Diets containing phytobiotics, l-arginine, vitamin E and captopril modulate ascites syndrome-related genes expression in broiler chickens exposed to low ambient temperature.

BACKGROUND: Our hypothesis centred on the potential to mitigate ascites outbreaks in birds exposed to cold stress by inhibiting pulmonary artery contraction through dietary intervention. OBJECTIVE: This study aimed to evaluate the effect of natural and synthetic medications on growth performance, ascites-related parameters and the expression of ascites-related genes in the lung tissue of broiler chickens under low ambient temperature. METHODS: We randomly assigned 450 one-day-old male Ross 308 chicks to six dietary treatments across five replicate pens, each containing 15 chicks. The treatments included a basal diet (control), and the basal diet was supplemented with hydroalcoholic extracts of sumac (HES, 200 mg/kg), Syrian mesquite (HEM, 200 mg/kg), l-arginine (40% above requirement), captopril (15 mg/kg) and vitamin E (100 mg/kg). RESULTS: Diets containing HEM, l-arginine and vitamin E resulted in increased average daily gain on days 8-14 and 0-28, whereas HES showed a similar effect only during days 8-14 compared to the control diet (p < 0.05). Additionally, feed additives decreased packed cell volume, left and right ventricle volumes and systolic blood pressure (p < 0.05). Moreover, chickens fed the control and l-arginine diets exhibited higher levels of angiotensin converting enzyme (ACE) mRNA in lung tissue compared to those fed HES, HEM and captopril (p < 0.05). Meanwhile, supplementation with HEM and l-arginine increased the expression of inducible nitric oxide synthase (iNOS) mRNA in lung tissue compared to other treatments (p < 0.05). Regarding Cu/Zn-superoxide dismutase (Cu/Zn-SOD) expression, feed additives increased mRNA level in lung tissue, except for captopril (p < 0.05). CONCLUSIONS: This study demonstrates that the plant extracts may reduce the incidence of ascites syndrome not only through their antioxidant properties but also by modulating the expression of ACE, iNOS and Cu/Zn-SOD genes.

Ascites-derived hsa-miR-181a-5p serves as a prognostic marker for gastric cancer-associated malignant ascites.

BACKGROUND: Peritoneal carcinomatosis was the main reason leading to gastric cancer (GC)-related death. We aimed to explore the roles of dysregulated microRNAs (miRNAs) and related immune regulation activities in GC-associated malignant ascites. METHODS: GSE126399 were downloaded from GEO database. Differentially expressed miRNAs in GC ascites samples was firstly screened, and critical miRNAs were further investigated by LASSO (least absolute shrinkage and selection operator) logistic regression and random forest (RF) algorithm. Receiver operating characteristic of critical miRNAs was also constructed. Moreover, functional analysis, immune cell infiltration associated with differentially expressed mRNAs were further analyzed. After selecting key modules by weighted gene co-expression network analysis, mRNAs related with survival performance and transcription factor (TF)-miRNA-mRNA network were constructed. RESULTS: Hsa-miR-181b-5p was confirmed as critical differentially expressed miRNAs in GC ascites. Then, the tumor samples were divided into high- and low- expression groups divided by mean expression levels of hsa-miR-181b-5p, and subjects with high hsa-miR-181b-5p levels had better survival outcomes. In total, 197 differentially expressed mRNAs associated with hsa-miR-181b-5p levels were obtained, and these mRNAs were mainly enriched in muscle activity and vascular smooth muscle contraction. Hsa-miR-181b-5 was positively related with activated CD4 T cells and negatively related with eosinophil. 17 mRNAs were selected as mRNAs significantly related with prognosis of GC, such as PDK4 and RAMP1. Finally, 75 TF-miRNA-mRNA relationships were obtained, including 15 TFs, hsa-miR-181b-5p, and five mRNAs. CONCLUSION: Our data suggest that the differentially expressed hsa-miR-181b-5p in ascites samples of GC patients may be a valuable prognostic marker and a potential target for therapeutic intervention, which should be validated in the near future.

Improving diagnostic accuracy of identifying gastric cancer patients with peritoneal metastases: tumor-guided cell-free DNA analysis of peritoneal fluid.

