Transcriptomic characterization and construction of M2 macrophage-related prognostic and immunotherapeutic signature in ovarian metastasis of gastric cancer.

BACKGROUND: Ovarian metastasis (OM) poses a major threat to the outcome of gastric cancer (GC) patients. Recently, immunotherapy emerged as a novel promising therapeutic strategy to treat late-stage GC, whereas its efficacy is influenced by tumor immune microenvironment (TIME). M2 macrophage, a key subset within TIME, plays dual immunosuppressive and pro-tumorigenic roles in cancer progression and is recognized as a potential therapeutic target. However, molecular mechanisms underlying OM remain elusive and the TIME-related prognostic and immunotherapeutic index for these patients is yet to establish. METHODS: Differential expressed genes (DEGs) between paired normal mucosa, primary GC and OM of patients from Fudan University Shanghai Cancer Center (FUSCC) cohort (n = 6) were identified by transcriptome sequencing, followed by the functional annotation of enriched hallmark pathways of DEGs between them. CIBERSORT was used to profile the relative expression level of 22 immune cell subsets in normal tissues, primary and metastatic tumors, followed by weighted gene coexpression network analysis (WGCNA) uncovering immune cell-correlated gene sets. The intersected genes between DEGs and M2 macrophage-related genes were processed by least absolute shrinkage and selection operator (LASSO) regression analysis to construct a predictive signature, M2GO, which was further validated by training set and test set of The Cancer Genome Atlas-Stomach Adenocarcinoma (TCGA-STAD), GSE62254 and GSE84437 cohorts. GC patients were divided into M2GO-high and -low subgroup according to the optimal cutoff value of the M2GO score. Furthermore, the clinical, molecular and immune features between M2GO-high and -low subgroups were analyzed. Clinical cohorts of immunotherapy were used to validate the predictive value of M2GO in regard to immunotherapy effectiveness. RESULTS: Transcriptomic sequencing and follow-up analyses of triple-matched normal tissues, primary and ovarian metastatic tumors identified distinctive sets of DEGs and enriched immune-, cancer- and metastasis-related pathways between them. Of note, M2 macrophage, a major immunosuppressive and pro-tumorigenic component within TIME, was significantly up-regulated in OMs. WGCNA and LASSO regression were applied to establish a novel OM- and M2 macrophage-related predictive signature, M2GO, based on M2 macrophage-related prognostic genes including GJA1, MAGED1 and SERPINE1. M2GO served as an independent prognostic factor of GC patients. Comprehensive molecular and immune characterization of M2GO-based subgroups uncovered their distinctive features in terms of enriched functional pathways, tumor mutation burden, key immune checkpoints, major regulators of natural immune cGAS-STING pathway, infiltrated subsets of immune cells and tumor immune exclusion/dysfunction (TIDE) score. Notably, the M2GO score was significantly lower in responsive group than non-responsive group (P < 0.05) in clinical cohort of metastatic GC patients undergoing immunotherapy. CONCLUSION: Transcriptomic characterization of paired normal mucosae, primary and ovarian metastatic tumors revealed their unique molecular and immune features. Follow-up analyses established a novel OM- and M2 macrophage-related signature, M2GO, which served as a promising prognostic and immunotherapeutic biomarker to distinguish the clinical outcome, molecular and immune features of GC patients and predict their differential responses to immunotherapy.

Direct prediction of carbapenem resistance in Pseudomonas aeruginosa by whole genome sequencing and metagenomic sequencing.

