Here, we showed that MACC1 promoted the transition from non-CSC to CSC in lung cancer. We found MACC1 was overexpressed in stemness enriched cells, enhancing the transition from no-CSCs to CSCs, while short-hairpin RNA-mediated Knockdown of MACC1 impaired this process. High-throughput sequencing and tumor specimen analysis revealed that MACC1 was negative correlated with Krüppel-like factor 4 (KLF4) expression level, which acts as a negative stemness regulator in lung cancer. Mechanistically, MACC1 delays the degradation of KLF4 mRNA by repressing the expression of microRNA-25, thereby promoting the KLF4 mRNA stabilization at the post-transcriptional level. Collectively, our findings may facilitate efforts to promote the development of precision targeted therapy for cancer stem cells in lung cancer.

EMSA-SELEX-seq method for analysis of binding site sequences in DNA-protein complexes

The BOB1 protein (OBF1, OCA-B) is a transcriptional coactivator of two POU domain proteins — OCT1, expressed in all cells, and lymphoid-specific OCT2. The interaction of BOB1 with OCT1/2 plays an important role in the regulation of immune responses in both physiological and pathological contexts. BOB1 is known to form a ternary complex with OCT1/2 bound to DNA in monomeric and certain dimeric configurations, changing the sequence specificity of the binding. To analyze DNA sequences from these complexes, in this work we proposed the EMSA-SELEX-seq method, based on the separation of OCT/BOB1 complexes of various compositions in a non-denaturing polyacrylamide gel (EMSA) followed by the isolation and amplification of the oligonucleotides that they contain (SELEX). Based on several rounds of the enrichment followed by the NGS sequencing and bioinformatics analysis, the DNA sequences were determined and the relevance of this approach was confirmed. Thus, the proposed EMSA-SELEX-seq method allows the analysis of DNA sequences in DNA-protein complexes with varying dimensions of its protein components.

Pasteurella multocida Serotype D Infection Induces Activation of the IL-17 Signaling Pathway in Goat Lymphocytes

(1) Background: Pasteurellosis is a global zoonotic bacterial disease, which has caused significant economic impacts in animal husbandry. Nevertheless, there is limited understanding of the immune response between goat peripheral blood lymphocytes (PBLs) and goat-derived Pasteurella multocida (P. multocida). (2) Methods: To investigate the immune response of host PBLs during infection with P. multocida type D, we established an in vitro cell model utilizing isolated primary goat PBLs. Utilizing this in vitro infection model, we employed an enzyme-linked immunosorbent assay (ELISA) to assess the cytokine profile variation in goat PBLs following infection. Meanwhile, RNA sequencing and quantitative PCR (qPCR) methods were employed to analyze the gene expression profile. (3) Results: The ELISA test results indicated that the expression levels of pro-inflammatory cytokines, such as IL-6, IFN-γ, CXCL10, and IL-17A, were significantly elevated within 12 h after infection with P. multocida. In contrast, the levels of the anti-inflammatory cytokine IL-10 were found to be reduced. RNA sequencing and functional enrichment analysis identified 2114 differentially expressed genes (DEGs) that were primarily associated with cytokine-cytokine receptor interactions, viral protein-cytokine interactions, and the IL-17 signaling pathway. Furthermore, protein-protein interaction (PPI) network analysis and qPCR highlighted CD86, CCL5, CD8A, CXCL8, CTLA4, TNF, CD274, IL-10, IL-6, CXCL10, IFNG, and IL-17A that were crucial for the response of PBLs to P. multocida infection. (4) Conclusions: This study systematically revealed the characteristics of PBLs in goats following infection with goat-derived P. multocida type D through the analysis of cytokines and gene expression, providing important theoretical insights for a deeper understanding of the defense mechanisms in goats against P. multocida.

