PDGFRß-Antagonistic Affibody-Mediated Tumor-Targeted Tumor Necrosis Factor-Alpha for Enhanced Radiotherapy in Lung Cancer.

The morbidity and mortality of lung cancer are still the highest among all malignant tumors. Radiotherapy plays an important role in clinical treatment of lung cancer. However, the effect of radiotherapy is not ideal due to the radiation resistance of tumor tissues. Abnormalities in tumor vascular structure and function affect blood perfusion, and oxygen transport is impeded, making tumor microenvironment hypoxic. Tumor hypoxia is the major cause of radiotherapy resistance. By promoting tumor vessel normalization and enhancing vascular transport function, tumor hypoxia can be relieved to reduce radiotherapy resistance and increase tumor radiotherapy sensitivity. In our previous study, a pericytes-targeted tumor necrosis factor alpha (named Z-TNFα) was first constructed and produced by genetically fusing the platelet-derived growth factor receptor ß (PDGFRß)-antagonistic affibody (ZPDGFRß) to the TNFα, and the Z-TNFα induced normalization of tumor vessels and improved the delivery of doxorubicin, enhancing tumor chemotherapy. In this study, the tumor vessel normalization effect of Z-TNFα in lung cancer was further clarified. Moreover, the tumor hypoxia improvement and radiosensitizing effect of Z-TNFα were emphatically explored in vivo. Inspiringly, Z-TNFα specifically accumulated in Lewis lung carcinoma (LLC) tumor graft and relieved tumor hypoxia as well as inhibited HIF-1α expression. As expected, Z-TNFα significantly increased the effect of radiotherapy in mice bearing LLC tumor graft. In conclusion, these results demonstrated that Z-TNFα is also a promising radiosensitizer for lung cancer radiotherapy.

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.

Periostin secreted from podoplanin-positive cancer-associated fibroblasts promotes metastasis of gastric cancer by regulating cancer stem cells via AKT and YAP signaling pathway.

Cancer-associated fibroblasts (CAFs) are heterogeneous stromal cells present in the tumor microenvironment (TME), which play a critical role in gastric cancer (GC) progression. Here, we examined a subset of CAFs with high podoplanin (PDPN) expression, which is correlated with tumor metastasis and poor survival in GC patients. Animal models of gastric cancer liver metastasis monitored by micro-PET/CT confirmed that periostin (POSTN) derived from PDPN(+) CAFs regulated CAFs' pro-migratory ability. Mechanistically, PDPN(+) CAFs secreted POSTN to modulate cancer stem cells (CSCs) through FAK/AKT phosphorylation. Furthermore, POSTN could also activate FAK/YAP signaling in GC cells to produce increased amounts of IL-6, which in turn induced phosphorylation of PI3K/AKT in PDPN(+) CAFs. Prolonged PI3K/AKT pathway activation in PDPN(+) CAFs maintains the production of POSTN and the effect on CSC enrichment and GC cell migration. In conclusion, our study demonstrated a positive feedback loop between PDPN(+) CAFs and CSCs during GC progression and suggested a selective target for GC treatment.

ICG-Dimeric Her2-Specific Affibody Conjugates for Tumor Imaging and Photothermal Therapy for Her2-Positive Tumors.

Human epidermal growth factor receptor 2 (Her2) is abundantly expressed in various solid tumors. The Her2-specific Affibody (ZHer2:2891) has been clinically tested in patients with Her2-positive breast cancer and is regarded as an ideal drug carrier for tumor diagnosis and targeted treatment. Indocyanine green (ICG) can be used as a photosensitizer for photothermal therapy (PTT), in addition to fluorescent dyes for tumor imaging. In this study, a dimeric Her2-specific Affibody (ZHer2) based on ZHer2:2891 was prepared using the E. coli expression system and then coupled to ICG through an N-hydroxysuccinimide (NHS) ester reactive group to construct a novel bifunctional protein drug (named ICG-ZHer2) for tumor diagnosis and PTT. In vitro, ICG-ZHer2-mediated PTT selectively and efficiently killed Her2-positive BT-474 and SKOV-3 tumor cells rather than Her2-negative HeLa tumor cells. In vivo, ICG-ZHer2 specifically accumulated in Her2-positive SKOV-3 tumor grafts rather than Her2-negative HeLa tumor grafts; high-contrast tumor optical images were obtained. However, Her2-negative HeLa tumor grafts were not detected. More importantly, ICG-ZHer2-mediated PTT exhibited a significantly enhanced antitumor effect in mice bearing SKOV-3 tumor grafts owing to the good photothermal properties of ICG-ZHer2. Of note, ICG-ZHer2 did not exhibit acute toxicity in mice during short-term treatment. Overall, our findings indicate that ICG-ZHer2 is a promising bifunctional drug for Her2-positive tumor diagnosis and PTT.