Carbapenem resistance is a major concern in the management of antibiotic-resistant Pseudomonas aeruginosa infections. The direct prediction of carbapenem-resistant phenotype from genotype in P. aeruginosa isolates and clinical samples would promote timely antibiotic therapy. The complex carbapenem resistance mechanism and the high prevalence of variant-driven carbapenem resistance in P. aeruginosa make it challenging to predict the carbapenem-resistant phenotype through the genotype. In this study, using whole genome sequencing (WGS) data of 1,622 P. aeruginosa isolates followed by machine learning, we screened 16 and 31 key gene features associated with imipenem (IPM) and meropenem (MEM) resistance in P. aeruginosa, including oprD(HIGH), and constructed the resistance prediction models. The areas under the curves of the IPM and MEM resistance prediction models were 0.906 and 0.925, respectively. For the direct prediction of carbapenem resistance in P. aeruginosa from clinical samples by the key gene features selected and prediction models constructed, 72 P. aeruginosa-positive sputum samples were collected and sequenced by metagenomic sequencing (MGS) based on next-generation sequencing (NGS) or Oxford Nanopore Technology (ONT). The prediction applicability of MGS based on NGS outperformed that of MGS based on ONT. In 72 P. aeruginosa-positive sputum samples, 65.0% (26/40) of IPM-insensitive and 63.2% (24/38) of MEM-insensitive P. aeruginosa were directly predicted by NGS-based MGS with positive predictive values of 0.897 and 0.889, respectively. By the direct detection of the key gene features associated with carbapenem resistance of P. aeruginosa, the carbapenem resistance of P. aeruginosa could be directly predicted from cultured isolates by WGS or from clinical samples by NGS-based MGS, which could assist the timely treatment and surveillance of carbapenem-resistant P. aeruginosa.

Identification of metabolic biomarkers associated with nonalcoholic fatty liver disease.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease. Metabolism-related genes significantly influence the onset and progression of the disease. Hence, it is necessary to screen metabolism-related biomarkers for the diagnosis and treatment of NAFLD patients. METHODS: GSE48452, GSE63067, and GSE89632 datasets including nonalcoholic steatohepatitis (NASH) and healthy controls (HC) analyzed in this study were retrieved from the Gene Expression Omnibus (GEO) database. First, differentially expressed genes (DEGs) between NASH and HC samples were obtained. Next, metabolism-related DEGs (MR-DEGs) were identified by overlapping DEGs and metabolism-related genes (MRG). Further, a protein-protein interaction (PPI) network was developed to show the interaction among MR-DEGs. Subsequently, the "Least absolute shrinkage and selection operator regression" and "Random Forest" algorithms were used to screen metabolism-related genes (MRGs) in patients with NAFLD. Next, immune cell infiltration and gene set enrichment analyses (GSEA) were performed on these metabolism-related genes. Finally, the expression of metabolism-related gene was determined at the transcription level. RESULTS: First, 129 DEGs related to NAFLD development were identified among patients with nonalcoholic steatohepatitis (NASH) and healthy control. Next, 18 MR-DEGs were identified using the Venn diagram. Subsequently, four genes, including AMDHD1, FMO1, LPL, and P4HA1, were identified using machine learning algorithms. Moreover, a regulatory network consisting of four genes, 25 microRNAs (miRNAs), and 41 transcription factors (TFs) was constructed. Finally, a significant increase in FMO1 and LPL expression levels and a decrease in AMDHD1 and P4HA1 expression levels were observed in patients in the NASH group compared to the HC group. CONCLUSION: Metabolism-related genes associated with NAFLD were identified, containing AMDHD1, FMO1, LPL, and P4HA1, which provide insights into diagnosing and treating patients with NAFLD.

Characterization of PANoptosis patterns predicts survival and immunotherapy response in gastric cancer.

Accumulating evidence suggests that pyroptosis, apoptosis and necroptosis are deeply involved in cancer immunity. However, the role of PANoptosis in gastric cancer (GC) remains unexplored. In this study, we identified three distinct PANoptosis patterns in 1316 patients with GC, each PANoptosis pattern has its own molecular, clinical and immunological features. A scoring system named PANscore was constructed to quantify the PANoptosis patterns of the individual patients with GC. The low PANscore group had a higher response rate to immunotherapy and had a better prognosis. The predictive ability of the PANscore for prognosis and immunotherapy response was validated in multiple independent cohorts. Pan-cancer analyses showed that high PANscore was significantly associated with low expression of immune checkpoints, high expression of TGF-ß, dense infiltration of cancer-associated fibroblasts and macrophage M2. In conclusion, characterization of PANoptosis patterns provides a roadmap for patient stratification, not only in GC but also across pan-cancer.

Estrogen-related receptors: novel potential regulators of osteoarthritis pathogenesis.