Exploring IAA biosynthesis and plant growth promotion mechanism for tomato root endophytes with incomplete IAA synthesis pathways

Exploring indoleacetic acid (IAA) biosynthesis pathways of plant growth promoting bacteria and their ability to synthesize IAA is crucial for understanding the promotion mechanism and for developing more efficient microbial fertilizer. In this study, 118 bacterial endophytic strains were isolated from tomato root and 68 isolates were identified as members of Bacillus and Pseudomonas genus. After screening abilities to synthesize IAA in vitro and promote plant growth for these identified Bacillus and Pseudomonas strains, 7 endophytic strains Bacillus sp. Y_21, B. paramycoides Y_29, B. albus Y_96, B. cereus Y_121, P. plecoglossicida Y_157, Bacillus sp. Y_165 and B. aryabhattai Y_170 strains showed a significant promotion role on wheat root or leaf, including 4 endophytic strains with the potential ability to utilize intermediate metabolites, such as tryptamine and indole acetamide, to produce IAA. Genomic sequencing analysis for selected representative plant growth promoting endophytes showed that IAA-producing bacteria B. cereus mr31 and P. putida Y_166 separately harbored a complete indoleacetamide (IAM) and complete tryptamine (TAM) pathway; whereas, Bacillus sp. Y_165 and B. aryabhattai Y_170, two strains producing IAA not using tryptophan but with indole-3-acetamide, had an incomplete IAM pathway. Fermentation experiments of three genome-sequenced strains using wheat or tomato root extracts as substrate, and combining with UHPLC–MS/MS analysis for wheat root extracts, demonstrated that Bacillus sp. Y_165 strain might produce IAA by using the plant-derived indole-3-acetamide. Our study first demonstrated a novel potential mechanism for the plant growth promoting endophytes to biosynthesize IAA using plant-derived intermediates. This additional mechanism has deepened our understanding of how bacterial endophytes promote plant health and resilience, offering valuable insights about how plants regulate IAA homeostasis within their internal tissues in association with bacterial endophytes.Graphical

Synergistic SDT/cuproptosis therapy for liver hepatocellular carcinoma: enhanced antitumor efficacy and specific mechanisms

The efficacy of sonodynamic therapy (SDT), an emerging approach for tumor treatment, is hindered by the high levels of the antioxidant glutathione (GSH) in the tumor microenvironment (TME). In this study, we constructed nanobubbles loaded with the sonosensitizer HMME and the tumor-targeting peptide RGD (HMME-RGD@C3F8 NBs) for synergistic SDT/cuproptosis therapy of liver hepatocellular carcinoma (LIHC) in combination with Elesclomol-Cu as cuproptosis inducers. Endogenous GSH is consumed by Cu2+ to modulate the complex TME, thereby amplifying oxidative stress and further improving SDT performance. Additionally, intracellular Cu2+ overload can induce cuproptosis, which is further amplified by SDT, to initiate irreversible protein toxicity. The specific mechanism of synergistic SDT/cuproptosis therapy in LIHC was investigated by RNA sequencing analysis. The synergistic SDT/cuproptosis therapy reprogrammed the TME to improve the efficacy of immune checkpoint inhibitor-based immunotherapy. Furthermore, a risk-scoring model was created and displayed significant promise in the prognosis of LIHC.Graphical

Integrated lipidomics and RNA-seq reveal prognostic biomarkers in well-differentiated and dedifferentiated retroperitoneal liposarcoma