Calcipotriol abrogates cancer-associated fibroblast-derived IL-8-mediated oxaliplatin resistance in gastric cancer cells via blocking PI3K/Akt signaling.

Activation of vitamin D receptor (VDR) in cancer-associated fibroblasts (CAFs) has been implicated in hesitating tumor progression and chemoresistance of several human malignancies. Yet, the role of VDR in CAF-induced chemotherapy resistance of gastric cancer (GC) cells remains elusive. In this study we first conducted immunohistochemistry analysis on tissue microarrays including 88 pairs of GC and normal mucosa samples, and provided clinical evidence that VDR was mainly expressed in gastric mucous cells but almost invisible in CAFs, and VDR expression was negatively correlated with malignant clinical phenotype and advanced stages, low VDR expression confers to poor overall survival rate of patients with GC. In a co-culture system of primary CAFs and cancer cells, we showed that treatment of HGC-27 and AGS GC cells with VDR ligand calcipotriol (Cal, 500 nM) significantly inhibited CAF-induced oxaliplatin resistance. By using RNA-sequencing and Human Cytokine Antibody Array, we demonstrated that IL-8 secretion from CAFs induced oxaliplatin resistance via activating the PI3K/AKT pathway in GC, whereas Cal treatment greatly attenuated the tumor-supportive effect of CAF-derived IL-8 on GC cells. Taken together, this study verifies the specific localization of VDR in GC tissues and demonstrates that activation of VDR abrogates CAF-derived IL-8-mediated oxaliplatin resistance in GC via blocking PI3K/Akt signaling, suggesting vitamin D supplementation as a potential strategy of enhancing the anti-tumor effect of chemotherapy in GC.

Fate of antibiotic resistance genes and bacteria in a coupled water-processing system with wastewater treatment plants and constructed wetlands in coastal eco-industrial parks.

In coastal eco-industrial zones, wastewater treatment plants (WWTPs) and constructed wetlands (CWs) can alleviate the challenge of water shortage and the negative effect of sewage discharge, while the problems of antibiotic resistance genes (ARGs) have not attracted enough attention. In this research, the Wafergen SmartChip system was adopted to investigate the ARG profiles in a coupled system combined WWTPs and CWs in a coastal industrial park. Potential risks of antibiotic resistance in chemical industrial wastewater were confirmed due to the higher abundance of target ARGs (> 107 copies/mL). General decline with partial enrichment in absolute and relative abundance of ARGs from the WWTPs to CWs revealed the effective removal of ARGs in the coupled system, while the fate of different ARG types varied greatly. Aminoglycoside and sulfonamide ARGs were detected with higher abundance (up to 5.34 ×107 and 3.61 ×107 copies/mL), especially aac(6')-Ib and sul1. Denitrification, secondary sedimentation, and acid hydrolysis contributed to the removal of aminoglycoside, sulfonamide, ß-lactamase, chloramphenicol, and multidrug ARGs. Catalytic ozonation contributed to the removal of tetracycline and MLSB ARGs. Subsurface CWs worked effectively for the removal of sulfonamide, tetracycline, and multidrug ARGs, especially tetX, cphA, tetG, and strB. Close correlations between ARGs and MGEs emphasized the vital roles of anthropogenic pollutants and horizontal gene transfer on the diffusion of ARGs. Actinobacteria, Bacteroidota, and Cyanobacteria were dominant in the CWs, while Proteobacteria, Firmicutes, and Planctomycetota were prevalent in the WWTPs. Redundancy analysis and variance partitioning analysis indicated that transposase and water quality posed greater influences on the distribution of ARGs. Co-occurrence network revealed that potential multiple antibiotic resistant pathogenic bacteria decreased in the CWs. The coupled system has a limited effect on the reduction of ARGs and potential ARG hosts, providing a comprehensive insight into the fate of ARGs in conventional water-processing systems.