Osteoarthritis (OA) is a chronic inflammatory disease that is associated with articular cartilage destruction, subchondral bone alterations, synovitis, and even joint deformity and the loss of joint function. Although current basic research on the pathogenesis of OA has made remarkable progress, our understanding of this disease still needs to be further improved. Recent studies have shown that the estrogen-related receptor (ERR) family members ERRα and ERRγ may play significant roles in the pathogenesis of OA. In this review, we refer to the latest research on ERRs and the pathogenesis of OA, elucidate the structure and physiopathological functions of the ERR orphan nuclear receptor family, and systematically examine the relationship between ERRs and OA at the molecular level. Moreover, we also discuss and predict the capacity of ERRs as potential targets in the clinical treatment of OA.

Guanine nucleotide-binding protein subunit beta-4 promotes gastric cancer progression via activating Erk1/2.

Gastric cancer (GC) is one of the most common and lethal malignancies worldwide, and its poor prognosis is mainly due to the rapid tumor progression including tumor invasion, distant metastasis, etc. Understanding the molecular mechanisms regulating GC progression lays the basis for the development of targeted therapeutic agents. Increasing evidence suggests that guanine nucleotide-binding protein subunit beta-4 (GNB4), a key subunit of heterotrimeric G protein, plays a crucial role in the initiation and progression of multiple malignancies. However, whether and how GNB4 promotes GC progression are still unknown. In this study, we found that GNB4 was highly expressed in GC tissues compared to that in non-tumor tissues and was significantly associated with tumor invasion depth, pathological stage and poor survival rate of GC patients. Both gain-of-function and loss-of-function studies revealed that GNB4 significantly enhanced GC cell growth and motility both in vitro and in vivo. Further studies revealed that GNB4 overexpression induced G1-S transition and promoted the process of epithelial-mesenchymal transformation. These tumor promoting effects were mediated by GNB4 which activates the Erk1/2 pathway through upregulating Erk1/2 phosphorylation, as U0126, an Erk1/2 phosphorylation inhibitor, could significantly inhibit GNB4-mediated cell proliferation, migration and invasion. In summary, GNB4 contributes to the proliferation and metastasis of GC cells by activating the Erk1/2 signaling pathway, and it may serve as a potential therapeutic target of GC.

HOTAIR enhanced paclitaxel and doxorubicin resistance in gastric cancer cells partly through inhibiting miR-217 expression.

Drug resistance is a big obstacle for clinical anti-tumor treatment outcome. However, the role of HOTAIR in drug resistance in gastric cancer (GC) remains unknown. In this study, we showed that overexpression of HOTAIR enhanced paclitaxel and doxorubicin resistance in GC cells. Furthermore, the expression of HOTAIR was upregulated in GC tissues and higher expression of HOTAIR was associated with late stage. In addition, we showed that miR-217 expression was lower in GC tissues compared with the paired non-tumour tissues and downregulated expression of miR-217 was correlated with late stage. Interestingly, the expression of miR-217 was negatively correlated with HOTAIR expression in GC tissues. Ectopic expression of HOTAIR increased GC cell proliferation, cell cycle, and migration. Elevated expression of HOTAIR suppressed miR-217 expression and enhanced GPC5 and PTPN14 expression. Furthermore, we demonstrated that overexpression of miR-217 suppressed paclitaxel and doxorubicin resistance in GC cells. Ectopic expression of HOTAIR promoted drug resistance and increased GC cell proliferation, cell cycle, and migration by targeting miR-217. These data suggested that overexpression of HOTAIR enhanced paclitaxel and doxorubicin resistance in GC cells through inhibiting miR-217 expression.

Ecotoxicological effects of fertilizers made from pulping waste liquor on earthworm Eisenia fetida.

In recent years, increasing efforts have focused on production of organic-inorganic compound fertilizers using ammonium sulfite pulping waste liquor. However, their ecological effects on soil have not been studied. In this study, earthworm Eisenia fetida was exposed to various doses (0, 0.13, 0.26 and 0.52 kg/m2) for different time (7, 14, 21, and 28 d) to evaluate the effects of fertilizers made from pulping waste liquor, including reactive oxygen species (ROS) generation, antioxidant enzymes activities, glutathione S-transferase enzyme (GST) activity, malondialdehyde (MDA) content and DNA damage. Results showed that there were significant increase of ROS and MDA levels after 14 d, inducing production of antioxidant enzymes (superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT)) as well as GST. Before 14 d, excess ROS and MDA caused damage to the DNA of earthworms, leading to gradual increase of Olive tail moment (OTM) in the comet assay. With the exposure time extended to 28 d, owing to the combined effects of elimination of free radicals by antioxidant enzymes and detoxification enzymes as well as self-repairing function of cells, ROS and MDA levels declined slightly and OTM gradually decreased. In summary, this study indicated that there was a toxicological effect on earthworms when fertilizers made from pulping waste liquor were applied to soil, which needs more attention.