Detection of peritoneal dissemination (PD) in gastric cancer (GC) patients remains challenging. The feasibility of tumor-guided cell-free DNA (cfDNA) detection in prospectively collected peritoneal fluid (ascites and peritoneal lavage) was investigated and compared to conventional cytology in 28 patients. Besides conventional cytology, next generation sequencing was performed on primary tumor DNA and cell-free DNA from peritoneal fluid. Patients were retrospectively grouped into: a positive group (with PD) and a negative group (without PD). Detectable mutations were found in the primary tumor of 68% (n = 19). Sensitivity of PD detection by tumor-guided cfDNA analysis was 91%, compared to 64% by conventional cytology. Within the positive group (n = 11), tumor-guided cfDNA was detected in all patients with ascites samples (4/4, 100%) and in 86% (6/7) of the lavage samples, opposed to 4/4 (100%) patients with ascites and 43% (3/7) with lavage by conventional cytology. Within the negative group (n = 8), conventional cytology was negative for all samples. In two patients, tumor-guided cfDNA was detected in peritoneal lavage fluid. Interestingly, these 2 patients developed PD within 6 months, suggesting a prognostic value of tumor-guided cfDNA detection. This study showed that tumor-guided cfDNA detection in peritoneal fluids of GC patients is feasible and superior to conventional cytology in detecting PD.

Effect of RNA interference with HIF-1α on the growth of pulmonary artery endothelial cells in broiler chickens.

Pulmonary artery remodeling is a characteristic feature of broiler ascites syndrome (BAS). Pulmonary artery endothelial cells (PAECs) regulated by HIF-1α play a critical role in pulmonary artery remodeling, but the underlying mechanisms of HIF-1α in BAS remain unclear. In this experiment, primary PAECs were cultured in vitro and were identified by coagulation factor VIII. After hypoxia and RNA interference, the mRNA and protein expression levels of HIF-1α and VEGF were determined by qPCR and Western blotting. The transcriptome profiles of PAECs were obtained by RNA sequencing. Our results showed that the positive rate of PAECs was more than 90%, hypoxia-induced promoted the proliferation and apoptosis of PAECs, and RNA interference significantly downregulated the expression of HIF-1α, inhibited the proliferation of PAECs, and promoted the apoptosis of PAECs. In addition, transcriptome sequencing analysis indicated that HIF-1α may regulate broiler ascites syndrome by mediating COL4A, vitronectin, vWF, ITGα8, and MKP-5 in the ECM, CAMs and MAPK pathways in PAECs. These studies lay the foundation for further exploration of the mechanisms of pulmonary artery remodeling, and HIF-1α may be a potentially effective gene for the prevention and treatment of BAS.

16S rRNA Next-Generation Sequencing May Not Be Useful for Examining Suspected Cases of Spontaneous Bacterial Peritonitis.

Background and Objectives: Ascites, often associated with liver cirrhosis, poses diagnostic challenges, particularly in detecting bacterial infections. Traditional methods have limitations, prompting the exploration of advanced techniques such as 16S rDNA next-generation sequencing (NGS) for improved diagnostics in such low-biomass fluids. The aim of this study was to investigate whether the NGS method enhances detection sensitivity compared to a conventional ascites culture. Additionally, we aimed to explore the presence of a microbiome in the abdominal cavity and determine whether it has a sterile condition. Materials and Methods: Ten patients with clinically suspected spontaneous bacterial peritonitis (SBP) were included in this study. A traditional ascites culture was performed, and all ascites samples were subjected to 16S ribosomal RNA gene amplification and sequencing. 16S rRNA gene sequencing results were interpreted by comparing them to positive and negative controls for each sample. Results: Differential centrifugation was applied to all ascites samples, resulting in very small or no bacterial pellets being harvested. The examination of the 16S amplicon sequencing libraries indicated that the target amplicon products were either minimally visible or exhibited lower intensity than their corresponding negative controls. Contaminants present in the reagents were also identified in the ascites samples. Sequence analysis of the 16S rRNA gene of all samples showed microbial compositions that were akin to those found in the negative controls, without any bacteria isolated that were unique to the samples. Conclusions: The peritoneal cavity and ascites exhibit low bacterial biomass even in the presence of SBP, resulting in a very low positivity rate in 16S rRNA gene sequencing. Hence, the 16S RNA sequencing method does little to enhance the rate of positive samples compared to traditional culture methods, including in SBP cases.

Ascites exosomal lncRNA PLADE enhances platinum sensitivity by inducing R-loops in ovarian cancer.