Determination of berberine-upregulated endogenous short-chain fatty acids through derivatization by 2-bromoacetophenone.

Short-chain fatty acids (SCFAs) are a major group of endogenous metabolites generated by the gut microbiota and have been reported to play an important role in physical health, such as improving energy metabolism. Here, using 2-bromoacetophenone as the derivatization reagent (BP, 10 mg/mL, 40 °C for 20 min), a sensitive liquid chromatography-tandem mass spectrometric method was established for the quantitative determination of seven short-chain fatty acids in plasma and feces. The analyses were performed on a C18 column in positive multiple reaction monitoring mode. Specificity, linearity, accuracy, precision, recovery, and stability were observed within the quantitative limits of biological sample analysis. The established method has largely improved the sensitivity by 200- to 2000-fold than that in gas chromatography (GC). Especially for butyrate, the lower quantitative limit of 1 ng/mL, 1600-fold higher in sensitivity than that of GC (1.6 µg/mL), ensured the accurate determination of its low level in blood or feces (88 ± 29 ng/mL in blood, 176 ± 18 µg/g in feces). Then, the validated method was applied for therapeutic studies of berberine in hyperlipidemia hamsters in vivo and screening of 13 compounds (including five metabolites of berberine and eight typical isoquinoline alkaloids) in vitro. After berberine treatment (oral, 200 mg/kg, 2 weeks) to hyperlipidemia hamsters, the levels of butyrate were significantly upregulated in blood (77 ± 10 ng/mL vs. 117 ± 13 ng/mL, *P < 0.05) and feces (132 ± 11 µg/g vs. 547 ± 57 µg/g, ***P < 0.001), which further verified butyrate as an active endogenous metabolite in coordination with berberine to lower the blood lipids. Additionally, the berberine metabolites (M1, M2, M3), as well as two isoquinoline alkaloids (tetrandrine and dauricine), could also obviously induce the production of SCFAs (butyrate, etc.) in gut microbiota. In total, we have successfully established a new derivative LC-MS/MS method for the targeted quantitative determination of seven SCFAs in biological samples. Graphical abstract.

In Vitro Study of the Metabolic Characteristics of Eight Isoquinoline Alkaloids from Natural Plants in Rat Gut Microbiota.

Gut microbiota is populated with an immense number of microorganisms, which can be regulated by dietary components and drugs to markedly affect the nutritional and health status of the host. Eight medicinal isoquinoline alkaloids from natural plants were cultured anaerobically with rat gut microbiota and an LC/MSn-IT-TOF technique was used to identify the resulting metabolites. Palmatine, tetrahydropalmatine, dauricine, and tetrandrine containing nitro-hexatomic isoquinoline rings could be easily transformed by the intestinal flora in vitro and a total of nine demethylated metabolites were detected. However, sinomenine, homoharringtonine, harringtonine, and galanthamine, which all contained benzazepine, could not undergo demethylation. Computer-assisted docking was used to analyze the binding between these compounds and sterol 14α-demethylase. The computational results demonstrated that hydrophobic interactions were the main driving force for binding, but the steric hindrance produced by the benzazepine structure resulted in a weak interaction between the hit compounds and the enzyme. This work illustrated that gut microbiota were important in the metabolism of isoquinoline alkaloids.

Interaction effects on cytochrome P450 both in vitro and in vivo studies by two major bioactive xanthones from Halenia elliptica D. Don.