Responses of atrazine degradation and native bacterial community in soil to Arthrobacter sp. strain HB-5.

The bioremediation of soil contaminated with organic pesticides is a safe and effective approach to remove pollutants from the soil. However, whether the invasion of foreign aid organisms affects the local organisms has received increasing attention in recent years. Therefore, the purpose of this study was to examine the degradation ability of atrazine by the strain HB-5 and evaluate its effects on natural bacterial communities in a miniature pot experiment. Results showed that HB-5 accelerated the degradation of atrazine and the degradation half-life of atrazine was 3.3 times less than the natural soil. Additionally, HB-5 increased the quantities of indigenous bacteria, the microbial biomass carbon and the Shannon, Simpson and McIntosh diversity indices of soil microbes in its early stage of use, But these parameters in soil treated with HB-5 decreased to values as low as those found in the control at the later stage of incubation. These suggested that the bacteria vanished as atrazine was completely removed. These results demonstrated that Arthrobacter sp. strain HB-5 had great potential and would be an effective and environmental friendly technique to remove atrazine from the contaminated soil.

Oxidative Damage and Genetic Toxicity Induced by DBP in Earthworms (Eisenia fetida).

Di-n-butyl phthalate (DBP) is one of the most ubiquitous plasticizers used worldwide. However, it has negatives effects on the soil, water, atmosphere, and other environmental media and can cause serious pollution. According to the artificial soil test and previous studies, this study was conducted to evaluate the toxicity of earthworms induced by DBP at different concentrations (0, 0.1, 1.0, 10, and 50 mg kg-1) on the 7th, 14th, 21st, and 28th days of exposure. The variations in the antioxidant activities of enzymes, such as catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and glutathione-S-transferase (GST), in the amounts of malondialdehyde (MDA) and reactive oxygen species (ROS) and in the amount of DNA damage were measured to evaluate the toxic impact of DBP in earthworms. Upon exposure to DBP, the SOD, CAT, POD, and GST activities were significantly increased, with the exception of the 0.1 mg kg-1 treatment dose. High concentrations of DBP (10 and 50 mg kg-1) induced superfluous ROS to be produced and caused the MDA content to increase significantly. Therefore, we proposed that DBP led to DNA damage in earthworm coelomocytes in a dose-dependent manner, which means that DBP is a source of oxidative damage and genetic toxicity in earthworms.

Patient-derived xenografts of gastrointestinal cancers are susceptible to rapid and delayed B-lymphoproliferation.

Patient-derived cancer xenografts (PDX) are widely used to identify and evaluate novel therapeutic targets, and to test therapeutic approaches in preclinical mouse avatar trials. Despite their widespread use, potential caveats of PDX models remain considerably underappreciated. Here, we demonstrate that EBV-associated B-lymphoproliferations frequently develop following xenotransplantation of human colorectal and pancreatic carcinomas in highly immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl /SzJ (NSG) mice (18/47 and 4/37 mice, respectively), and in derived cell cultures in vitro. Strikingly, even PDX with carcinoma histology can host scarce EBV-infected B-lymphocytes that can fully overgrow carcinoma cells during serial passaging in vitro and in vivo. As serial xenografting is crucial to expand primary tumor tissue for biobanks and cohorts for preclinical mouse avatar trials, the emerging dominance of B-lymphoproliferations in serial PDX represents a serious confounding factor in these models. Consequently, repeated phenotypic assessments of serial PDX are mandatory at each expansion step to verify "bona fide" carcinoma xenografts.

Pyruvate kinase is necessary for Brucella abortus full virulence in BALB/c mouse.