Cisplatin resistance is a major cause of therapeutic failure in patients with high-grade serous ovarian cancer (HGSOC). Long noncoding RNAs (lncRNAs) have emerged as key regulators of human cancers; however, their modes of action in HGSOC remain largely unknown. Here, we provide evidence to demonstrate that lncRNA Platinum sensitivity-related LncRNA from Ascites-Derived Exosomes (PLADE) transmitted by ascites exosomes enhance platinum sensitivity in HGSOC. PLADE exhibited significantly decreased expression in ascites exosomes and tumor tissues, as well as in the corresponding metastatic tumors from patients with HGSOC cisplatin-resistance. Moreover, HGSOC patients with higher PLADE expression levels exhibited longer progression-free survival. Gain- and loss-of-function studies have revealed that PLADE promotes cisplatin sensitivity by suppressing cell proliferation, migration and invasion, and enhancing apoptosis in vitro and in vivo. Furthermore, the functions of PLADE in increasing cisplatin sensitivity were proven to be transferred by exosomes to the cultured recipient cells and to the adjacent tumor tissues in mouse models. Mechanistically, PLADE binds to and downregulates heterogeneous nuclear ribonucleoprotein D (HNRNPD) by VHL-mediated ubiquitination, thus inducing an increased amount of RNA: DNA hybrids (R-loop) and DNA damage, consequently promoting cisplatin sensitivity in HGSOC. Collectively, these results shed light on the understanding of the vital roles of long noncoding RNAs in cancers.

Regulations of Exosomal-Transmitted AFAP1-AS1 LncRNA on Ovarian Cancer Cell Migration and Invasion.

BACKGROUND: Adolescent ovarian cancer (OC) has high malignancy. Long non-coding RNAs (lncRNAs) have been implicated in the pathogenesis of various malignancies, but their role in adolescent OC remains poorly understood. This study aims to assess the modulatory role of Exosome-transmitted lncRNA Actin filament-associated protein 1 Antisense RNA 1 (AFAP1-AS1) on the activity of OC cells. METHODS: We recruited a cohort of 40 adolescent patients diagnosed with OC and a control group of 40 healthy individuals. Serum samples were collected from both groups prior surgical intervention. Exosomes from peripheral blood and ascites were collected via differential centrifugation. The expression levels of AFAP1-AS1 in OC tissues and cell lines (IOSE-80, CAOV3, and SKOV3) were quantified using quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The exosomal particle size and surface markers were characterized through nanoparticle tracking analysis and transmission electron microscopy. Furthermore, siRNA-mediated knockdown of AFAP1-AS1 was performed in IOSE-80, CAOV3, and SKOV3 cell lines. Functional assays, including wound-healing experiments and Transwell migration assays, were conducted to evaluate cellular migration and metastasis. RESULTS: Our findings demonstrate that the expression of AFAP1-AS1 is significantly upregulated in OC patients' serum exosomes and ascitic fluid, correlating with unfavorable pathological features such as advanced federation international of gynecology and obstetrics (FIGO) stage and larger tumor diameter. In-vitro experiments revealed that OC cell lines and primary human OC cells showed enhanced proliferation and metastasis when exposed to ascites-derived exosomes enriched in AFAP1-AS1. Importantly, we observed that AFAP1-AS1 can be transmitted to neighboring cells via exosomal pathways. Additionally, exosomes isolated from ascites treated with siRNA targeting AFAP1-AS1 can inhibit cellular migration and invasion. CONCLUSIONS: Our data provide evidence for the oncogenic role of AFAP1-AS1, which is transmitted via exosomes. This finding has significant implications for understanding the molecular mechanisms of AFAP1-AS1 in the pathogenesis of adolescent ovarian cancer.

Factors Affecting Performance of DNA Methylation as a Potential Biomarker in Ascites for Peritonitis and Peritoneal Carcinomatosis.