The major components, 1-hydroxy-2,3,5-trimethoxy-xanthone (HM-1) and 1,5-dihydroxy-2,3-dimethoxy-xanthone (HM-5) isolated from Halenia elliptica D. Don (Gentianaceae), could cause vasodilatation in rat coronary artery with different mechanisms. In this work, high-performance liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LCMS-IT-TOF) was used to clarify the metabolic pathways, and CYP450 isoform involvement of HM-1 and HM-5 were also studied in rat. At the same time, in vivo inhibition effects of HM-1 and ethyl acetate extracts from origin herb were studied. Three metabolites of HM-5 were found in rat liver microsomes (RLMs); demethylation and hydroxylation were the major phase I metabolic reactions for HM-5. Multiple CYP450s were involved in metabolism of HM-1 and HM-5. The inhibition study showed that HM-5 inhibited Cyp1a2, 2c6 and 2d2 in RLMs. HM-1 inhibited activities of Cyp1a2, Cyp2c6 and Cyp3a2. In vivo experiment demonstrated that both HM-1 and ethyl acetate extracts could inhibit Cyp3a2 in rats. In conclusion, the metabolism of xanthones from the origin herb involved multiple CYP450 isoforms; in vitro, metabolism of HM-5 was similar to that of its parent drug HM-1, but their inhibition effects upon CYP450s were different; in vivo, Cyp3a2 could be inhibited by HM-1 and ethyl acetate extracts.

In Vivo Metabolite Profiling of a Purified Ellagitannin Isolated from Polygonum capitatum in Rats.

Ellagitannin is a common compound in food and herbs, but there are few detailed studies on the metabolism of purified ellagitannins. FR429 is a purified ellagitannin with antitumor potential, which is from Polygonum capitatum Buch.-Ham.ex D. Don. The present study was designed to investigate the metabolic profiles of FR429 in rats in vivo. Using liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LC/MS(n)-IT-TOF), total eight metabolites were found in rat bile and urine after intravenous administration of FR429, but could not be detected in plasma. These metabolites were ellagic acid, mono-methylated FR429, ellagic acid methyl ether glucuronide, ellagic acid methyl ether diglucuronide, ellagic acid dimethyl ether glucuronide, and ellagic acid dimethyl ether diglucuronide. It was concluded that methylation and subsequent glucuronidation were the major metabolic pathways of FR429 in rats in vivo. This is the first report on the in vivo metabolism of the purified ellagitannin in rats.

Effect of Berberine on promoting the excretion of cholesterol in high-fat diet-induced hyperlipidemic hamsters.

BACKGROUND: Berberine (BBR), as a new medicine for hyperlipidemia, can reduce the blood lipids in patients. Mechanistic studies have shown that BBR activates the extracellular-signal regulated kinase pathway by stabilizing low-density-lipoprotein receptor mRNA. However, aside from inhibiting the intestinal absorption of cholesterol, the effects of BBR on other metabolic pathways of cholesterol have not been reported. This study aimed to investigate the action of BBR on the excretion of cholesterol in high-fat diet-induced hyperlipidemic hamsters. METHODS: Golden hamsters were fed a high-fat diet (HFD) for 6 weeks to induce hyperlipidemia, followed by oral treatment with 50 and 100 mg/kg/day of BBR or 10 and 30 mg/kg/day of lovastatin for 10 days, respectively. The levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), transaminases, and total bile acid in the serum, liver, bile and feces were measured using an enzyme-linked immunosorbent assay. The cholesterol (as well as coprostanol) levels in the liver, bile and feces were determined by gas chromatography-mass spectrometry. RESULTS: The HFD hamsters showed significantly hyperlipidemic characteristics compared with the normal hamsters. Treatment with BBR for 10 days reduced the serum TC, TG and LDL-C levels in HFD hamsters by 44-70, 34-51 and 47-71%, respectively, and this effect was both dose- and time-dependent. Initially, a large amount of cholesterol accumulated in the hyperlipidemic hamster livers. After BBR treatment, reductions in the liver cholesterol were observed by day 3 and became significant by day 7 at both doses (P < 0.001). Meanwhile, bile cholesterol was elevated by day 3 and significantly increased at day 10 (P < 0.001). BBR promoted cholesterol excretion from the liver into the bile in hyperlipidemic hamsters but not in normal hamsters, and these results provide a link between the cholesterol-lowering effect of BBR with cholesterol excretion into the bile. CONCLUSIONS: We conclude that BBR significantly promoted the excretion of cholesterol from the liver to the bile in hyperlipidemic hamsters, which led to large decreases in the serum TC, TG and LDL-C levels. Additionally, compared with lovastatin, the BBR treatment produced no obvious side effects on the liver function.