BackgroundRetroperitoneal liposarcoma (RLPS) is a mesenchymal malignant tumor characterized by different degrees of adipocytic differentiation. Well-differentiated liposarcoma (WDLPS) and dedifferentiated liposarcoma (DDLPS) are two of the most common subtypes of RLPS, exhibiting clear differences in biological behaviors and clinical prognosis. The metabolic features and genomic characteristics remain unclear.MethodsThis study employed lipidomic and RNA-seq analyses of RLPS tissues from 19 WDLPS and 29 DDLPS patients. Western blot and immunohistochemistry staining were performed to verify the tumor tissue protein levels of TIMP1, FN1, MMP11, GPNMB, and ECM1. Enzyme-linked immunosorbent assay (ELISA) was performed to evaluate different serum protein levels in 128 blood samples from patients with RLPS. Multivariate analysis was performed to identify the most crucial variables associated with overall survival (OS) and recurrence-free survival (RFS) of the RLPS patients.ResultsLipidomic analysis revealed a significant difference in lipid metabolism, particularly in phosphatidylcholines and triacylglycerides metabolism. RNA sequencing analysis revealed that 1,630 differentially expressed genes (DEGs) were significantly enriched in lipid metabolism, developmental process, and extracellular matrix (ECM) pathways. Integrated lipidomic and transcriptomic analysis identified 29 genes as potential biomarkers between WDLPS and DDLPS. Among the 29 DEGs, we found that TIMP1, FN1, MMP11, GPNMB, and ECM1 were increased in DDLPS tumor tissues than in WDLPS tumor tissues. The receiver operating characteristic (ROC) curve showed high specificity and sensitivity in diagnosing patients using a five-gene combination (AUC = 0.904). ELISA revealed a significant increase in the serum levels of ECM1 and GPNMB in patients with DDLPS compared to patients with WDLPS. ECM1 increased progressively across different FNCLCC Grades, correlating negatively with RFS (P = 0.043). GPNMB levels showed a negative correlation with OS (P = 0.019).ConclusionsOur study reveals different lipid metabolism, several transcriptional pathways between WDLPS and DDLPS, and examines several serum markers associated with the prognosis of RLPS. These findings provide a vital basis for future endeavors in diagnosing and predicting the prognosis of retroperitoneal liposarcoma with different differentiations.

Intestinal metaplasia key molecules and UPP1 activation via Helicobacter pylori /NF-kB: drivers of malignant progression in gastric cancer

Gastric cancer (GC) remains a significant global health challenge due to its high morbidity and mortality rates. The development of GC is a multi-hit process and the exploration of precancerous lesions is crucial. To elucidate the molecular and cellular dynamics underlying gastric carcinogenesis, we conducted an integrative single-cell RNA sequencing analysis of 26,028 high-quality cells from gastric antral mucosa biopsies across various stages, including non-atrophic gastritis, chronic atrophic gastritis, intestinal metaplasia, and early gastric cancer. By constructing a detailed single-cell atlas, we identified distinct epithelial cell subpopulations and their corresponding molecular signatures. We focused on the biological link between gastric epithelial cells and cancer cells. Notably, we observed that gland mucous cells acquired an intestinal-like stem cell phenotype during metaplasia, with MUC6, MUC2 and OLFM4 emerging as the specific markers for unique endocrine cells in early malignant lesions. Additionally, our analysis highlighted UPP1 as a key oncogene, with its expression progressively increasing from normal epithelial cells to malignant cells. UPP1 upregulation was shown to promote GC cell proliferation and migration, implicating it in the oncogenic process. Further, we explored the impact of Helicobacter pylori infection on gene expression, revealing that Helicobacter pylori infection upregulates UPP1 via the NF-κB pathway. Our cell-cell communication analysis underscored the significant role of the Macrophage migration inhibitory factor pathway in the tumor microenvironment, contributing to GC progression. Various key molecules involved in intestinal metaplasia, along with UPP1 and the Macrophage migration inhibitory factor pathway, collectively illustrate the multifaceted nature and complexity of gastric cancer evolution, highlighting the cumulative impacts that drive tumorigenesis.

Exosome-delivered NR2F1-AS1 and NR2F1 drive phenotypic transition from dormancy to proliferation in treatment-resistant prostate cancer via stabilizing hormonal receptors

Cancer cells acquire the ability to reprogram their phenotype in response to targeted therapies, yet the transition from dormancy to proliferation in drug-resistant cancers remains poorly understood. In prostate cancer, we utilized high-plasticity mouse models and enzalutamide-resistant (ENZ-R) cellular models to elucidate NR2F1 as a key factor in lineage transition and ENZ resistance. Depletion of NR2F1 drives ENZ-R cells into a relative dormancy state, characterized by reduced proliferation and heightened drug resistance, while NR2F1 overexpression yields contrasting outcomes. Transcriptional sequencing analysis of NR2F1-silenced prostate cancer cells and tissues from the Cancer Genome Atlas-prostate cancer and SU2C cohorts indicated exosomes as the most enriched cell component, with pathways implicated in steroid hormone biosynthesis and drug metabolism. Moreover, NR2F1-AS1 forms a complex with SRSF1 to upregulate NR2F1 expression, facilitating its binding with ESR1 to sustain hormonal receptor expression and enhance proliferation in ENZ-R cells. Furthermore, HnRNPA2B1 interacts with NR2F1 and NR2F1-AS1, assisting their packaging into exosomes, wherein exosomal NR2F1 and NR2F1-AS1 promote the proliferation of dormant ENZ-R cells. Our works offer novel insights into the reawaking of dormant drug-resistant cancer cells governed by NR2F1 upregulation triggered by exosome-derived NR2F1-AS1 and NR2F1, suggesting therapeutic potential for phenotype reversal.Graphical