BACKGROUND AND AIMS: Despite limited sensitivity, the gold standard for the diagnosis of malignant cells in ascites is still cytology. The aim of this prospective proof-of-principle study was to evaluate DNA methylation as a molecular tool for the differential diagnosis of benign and malignant ascites. METHODS: A cohort of 79 patients with malignant and non-malignant ascites was prospectively enrolled. Ascites was assessed by cytopathological and laboratory examination. Cell pellets obtained by centrifugation were analyzed for differences in DNA methylation of of long interspersed nuclear element-1 (LINE-1) and microRNA-137. Quantitative determination of methylation in bisulfite-converted DNA was performed by pyrosequencing. In a subsequent stage, we compared our data to previously published data in the field following systematic review of the literature. RESULTS: Methylation status of studied LINE-1 and microRNA-137 could be reliably detected in all samples. Systematic evaluation revealed reliable reproducibility with satisfactory short- and long-term stability against degradation. Ascites from patients with a malignancy had a significantly higher methylation level of microRNA-137 compared with patients without tumor disease, whereas patients with peritonitis had significantly decreased methylation of microRNA-137. In contrast, differences in the measurement of the methylation status of LINE-1 could only be detected between patients with portal hypertension and a combination of malignant and infectious ascites. Inflammatory cells reflecting peritonitis correlated to DNA methylation changes. CONCLUSIONS: Analysis of DNA methylation in ascites is technically feasible, well reproducible and may lead to identification of potential biomarkers for peritoneal carcinomatosis and other conditions. Inflammatory cells due to peritonitis may also be associated with DNA methylation changes and need to be considered in future studies. Profiling studied under standardized conditions will be needed to identify the appropriate biomarkers for differential diagnosis of ascites.

Cancer cell genotype associated tumor immune microenvironment exhibits differential response to therapeutic STING pathway activation in high-grade serous ovarian cancer.

BACKGROUND: High-grade serous ovarian carcinoma (HGSC) is the most lethal gynecologic malignancy characterized by resistance to chemotherapy and high rates of recurrence. HGSC tumors display a high prevalence of tumor suppressor gene loss. Given the type 1 interferon regulatory function of BRCA1 and PTENgenes and their associated contrasting T-cell infiltrated and non-infiltrated tumor immune microenvironment (TIME) states, respectively, in this study we investigated the potential of stimulator of interferon genes (STING) pathway activation in improving overall survival via enhancing chemotherapy response, specifically in tumors with PTEN deficiency. METHODS: Expression of PTEN protein was evaluated in tissue microarrays generated using pretreatment tumors collected from a cohort of 110 patients with HGSC. Multiplex immunofluorescence staining was performed to determine spatial profiles and density of selected lymphoid and myeloid cells. In vivo studies using the syngeneic murine HGSC cell lines, ID8-Trp53 -/-; Pten -/- and ID8-Trp53 -/-; Brca1 -/-, were conducted to characterize the TIME and response to carboplatin chemotherapy in combination with exogenous STING activation therapy. RESULTS: Patient tumors with absence of PTEN protein exhibited a significantly decreased disease specific survival and intraepithelial CD68+ macrophage infiltration as compared with intact PTEN expression. In vivo studies demonstrated that Pten-deficient ovarian cancer cells establish an immunosuppressed TIME characterized by increased proportions of M2-like macrophages, GR1+MDSCs in the ascites, and reduced effector CD8+ cytotoxic T-cell function compared with Brca1-deficient cells; further, tumors from mice injected with Pten-deficient ID8 cells exhibited an aggressive behavior due to suppressive macrophage dominance in the malignant ascites. In combination with chemotherapy, exogenous STING activation resulted in longer overall survival in mice injected with Pten-deficient ID8 cells, reprogrammed intraperitoneal M2-like macrophages derived from Pten-deficient ascites to M1-like phenotype and rescued CD8+ cytotoxic T-cell activation. CONCLUSIONS: This study reveals the importance of considering the influence of cancer cell intrinsic genetic alterations on the TIME for therapeutic selection. We establish the rationale for the optimal incorporation of interferon activating therapies as a novel combination strategy in PTEN-deficient HGSC.

Single-cell sequencing of ascites fluid illustrates heterogeneity and therapy-induced evolution during gastric cancer peritoneal metastasis.