Biotransformation and in vitro metabolic profile of bioactive extracts from a traditional Miao-nationality herbal medicine, Polygonum capitatum.

Polygonum capitatum Buch.-Ham.ex D. Don, a traditional Miao-nationality herbal medicine, has been widely used in the treatment of various urologic disorders. Recent pharmacological studies demonstrated that a pure compound, FR429, isolated from the ethanol extracts of P. capitatum could selectively inhibit the growth of four hepatocellular carcinoma (HCC) cell lines in a dose-dependent manner. Thus, P. capitatum probably exhibits potential antitumor activity. However, there is very little information on the metabolism of substances present in P. capitatum extracts. In this study, gallic acid, quercetrin, ethanol extracts and ethyl acetate fraction of ethnolic extract (EtOAc fraction) of P. capitatum were cultured anaerobically with rat intestinal bacteria. A highly sensitive and selective liquid chromatography electrospray ionization-ion trap-time of fight mass spectrometry (LC/MSn-IT-TOF) technique was employed to identify and characterize the resulting metabolites. A total of 22 metabolites (M1-M22), including tannins, phenolic acids and flavonoids, were detected and characterized. The overall results demonstrated that the intestinal bacteria played an important role in the metabolism of P. capitatum, and the main metabolic pathways were hydrolysis, reduction and oxidation reactions. Our results provided a basis for the estimation of the metabolic transformation of P. capitatum in vivo.

Significance of dysregulated M2 macrophage and ESR2 in the ovarian metastasis of gastric cancer.

Background: Prognosis of gastric cancer (GC) patients with ovarian metastasis (OM) remains poor. We hereby characterized the role of tumor immune microenvironment (TIME) and identified potential key regulators in the OM with the aim of understanding its molecular basis to develop novel therapeutic targets. Methods: Transcriptomic analyses of paired primary and ovarian metastatic lesions of seven GC patients from Fudan University Shanghai Cancer Center uncovered and functionally annotated their differentially expressed genes (DEGs). CIBERSORT analysis revealed differential TIME between primary GCs and OMs, which was further validated by multiplex immunofluorescence (mIF). Unique overexpression of candidate regulator in OMs was validated by an immunohistochemical (IHC) staining-based cohort study and in vitro cell growth, migration and invasion assays were conducted to characterize its function in GC progression. Results: Functional enrichment analyses of DEGs between GCs and matched OMs revealed multiple significantly dysregulated immune-related and cancer-related pathways. Distinctive subsets of immune cells, especially M2 macrophage, were selectively enriched in metastatic lesions. mIF-based quantification further validated the overexpression of CD68+CD206+ M2 macrophage in the OMs. Estrogen receptor 2 (ESR2), which encodes estrogen receptor ß (ERß), was not only potentially correlated with M2 macrophage but also overexpressed in the OM of GC. ESR2 was up-regulated in cancerous tissue and its high expression correlated with younger age, more advanced lymph node metastasis and pathological stage, as well as a worse patient survival. IHC staining of ERß in the cohort of paired primary and metastatic GCs validated its selective overexpression in OMs. Small-interfering RNAs (siRNAs)-induced knockdown of ESR2 significantly inhibited the invasion and migration of both AGS and HGC-27 GC cell lines. Conclusions: Comparative RNA-sequencing analysis revealed the dysregulated TIME, M2 macrophage in particular, between primary GC and OM. ESR2 potentially correlated with M2 macrophage and played pro-oncogenic roles in GC progression and metastasis.

Subcutaneously transplanted xenogeneic protein recruits treg cells and M2 macrophages to induce browning of inguinal white adipose tissue.