Brucellosis, caused by a facultative intracellular pathogen Brucella, is one of the most prevalent zoonosis worldwide. Host infection relies on several uncanonical virulence factors. A recent research hotpot is the links between carbon metabolism and bacterial virulence. In this study, we found that a carbon metabolism-related pyruvate kinase (Pyk) encoded by pyk gene (locus tag BAB_RS24320) was associated with Brucella virulence. Determination of bacterial growth curves and resistance to environmental stress factors showed that Pyk plays an important role in B. abortus growth, especially under the conditions of nutrition deprivation, and resistance to oxidative stress. Additionally, cell infection assay showed that Pyk is necessary for B. abortus survival and evading fusion with lysosomes within RAW264.7 cells. Moreover, animal experiments exhibited that the Pyk deletion significantly reduced B. abortus virulence in a mouse infection model. Our results elucidated the role of the Pyk in B. abortus virulence and provided information for further investigation of Brucella virulence associated carbon metabolism.

CPT1C-positive cancer-associated fibroblast facilitates immunosuppression through promoting IL-6-induced M2-like phenotype of macrophage.

Cancer-associated fibroblasts (CAFs) exhibit remarkable phenotypic heterogeneity, with specific subsets implicated in immunosuppression in various malignancies. However, whether and how they attenuate anti-tumor immunity in gastric cancer (GC) remains elusive. CPT1C, a unique isoform of carnitine palmitoyltransferase pivotal in regulating fatty acid oxidation, is briefly indicated as a protumoral metabolic mediator in the tumor microenvironment (TME) of GC. In the present study, we initially identified specific subsets of fibroblasts exclusively overexpressing CPT1C, hereby termed them as CPT1C+CAFs. Subsequent findings indicated that CPT1C+CAFs fostered a stroma-enriched and immunosuppressive TME as they correlated with extracellular matrix-related molecular features and enrichment of both immunosuppressive subsets, especially M2-like macrophages, and multiple immune-related pathways. Next, we identified that CPT1C+CAFs promoted the M2-like phenotype of macrophage in vitro. Bioinformatic analyses unveiled the robust IL-6 signaling between CPT1C+CAFs and M2-like phenotype of macrophage and identified CPT1C+CAFs as the primary source of IL-6. Meanwhile, suppressing CPT1C expression in CAFs significantly decreased IL-6 secretion in vitro. Lastly, we demonstrated the association of CPT1C+CAFs with therapeutic resistance. Notably, GC patients with high CPT1C+CAFs infiltration responded poorly to immunotherapy in clinical cohort. Collectively, our data not only present the novel identification of CPT1C+CAFs as immunosuppressive subsets in TME of GC, but also reveal the underlying mechanism that CPT1C+CAFs impair tumor immunity by secreting IL-6 to induce the immunosuppressive M2-like phenotype of macrophage in GC.

Inhibiting the "isolated island" effect in simulated bone defect repair using a hollow structural scaffold design.

The treatment of bone tissue defects remains a complicated clinical challenge. Recently, the bone tissue engineering (BTE) technology has become an important therapeutic approach for bone defect repair. Researchers have improved the scaffolds, cells, and bioactive factors used in BTE through various existing bone repair material preparation strategies. However, due to insufficient vascularization, inadequate degradation, and fibrous wrapping, most BTE scaffolds impede new bone ingrowth and the reconstruction of grid-like connections in the middle and late stages of bone repair. These non-degradable scaffolds become isolated and disordered like independent "isolated islands", which leads to the failure of osteogenesis. Consequently, we hypothesized that the "island effect" prevents successful bone repair. Accordingly, we proposed a new concept of scaffold modification-osteogenesis requires a bone temporary shelter (also referred to as the empty shell osteogenesis concept). Based on this concept, we consider that designing hollow structural scaffolds is the key to mitigating the "isolated island" effect and enabling optimal bone regeneration and reconstruction.

Metagenomics next-generation sequencing (mNGS) reveals emerging infection induced by Klebsiella pneumoniaeniae.