Peritoneal metastasis is the leading cause of death for gastrointestinal cancers. The native and therapy-induced ascites ecosystems are not fully understood. Here, we characterize single-cell transcriptomes of 191,987 ascites cancer/immune cells from 35 patients with/without gastric cancer peritoneal metastasis (GCPM). During GCPM progression, an increase is seen of monocyte-like dendritic cells (DCs) that are pro-angiogenic with reduced antigen-presenting capacity and correlate with poor gastric cancer (GC) prognosis. We also describe the evolution of monocyte-like DCs and regulatory and proliferative T cells following therapy. Moreover, we track GC evolution, identifying high-plasticity GC clusters that exhibit a propensity to shift to a high-proliferative phenotype. Transitions occur via the recently described, autophagy-dependent plasticity program, paligenosis. Two autophagy-related genes (MARCKS and TXNIP) mark high-plasticity GC with poorer prognosis, and autophagy inhibitors induce apoptosis in patient-derived organoids. Our findings provide insights into the developmental trajectories of cancer/immune cells underlying GCPM progression and therapy resistance.

Identification of long noncoding RNA NEAT1 as a key gene involved in the extramedullary disease of multiple myeloma by bioinformatics analysis.

OBJECTIVE: Long non-coding RNAs (lncRNAs) are involved in tumorigenesis and play a key role in cancer progression. To determine whether lncRNAs are involved in extramedullary disease of multiple myeloma (EMD), we analyzed the expression profile of lncRNAs in EMD. METHODS: Three pairs of EMD patients and their intramedullary MM cells were screened by microarray first. We extracted data from gene chips and made an identification of lncRNAs and mRNAs with significant differences between EMD group and non EMD group. WGCNA confirmed the EMD related gene module and drew a heat map to further determine the key gene lncRNA-NEAT1. In the meantime, bone marrow and extramedullary samples (hydrothorax and ascites) were collected from 2 MM patients and subjected to single-cell RNA-seq. Single cell Transcriptome analysis was conducted to verify the gene expression difference of malignant plasma cells derived from intramedullary and extramedullary. Then we verified high expression level of lncRNA-NEAT1 in EMD patients by using quantitative real-time PCR (qRT-PCR) and analyzed the correlation between expression patterns and survival and molecular genetics analysis of the LncRNA (NEAT1) involved in MM patients. At last, cell experiments were conducted to observe the effects of down-regulation of NEAT1on the proliferation, cell cycle and PTEN pathway related proteins of multiple myeloma cell lines U266 and RPMI8226. RESULTS: We identified one of the EMD related key gene is lncRNA-NEAT1. Compared with patients without extramedullary lesions, intramedullary MM cells in EMD patients expressed NEAT1 highly. The outcome of parallel single-cell RNA sequencing (RNA-seq) revealed NEAT1 level of plasma cells came from pleural effusion /ascites increased significantly compared with myeloma-stricken bone marrow. By survival and molecular genetic analysis, NEAT1 gene expression was not associated with OS and PFS in MM patients. However, the expression of NEAT1 is related to adverse therapeutic reactions and the progression of MM. We found that the expressions of NEAT1 were negatively associated with albumin levels and were positively associated with gain of chromosome 1q, IGH-CCND1, IGH@-FGFR3/WHSC1,and IGH-MAF gene fusion, respectively. At the level of cell experiment, CCK-8, soft agar clone formation experiment and CFSE staining showed that down regulating NEAT1 could inhibit the proliferation of U266 and RPMI8226 cells; Cell cycle detection showed that down-regulation of NEAT1 would interfere with the cell cycle process, and RPMI 8226 cells were blocked in G1 phase. Western blot analysis showed that when the expression of NEAT1 was down regulated in U266 and RPMI 8226 cells, the expression of PTEN and p-PTEN (phosphorylated phosphatase and tensin homologue) was up-regulated, and the expression of PI3K, p-PI3K (human phosphorylated inositol 3 kinase), Akt, p-Akt (phosphorylated protein kinase B). DISCUCCION AND CONCLUSION: This study provides novel insights into the lncRNA-NEAT1 and reveals that NEAT1 maybe a potential lncRNA biomarkers in EMD.

Single-Cell Sequencing of Malignant Ascites Reveals Transcriptomic Remodeling of the Tumor Microenvironment during the Progression of Epithelial Ovarian Cancer.