OBJECTIVE: To study whether subcutaneously embedding xenogeneic protein threads or synthetic polymer absorbable threads can improve obesity phenotypes and metabolic conditions, and to further explore its underlying mechanism. METHODS: Thirty-six 8-week-old ob/ob mice were randomly allocated to three groups, respectively, receiving catgut embedding, PGA thread embedding or sham treatment bilaterally to the groin. Individual parameters including weight, food intake, and core temperature are recorded and metabolism assessment, energy expenditure analysis, and PET/CT scanning are also performed at fixed timepoints. After surgical incision, the inguinal white adipose tissue was histologically examined and its expression profile was tested and compared among groups 4 weeks and 12 weeks after operation. RESULTS: Catgut embedding reduced weight gain and improved metabolic status in ob/ob mice. Browning of bilateral inguinal WAT (white adipose tissue) was induced after catgut embedding, with massive infiltration of Treg cells and M2 macrophages in the tissue slices of fat pads. IL-10 and TGF-ß released by Treg cells targeted macrophages and the induced M2 macrophages increased the expression of thermogenic and anti-inflammatory genes in fat. The secretion of catecholamines by polarized M2 macrophages led to the activation of ß3-AR-related pathways in adipocytes and the browning of adipose tissue. CONCLUSIONS: Abdominal subcutaneous catgut embedding has the potential to combat obesity through the induction of WAT browning mediated by infiltrated Treg cells and macrophages.

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.

Antibiotic resistance genes in constructed wetlands: Driving indicators and risk assessment.

Constructed wetlands (CWs) were responsible for the in-depth purification of wastewater, providing an ideal environment for the transport, acquisition, and dissemination of antibiotic resistance genes (ARGs). A better understanding of influencing factors and risks of ARGs in CWs was deemed indispensable. In this research, the abundance of ARGs and mobile genetic elements (MGEs) was determined to be higher in summer and spring, ranging from 53.7 to 8.51 × 106 and 30.9-6.02 × 106 copies/mL, respectively. Seasonal variation significantly influenced the abundance of ARGs and MGEs, as well as the co-occurrence patterns among ARGs, MGEs and bacteria. However, the environmental gradients, from the influent (CW01) to the effluent (CW10), did not impose significant effects on the abundance of ARGs and MGEs. Furthermore, the ratios of pathogenic bacteria to ARG hosts and ARG risks index decreased by 50.4% and 88.54% along with the environmental gradients, indicating that CWs could act as barriers to the transfer of ARGs. Partial least squares-path modeling (PLSPM) revealed that temperature was the main driving factor of ARGs, followed by MGEs, stable and differential bacteria. This finding effectively and innovatively explored the driving indicators for the variations and risks of ARGs caused by spatial-temporal variations, providing new insights into the evaluation and control of ARGs in CWs.

Tumor-infiltrating CD226+CD8+ T cells are associated with postoperative prognosis and adjuvant chemotherapeutic benefits in gastric cancer patients.

PURPOSE: Defining the phenotypic characteristics of CD8+ T cell subsets in gastric cancer (GC) can help remodel the immune microenvironment of the tumor, thereby improving patient prognosis. CD226 has recently been shown to regulate the activity of CD8+ T cell in several malignancies. However, the clinical relevance of CD226+CD8+ T cells in GC remains unclear. METHODS: Fudan University Shanghai Cancer Center (FUSCC) cohort (n = 316), The Cancer Genome Atlas (TCGA) cohort (n = 407), KUGH/KUCM cohort (n = 202), and Asian Cancer Research Group (ACRG) cohort (n = 300) were included in prognosis and response to adjuvant chemotherapy (ACT) analyses. Flow cytometry and multiplex immunostaining were used to characterize CD226+CD8+ T cells. RESULTS: CD226+CD8+ T cells predicted favorable outcomes in patients undergoing curative resection for GC. GC patients with high CD226+CD8+ T cell infiltration benefitted more from adjuvant chemotherapy. CD155 is upregulated in GC tissues and is associated with decreased intra-tumoral CD226+CD8+ T cell infiltration. The combination of intra-tumoral CD226+CD8+ T cells and CD155 is a strong prognostic predictor in patients with GC. CONCLUSION: CD226+CD8+ T cells may represent a novel therapeutic target and a useful marker of prognosis and therapeutic response in patients with GC.

Apocynum venetum leaf extract alleviated doxorubicin-induced cardiotoxicity by regulating organic acid metabolism in gut microbiota.