OBJECTIVES: The increasing emergence of hypervirulent Klebsiella pneumoniae (hv-Kp) and carbapenem-resistant K. pneumoniae (CR-Kp) is a serious and substantial public health problem. The use of the last resort antimicrobials, tigecycline and polymyxin to combat infections is complicated by the expanding repertoire of newly-identified CR-hvKp. The transmission and co-occurrence of the corresponding antimicrobial resistance and virulence determinants are largely unknown. The aim of this study was to investigate the dissemination and dynamics of CR-Kp and its antibiotic resistance in a hospitalised patient. METHODS: Metagenomic next-generation sequencing (mNGS) was conducted for different specimens collected from an elderly male hospitalised patient. CR-Kp strains were examined using antibiotic susceptibility and string testing. Antimicrobial and virulence genes were annotated using whole-genome sequencing (WGS). RESULTS: A clinical case of a patient infected with a variety of CR-Kp isolates was reported. The co-occurrence of KPC-2 and NDM-1 in the patient was revealed. The CR-Kp isolates, such as BALF2, and Sputum T1 and T3, were classified into ST11 and ST147, respectively. The genetic signature (iuc operon) of hypervirulence was identified in strain T1, although string testing indicated its intermediate virulence. CONCLUSIONS: In this study, multiple infections of CR-Kp isolates were revealed by mNGS, and their dissemination was attributed to plasmid variations, mgrB inactivation and integrative conjugative elements (ICEs). Furthermore, the finding indicated one likely convergence to form CR-hvKp, different from acquisition of carbapenem-resistance determinants in hvKp. A combination of mNGS and WGS is beneficial for clinical diagnosis and anti-infection therapy, and facilitates a better understanding of genetic variants conferring antimicrobial and virulence properties.

Dysbiosis of lower respiratory tract microbiome are associated with proinflammatory states in non-small cell lung cancer patients.

BACKGROUND: The lung has a sophisticated microbiome, and respiratory illnesses are greatly influenced by the lung microbiota. Despite the fact that numerous studies have shown that lung cancer patients have a dysbiosis as compared to healthy people, more research is needed to explore the association between the microbiota dysbiosis and immune profile within the tumor microenvironment (TME). METHODS: In this study, we performed metagenomic sequencing of tumor and normal tissues from 61 non-small cell lung cancer (NSCLC) patients and six patients with other lung diseases. In order to characterize the impact of the microbes in TME, the cytokine concentrations of 24 lung tumor and normal tissues were detected using a multiple cytokine panel. RESULTS: Our results showed that tumors had lower microbiota diversity than the paired normal tissues, and the microbiota of NSCLC was enriched in Proteobacteria, Firmicutes, and Actinobacteria. In addition, proinflammatory cytokines such as IL-8, MIF, TNF- α, and so on, were significantly upregulated in tumor tissues. CONCLUSION: We discovered a subset of bacteria linked to host inflammatory signaling pathways and, more precisely, to particular immune cells. We determined that lower airway microbiome dysbiosis may be linked to the disruption of the equilibrium of the immune system causing lung inflammation. The spread of lung cancer may be linked to specific bacteria.

Three-Dimensional-Printed Spherical Hollow Structural Scaffolds for Guiding Critical-Sized Bone Regeneration.

The treatment of bone tissue defects continues to be a complex medical issue. Recently, three-dimensional (3D)-printed scaffold technology for bone tissue engineering (BTE) has emerged as an important therapeutic approach for bone defect repair. Despite the potential of BTE scaffolds to contribute to long-term bone reconstruction, there are certain challenges associated with it including the impediment of bone growth within the scaffolds and vascular infiltration. These difficulties can be resolved by using scaffold structural modification strategies that can effectively guide bone regeneration. This study involved the preparation of biphasic calcium phosphate spherical hollow structural scaffolds (SHSS) with varying pore sizes using 3D printing (photopolymerized via digital light processing). The chemical compositions, microscopic morphologies, mechanical properties, biocompatibilities, osteogenic properties, and impact on repairing critical-sized bone defects of SHSS were assessed through characterization analyses, in vitro cytological assays, and in vivo biological experiments. The results revealed the biomimetic properties of SHSS and their favorable biocompatibility. The scaffolds stimulated cell adhesion, proliferation, differentiation, and migration and facilitated the expression of osteogenic genes and proteins, including Col-1, OCN, and OPN. Furthermore, they could effectively repair a critical-sized bone defect in a rabbit femoral condyle by establishing an osteogenic platform and guiding bone regeneration in the defect region. This innovative strategy presents a novel therapeutic approach for assessing critical-sized bone defects.