Epithelial ovarian cancer (EOC) is the main cause of mortality among gynecological malignancies worldwide. Although patients with EOC undergo aggregate treatment, the prognosis is often poor. Peritoneal malignant ascites is a distinguishable clinical feature in EOC patients and plays a pivotal role in tumor progression and recurrence. The mechanisms of the tumor microenvironment (TME) in ascites in the regulation of tumor progression need to be explored. We comprehensively analyzed the transcriptomes of 4680 single cells from five EOC patients (three diagnostic samples and two recurrent samples) derived from Gene Expression Omnibus (GEO) databases. Batch effects between different samples were removed using an unsupervised deep embedding single-cell cluster algorithm. Subcluster analysis identified the different phenotypes of cells. The transition of a malignant cell state was confirmed using pseudotime analysis. The landscape of TME in malignant ascites was profiled during EOC progression. The transformation of epithelial cancer cells into mesenchymal cells was observed to lead to the emergence of related anti-chemotherapy and immune escape phenotypes. We found the activation of multiple biological pathways with the transition of tumor-associated macrophages and fibroblasts, and we identified the infiltration of CD4+CD25+ T regulatory cells in recurrent samples. The cell adhesion molecules mediated by integrin might be associated with the formation of the tumorsphere. Our study provides novel insights into the remodeling of the TME heterogeneity in malignant ascites during EOC progression, which provides evidence for identifying novel therapeutic targets and promotes the development of ovarian cancer treatment.

Integrated analysis of ascites and plasma extracellular vesicles identifies a miRNA-based diagnostic signature in ovarian cancer.

Ovarian cancer is mostly diagnosed at advantaged stages due to the lack of early diagnostic biomarkers. The common metastasis pattern is characterized by peritoneal dissemination with a formation of malignant ascites. Extracellular vesicles (EVs) are emerging as promising clinical biomarkers in liquid biopsy. Here, we aimed to investigate robust liquid biopsy-based EV miRNA biomarkers for ovarian cancer diagnosis and metastasis regulation. EVs were isolated from malignant ascites and plasma of ovarian cancer patients as well as the benign control counterparts of patients with benign gynecologic diseases. EV small RNA sequencing identified a panel of eight miRNAs (miR-1246, miR-1290, miR-483, miR-429, miR-34b-3p, miR-34c-5p, miR-145-5p, miR-449a) based on dysregulated miRNAs overlapped in the ascites and plasma subset. The ovarian cancer EV miRNA (OCEM) signature developed based on these eight miRNAs demonstrated high diagnostic accuracy in our in-house dataset and multiple public datasets across diverse clinical samples (blood, tissue and urine). In addition, malignant ascites-derived EVs could significantly facilitate the aggressive property of ovarian cancer cells and boost the growth of ascites-derived organoids. Notably, miR-1246 and miR-1290 shuttled in malignant ascites-derived EVs were identified to promote the invasion and migration of ovarian cancer cells through regulating a common target RORα.

Hsa_circ_0000497 and hsa_circ_0000918 contributed to peritoneal metastasis of ovarian cancer via ascites.

PURPOSE: As a common complication of epithelial ovarian cancer (EOC), malignant ascites contributes to the peritoneal metastasis of EOC. CircRNAs play essential roles in tumor metastasis. However, no circRNAs have been reported to be involved in EOC peritoneal metastasis via ascites. METHODS: Total of 22 samples from 9 EOC patients containing primary lesions (T), tumor cells from ascites (ASC), and metastatic lesions (M) were included for RNA sequencing to identify differentially expressed circRNAs and mRNAs among different tumors. Bioinformatic analyses, including single-sample Gene Set Enrichment Analysis and soft cluster analysis, were performed to find circRNAs potentially correlated with ascitic metastasis. Wound healing and transwell analysis were performed to evaluate tumor cells metastasis in vitro. Quantitative real-time PCR and western-blot were used for gene expression evaluation. RESULTS: According to transcriptomic analysis, ASC showed mesenchymal phenotype while T and M showed epithelial phenotype. 10 circRNAs were differentially expressed among ASC, T, and M. Among them, hsa_circ_0000497 and hsa_circ_0000918 were significantly up-regulated in ASC. Functional analysis showed that both hsa_circ_0000497 and hsa_circ_0000918 promoted metastasis of EOC via epithelial-mesenchymal transition (EMT) in vitro. The regulatory network construction identified 8 miRNAs and 19 mRNAs, and 7 miRNAs and 17 mRNAs as potential downstream target genes of hsa_circ_0000497 and hsa_circ_0000918, respectively, which may play pivotal roles in EOC ascitic metastasis. CONCLUSIONS: circRNAs (hsa_circ_0000497 and hsa_circ_0000918) contribute to metastasis of EOC via ascites by regulating EMT. These circRNAs may serve as novel potential therapeutic targets or prognostic biomarkers for EOC peritoneal metastasis.