Deciphering pathogenicity and virulence of the first Staphylococcus debuckii isolate from diabetic foot osteomyelitis

IntroductionThis study identifies Staphylococcus debuckii as a new coagulase-negative staphylococcal species isolated from diabetic foot osteomyelitis (DFOM) and provides an in-depth analysis of its pathogenic and virulence profile, as well as demonstrating its potential to cause infection.MethodsThe S. debuckii NSD001 strain was examined for its planktonic growth, biofilm production, and phagocytosis rates in murine macrophages compared to S. aureus NSA739. Additionally, persistence and replication within human osteoblasts were investigated, while the zebrafish embryo model was employed to assess virulence. Genomic sequencing and bioinformatic analysis were also conducted to identify genes associated with virulent potential.Results and DiscussionS. debuckii NSD001 exhibited robust planktonic growth and significant biofilm production, highlighting its capacity to initiate and maintain an infection, and demonstrated similar rates of phagocytosis as S. aureus NSA739 in murine macrophages, suggesting a mechanism for evading initial host defenses. The strain persisted and replicated within human osteoblasts, indicative of a strategy for intracellular survival and facilitation of chronic osteomyelitis. The zebrafish embryo model revealed a slower, yet fatal, virulence profile for S. debuckii NSD001 compared to the rapid lethality induced by S. aureus NSA739. Genomic sequencing and bioinformatic analysis uncovered various genes corroborating its virulence. S. debuckii NSD001 poses a significant concern in DFOM due to its ability to form biofilms and survive within host cells, presenting challenges for current treatment strategies. This underscores the need for updated clinical protocols and increased awareness among healthcare professionals to effectively manage infections caused by this emerging pathogen.

SmHSFA8 Enhances the Heat Tolerance of Eggplant by Regulating the SmEGY3-SmCSD1 Module and Promoting SmF3H-mediated Flavonoid Biosynthesis.

High temperature (HT) is a major environmental factor that restrains eggplant growth and production. Heat shock factors (HSFs) play a vital role in the response of plants to high-temperature stress (HTS). However, the molecular mechanism by which HSFs regulate heat tolerance in eggplants remains unclear. Previously, we reported that SmEGY3 enhanced the heat tolerance of eggplant. Herein, SmHSFA8 activated SmEGY3 expression and interacted with SmEGY3 protein to enhance the activation function of SmEGY3 on SmCSD1. Virus-induced gene silencing (VIGS) and overexpression assays suggested that SmHSFA8 positively regulated heat tolerance in plants. SmHSFA8 enhanced the heat tolerance of tomato plants by promoting SlEGY3 expression, H2O2 production and H2O2-mediated retrograde signalling pathway. DNA affinity purification sequencing (DAP-seq) analysis revealed that SmHSPs (SmHSP70, SmHSP70B and SmHSP21) and SmF3H were candidate downstream target genes of SmHSFA8. SmHSFA8 regulated the expression of HSPs and F3H and flavonoid content in plants. The silencing of SmF3H by VIGS reduced the flavonoid content and heat tolerance of eggplant. In addition, exogenous flavonoid treatment alleviated the HTS damage to eggplants. These results indicated that SmHSFA8 enhanced the heat tolerance of eggplant by activating SmHSPs exprerssion, mediating the SmEGY3-SmCSD1 module, and promoting SmF3H-mediated flavonoid biosynthesis.