Rapid inference of antibiotic resistance and susceptibility for Klebsiella pneumoniae by clinical shotgun metagenomic sequencing.

OBJECTIVES: The study aimed to develop a genotypic antimicrobial resistance testing method for Klebsiella pneumoniae using metagenomic sequencing data. METHODS: We utilized Lasso regression on assembled genomes to identify genetic resistance determinants for six antibiotics (Gentamicin, Tobramycin, Imipenem, Meropenem, Ceftazidime, Trimethoprim/Sulfamethoxazole). The genetic features were weighted, grouped into clusters to establish classifier models. Origin species of detected antibiotic resistant gene (ARG) was determined by novel strategy integrating "possible species", "gene copy number calculation" and "species-specific kmers". The performance of the method was evaluated on retrospective case studies. RESULTS: Our study employed machine learning on 3928 K. pneumoniae isolates, yielding stable models with AUCs > 0.9 for various antibiotics. GenseqAMR, a read-based software, exhibited high accuracy (AUC 0.926-0.956) for short-read datasets. The integration of a species-specific kmer strategy significantly improved ARG-species attribution to an average accuracy of 96.67%. In a retrospective study of 191 K. pneumoniae-positive clinical specimens (0.68%-93.39% genome coverage), GenseqAMR predicted 84.23% of AST results on average. It demonstrated 88.76%-96.26% accuracy for resistance prediction, offering genotypic AST results with a shorter turnaround time (mean ± SD: 18.34 ± 0.87 hours) than traditional culture-based AST (60.15 ± 21.58 hours). Furthermore, a retrospective clinical case study involving 63 cases showed that GenseqAMR could lead to changes in clinical treatment for 24 (38.10%) cases, with 95.83% (23/24) of these changes deemed beneficial. CONCLUSIONS: In conclusion, GenseqAMR is a promising tool for quick and accurate AMR prediction in Klebsiella pneumoniae, with the potential to improve patient outcomes through timely adjustments in antibiotic treatment.

Construction of ovarian metastasis-related immune signature predicting prognosis of gastric cancer patients.

BACKGROUND: Ovarian metastasis (OM) results in poor survival of gastric cancer (GC) patients. While immunotherapy has emerged as a promising approach for late-stage GC, validated immune-related prognostic signatures still remain in need. In this study, we constructed an ovarian metastasis- and immune-related prognostic signature (OMIRPS), characterized the molecular and immune features of OMIRPS-categorized subgroups and predicted their potential response to immunotherapy. METHODS: Three individual cohorts were used to construct and evaluate OMIRPS: RNA-seq of matched primary GC and OM from Fudan University Shanghai Cancer Center (FUSCC) (discovery cohort, n = 4), The Cancer Genome Atlas (TCGA) (training cohort, n = 544) and GSE84437 (validation cohort, n = 433). Differentially expressed genes (DEGs) identified between primary GC and OM and immune-related genes (IRGs) from the ImmPort and InnateDB databases were used to identify immune-related prognostic hub genes, which were further used to construct OMIRPS by using LASSO regression analysis. Prognosis, molecular characteristics, immune features, and differential immunotherapy efficacy between different OMIRPS subgroups were analyzed. RESULTS: Functional analyses of DEGs revealed the significance of immune-related signatures and pathways in the OM. Immune-related prognostic hub genes including TNFRSF18, CARD11, BCL11B, NRP1, BNIP3L, and ATF3 were utilized to construct OMIRPS, which was identified as an independent prognostic factor. Comprehensive analyses unveiled the distinctive molecular and immune characteristics of OMIRPS-high and -low subgroup in regard to enriched pathways, mutation rate, tumor mutation burden, microsatellite instability status, infiltrated immune cell, immune exclusion score, and the prediction of immunotherapy efficacy. Additionally, OMIRPS was associated with Immune Subtypes with borderline significance. CONCLUSIONS: RNA-seq of paired primary and ovarian metastatic tumors unveiled the significance of immune-related pathways and tumor immune microenvironment in OM. OMIRPS served as a promising biomarker to predict the prognosis of GC patients and distinguish the molecular features, immune characteristics, and efficacy of immunotherapy between different subgroups.