Integrated molecular profiling of patient-derived ovarian cancer models identifies clinically relevant signatures and tumor vulnerabilities.

High-grade serous ovarian carcinoma (HGSOC) is a highly aggressive and intractable neoplasm, mainly because of its rapid dissemination into the abdominal cavity, a process that is favored by tumor-associated peritoneal ascites. The precise molecular alterations involved in HGSOC onset and progression remain largely unknown due to the high biological and genetic heterogeneity of this tumor. We established a set of different tumor samples (termed the As11-set) derived from a single HGSOC patient, consisting of peritoneal ascites, primary tumor cells, ovarian cancer stem cells (OCSC) and serially propagated tumor xenografts. The As11-set was subjected to an integrated RNA-seq and DNA-seq analysis which unveiled molecular alterations that marked the different types of samples. Our profiling strategy yielded a panel of signatures relevant in HGSOC and in OCSC biology. When such signatures were used to interrogate the TCGA dataset from HGSOC patients, they exhibited prognostic and predictive power. The molecular alterations also identified potential vulnerabilities associated with OCSC, which were then tested functionally in stemness-related assays. As a proof of concept, we defined PI3K signaling as a novel druggable target in OCSC.

SNP-based breeding for broiler resistance to ascites and evaluation of correlated production traits.

BACKGROUND: The goal of this study was to evaluate marker-assisted selection (MAS) in broiler chickens using previously mapped gene regions associated with ascites syndrome incidence. The second-generation MAS products were assessed for impact on ascites phenotype and whether there were associated changes in important production traits. Previously, we used whole genome resequencing (WGR) to fine-map 28 chromosomal regions as associated with ascites phenotype in our experimental ascites broiler line (Relaxed, REL) based on a hypobaric chamber challenge. Genotypes for single nucleotide polymorphisms (SNPs) in mapped regions on chromosomes 2 and 22, were used for MAS in our REL line. After two generations, birds homozygous for the genotypes associated with resistance for both chromosomal regions were established. The MAS F2 generation was then compared to the REL line for ascites susceptibility and 25 production traits. RESULTS: Selection based on SNPs in the carboxypeptidase Q (CPQ, Gga2) and leucine rich repeat transmembrane neuronal 4 (LRRTM4, Gga22) gene regions resulted in a sex- and simulated altitude- dependent reduction of ascites incidence in two F2 cohorts of the MAS line. Comparisons of the F2 MAS and REL lines for production traits when reared at ambient pressure found no significant negative impacts for feed intake (FI), feed conversion ratio (FCR), or deboned part yields for either sex for two F2 cohorts. There were, however, improvements in the MAS for full-trial body weight gain (BWG), FCR, absolute and relative tender weights, and relative drumstick weight. CONCLUSIONS: These results validate the mapping of the 28 chromosomal regions and demonstrate that fine mapping by WGR is an effective strategy for addressing a complex trait; it also stands as the first successful SNP-based selection program against a complex disease trait, such as ascites. The MAS line is comparable and, in some instances, superior, in growth performance to the REL control while being more resistant to ascites. This study indicates that MAS based on WGR can provide significant breeding potential in agricultural systems.

Comparative Analysis of Tumor-Associated microRNAs and Tetraspanines from Exosomes of Plasma and Ascitic Fluids of Ovarian Cancer Patients.

Ovarian cancer (OC) is one of the most common and fatal types of gynecological cancer. In the early phase of OC detection, the current treatment and diagnostic methods are not efficient and sensitive enough. Therefore, it is crucial to explore the mechanisms of OC metastasis and discover valuable factors for early diagnosis of female cancers and novel therapeutic strategies for metastasis. Exosomes are known to be involved in the development, migration, and invasion of cancer cells, and their cargo could be useful for the non-invasive biopsy development. CD151- and Tspan8-positive exosomes are known to support the degradation of the extracellular matrix, and are involved in stroma remodeling, angiogenesis and cell motility, as well as the association of miR-24 and miR-101 with these processes. The objective of this study was to explore the relationship of these components of exosomal cargo, in patients with OC, to clarify the clinical significance of these markers in liquid biopsies. The levels of tetraspanins Tspan8+ and CD151+ exosomes were significantly higher in plasma exosomes of OC patients compared with healthy females (HFs). The relative levels of miR-24 and miR-101 in plasma exosomes of HFs were significantly higher than in plasma exosomes of OC patients, while the levels of these microRNAs in exosomes from plasma and ascites of ill females showed no difference. Our study revealed a strong direct correlation between the change in the ascites exosomes CD151+Tspan8+ subpopulation level and the expression levels of the ascites (R = 0.81, p < 0.05) and plasma exosomal miR-24 (R = 0.74, p < 0.05) in OC patients, which confirms the assumption that exosomal cargo act synergistically to increase cellular motility, affecting cellular processes and signaling. Bioinformatics analysis confirmed the involvement of CD151 and Tspan8 tetraspanins and genes controlled by miR-24-3p and miR-101 in signaling pathways, which are crucial for carcinogenesis, demonstrating that these tetraspanins and microRNAs are potential biomarkers for OC screening, and predictors of poor clinicopathological behavior in tumors.