Apocynum venetum leaf is commonly utilized for its beneficial effects in reducing blood pressure, inducing sedation, promoting diuresis, anti-aging, and cardioprotection, which also exhibit positive effects on the gut microbiota. The gut microbiota plays a role as an endocrine organ by producing bioactive metabolites that can directly or indirectly impact host physiology, specifically cardiovascular diseases. In this study, main chemical components of A. venetum leaf extract (AVLE) were identified by LC-MS, and an orally administered AVLE was employed to treat mice with doxorubicin (Dox)-induced cardiotoxicity. The results showed that AVLE contained hyperoside and oganic acids. The pharmacological findings revealed that AVLE regulated the gut microbiota, resulting in a significant increase in the levels of two organic acids, indole-3-propionic acid (IPA) and acetic acid (AA). Both IPA and AA exhibited the ability to reduce BNP, CK, and LDH levels in mice with Dox-induced cardiotoxicity. Moreover, IPA demonstrated an improvement in Dox-induced cardiac injury by inhibiting apoptosis, while AA promoted increased secretion of ghrelin through the parasympathetic nervous system, subsequently reducing cardiac fibrosis by decreasing collagen I, collagen III, and activin A. Hence, our study demonstrates that AVLE exerts a beneficial cardioprotective effect by modulating the gut microbiota, providing a potential novel target for the treatment and prevention of Dox-induced cardiotoxicity.

Taxonomy and Phylogeny of Novel and Extant Taxa in Pleosporales Associated with Mangifera indica from Yunnan, China (Series I).

Pleosporales is the largest fungal order with a worldwide distribution in terrestrial and aquatic environments. During investigations of saprobic fungi associated with mango (Mangifera indica) in Baoshan and Honghe, Yunnan, China, fungal taxa belonging to pleosporales were collected. Morphological examinations and phylogenetic analyses of ITS, LSU, SSU, rpb2 and tef1-α loci were used to identify the fungal taxa. A new genus, Mangifericomes; four new species, namely Mangifericomes hongheensis, Neomassaria hongheensis, Paramonodictys hongheensis, and Paramonodictys yunnanensis; and six new host and country records, namely Byssosphaeria siamensis, Crassiparies quadrisporus, Paradictyoarthrinium aquatica, Phaeoseptum mali, Torula fici, and Vaginatispora amygdali, are introduced. Photoplates, full descriptions, and phylogenetic trees to show the placement of new and known taxa are provided.

Diagnostic and Prognostic Values of KRAS Mutations on EUS-FNA Specimens and Circulating Tumor DNA in Patients With Pancreatic Cancer.

INTRODUCTION: The ability of carbohydrate antigen 19-9 (CA19-9) to differentiate pancreatic cancer from other benign pancreatic lesions is unsatisfactory. This study explored the diagnostic value of KRAS gene mutations and plasma circulating tumor DNA (ctDNA) in patients with pancreatic cancer. METHODS: The prospective cohort study comprised 149 consecutive patients with solid pancreatic lesions who underwent endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA). KRAS subtype mutations were analyzed by digital droplet PCR (ddPCR) in EUS-FNA histopathology tissue samples, and blood samples were sent for plasma ctDNA analysis. The final diagnosis was based on surgical resection pathology or follow-up for at least 2 years. RESULTS: Adding KRAS mutation ddPCR increased the sensitivity and accuracy of EUS-FNA from 71.4% to 91.6% (P < 0.001) and 75.8% to 88.6% (P < 0.001), respectively. By comparison, the sensitivities of circulating biomarkers ctDNA and CA19-9 were only 35.2% and 71.2%. The area under the curve of the receiver operating characteristic curve (AUC) of EUS-FNA and KRAS ddPCR combination was >0.90 for distinguishing pancreatic cancer from benign lesions, whereas the AUC of EUS-FNA and CA19-9 combination was 0.83. The median survival time was significantly shorter in patients with G12D KRAS mutations than that in patients with other mutations (180 vs 240 days, P < 0.001). DISCUSSION: FNA tissue sample KRAS mutation analysis in tissues significantly improves the diagnostic accuracy of cyto/histopathological evaluation in EUS-FNA samples. The combination of EUS-FNA and tissue sample KRAS ddPCR provided a more accurate method for pancreatic cancer diagnosis, superior to the combination of EUS-FNA and CA19-9/ctDNA. G12D KRAS mutations in pancreatic cancer were independently associated with poor overall survival.