Single cell-derived spheroids capture the self-renewing subpopulations of metastatic ovarian cancer.

High Grade Serous Ovarian cancer (HGSOC) is a major unmet need in oncology, due to its precocious dissemination and the lack of meaningful human models for the investigation of disease pathogenesis in a patient-specific manner. To overcome this roadblock, we present a new method to isolate and grow single cells directly from patients' metastatic ascites, establishing the conditions for propagating them as 3D cultures that we refer to as single cell-derived metastatic ovarian cancer spheroids (sMOCS). By single cell RNA sequencing (scRNAseq) we define the cellular composition of metastatic ascites and trace its propagation in 2D and 3D culture paradigms, finding that sMOCS retain and amplify key subpopulations from the original patients' samples and recapitulate features of the original metastasis that do not emerge from classical 2D culture, including retention of individual patients' specificities. By enabling the enrichment of uniquely informative cell subpopulations from HGSOC metastasis and the clonal interrogation of their diversity at the functional and molecular level, this method provides a powerful instrument for precision oncology in ovarian cancer.

Ascites in ovarian cancer: MicroRNA deregulations and their potential roles in ovarian carcinogenesis.

Ovarian cancer comprises the most lethal gynecologic malignancy and is accompanied by the high potential for the incidence of metastasis, recurrence and chemotherapy resistance, often associated with a formation of ascitic fluid. The differentially expressed ascites-derived microRNAs may be linked to ovarian carcinogenesis. The article focuses on a number of miRNAs that share a common expression pattern as determined by independent studies using ascites samples and with regard to their functions and outcomes in experimental and clinical investigations.Let-7b and miR-143 have featured as tumor suppressors in ovarian cancer, which is in line with data on other types of cancer. Although two miRNAs, i.e. miR-26a-5p and miR-145-5p, act principally as tumor suppressor miRNAs, they occasionally exhibit oncogenic roles. The performance of miR-95-3p, upregulated in ascites, is open to debate given the current lack of supportive data on ovarian cancer; however, data on other cancers indicates its probable oncogenic role. Different findings have been reported for miR-182-5p and miR-200c-3p; in addition to their presumed oncogenic roles, contrasting findings have indicated their ambivalent functions. Further research is required for the identification and evaluation of the potential of specific miRNAs in the diagnosis, prediction, treatment and outcomes of ovarian cancer patients.

rRNA Analysis Based on Long-Read High-Throughput Sequencing Reveals a More Accurate Diagnostic for the Bacterial Infection of Ascites.

Traditional pathogenic diagnosis presents defects such as a low positivity rate, inability to identify uncultured microorganisms, and time-consuming nature. Clinical metagenomics next-generation sequencing can be used to detect any pathogen, compensating for the shortcomings of traditional pathogenic diagnosis. We report third-generation long-read sequencing results and second-generation short-read sequencing results for ascitic fluid from a patient with liver ascites and compared the two types of sequencing results with the results of traditional clinical microbial culture. The distribution of pathogenic microbial species revealed by the two types of sequencing results was quite different, and the third-generation sequencing results were consistent with the results of traditional microbial culture, which can effectively guide subsequent treatment. Short reads, the lack of amplification, and enrichment to amplify signals from trace pathogens, and host background noise may be the reasons for the high error in the second-generation short-read sequencing results. Therefore, we propose that long-read-based rRNA analysis technology is superior to the short-read shotgun-based metagenomics method in the identification of pathogenic bacteria.