TOTEM: a multi-cancer detection and localization approach using circulating tumor DNA methylation markers.

BACKGROUND: Detection of cancer and identification of tumor origin at an early stage improve the survival and prognosis of patients. Herein, we proposed a plasma cfDNA-based approach called TOTEM to detect and trace the cancer signal origin (CSO) through methylation markers. METHODS: We performed enzymatic conversion-based targeted methylation sequencing on plasma cfDNA samples collected from a clinical cohort of 500 healthy controls and 733 cancer patients with seven types of cancer (breast, colorectum, esophagus, stomach, liver, lung, and pancreas) and randomly divided these samples into a training cohort and a testing cohort. An independent validation cohort of 143 healthy controls, 79 liver cancer patients and 100 stomach cancer patients were recruited to validate the generalizability of our approach. RESULTS: A total of 57 multi-cancer diagnostic markers and 873 CSO markers were selected for model development. The binary diagnostic model achieved an area under the curve (AUC) of 0.907, 0.908 and 0.868 in the training, testing and independent validation cohorts, respectively. With a training specificity of 98%, the specificities in the testing and independent validation cohorts were 100% and 98.6%, respectively. Overall sensitivity across all cancer stages was 65.5%, 67.3% and 55.9% in the training, testing and independent validation cohorts, respectively. Early-stage (I and II) sensitivity was 50.3% and 45.7% in the training and testing cohorts, respectively. For cancer patients correctly identified by the binary classifier, the top 1 and top 2 CSO accuracies were 77.7% and 86.5% in the testing cohort (n = 148) and 76.0% and 84.0% in the independent validation cohort (n = 100). Notably, performance was maintained with only 21 diagnostic and 214 CSO markers, achieving a training AUC of 0.865, a testing AUC of 0.866, and an integrated top 2 accuracy of 83.1% in the testing cohort. CONCLUSIONS: TOTEM demonstrates promising potential for accurate multi-cancer detection and localization by profiling plasma methylation markers. The real-world clinical performance of our approach needs to be investigated in a much larger prospective cohort.

Hsa_circ_0102899 promotes epithelial–mesenchymal transition in non-small cell lung cancer

Objective The significance of circular RNAs (circRNAs) has been identified in the progression of non-small cell lung cancer (NSCLC). Consistently, our study probed the functional actions of hsa_circ_0102899 (circ_0102899) in NSCLC cells. Methods circ_0102899 expression was checked in NSCLC tissues, as well as its correlation with clinical characteristics of patients, Using A459 cells, transfection to alter gene expression was performed, thus measuring the changes of proliferation, apoptosis, migration, and apoptosis, as well as epithelial–mesenchymal transition (EMT)-related proteins. circ_0102899’s effects in vivo were validated by tumor xenograft assay. Finally, the regulatory mechanism of circ_0102899 was investigated. Results circ_0102899 indicated a high-expression level in NSCLC tissues which was associated with NSCLC tumor characteristics. Functionally, circ_0102899 knockdown not only inhibited the growth and EMT process of NSCLC cells, but also inhibited tumor formation in vivo. In terms of the regulatory mechanism, circ_0102899 had a binding to miR-885-5p to target eukaryotic translation initiation factor 4γ2 (EIF4G2). circ_0102899 mediated miR-885–5/EIF4G2 axis to accelerate the process of cell malignant behavior in NSCLC. Conclusion circ_0102899 promotes EMT and metastasis in NSCLC by regulating the miR-885-5p/EIF4G2 axis (AU)

Hsa_circ_0102899 promotes epithelial-mesenchymal transition in non-small cell lung cancer.

OBJECTIVE: The significance of circular RNAs (circRNAs) has been identified in the progression of non-small cell lung cancer (NSCLC). Consistently, our study probed the functional actions of hsa_circ_0102899 (circ_0102899) in NSCLC cells. METHODS: circ_0102899 expression was checked in NSCLC tissues, as well as its correlation with clinical characteristics of patients, Using A459 cells, transfection to alter gene expression was performed, thus measuring the changes of proliferation, apoptosis, migration, and apoptosis, as well as epithelial-mesenchymal transition (EMT)-related proteins. circ_0102899's effects in vivo were validated by tumor xenograft assay. Finally, the regulatory mechanism of circ_0102899 was investigated. RESULTS: circ_0102899 indicated a high-expression level in NSCLC tissues which was associated with NSCLC tumor characteristics. Functionally, circ_0102899 knockdown not only inhibited the growth and EMT process of NSCLC cells, but also inhibited tumor formation in vivo. In terms of the regulatory mechanism, circ_0102899 had a binding to miR-885-5p to target eukaryotic translation initiation factor 4γ2 (EIF4G2). circ_0102899 mediated miR-885-5/EIF4G2 axis to accelerate the process of cell malignant behavior in NSCLC. CONCLUSION: circ_0102899 promotes EMT and metastasis in NSCLC by regulating the miR-885-5p/EIF4G2 axis.

LncRNA FOXP4-AS promotes the progression of non-small cell lung cancer by regulating the miR-3184-5p/EIF5A axis.

Long non coding RNA FOXP4-AS1 exerted crucial functions in various human cancers, while its role in non-small cell lung cancer (NSCLC) remains unclear. A total of 30 pairs of NSCLC tissues and matched adjacent normal tissues were used to evaluate the expression of FOXP4-AS1 and miR-3184-5p. Cell proliferation was assessed by CCK-8 assay and colony formation assay. Cell apoptosis was measured by flow cytometry. Bioinformatic analysis and luciferase reporter assay were performed to determine the regulatory relationship among FOXP4-AS1, miR-3184-5p and EIF5A. The xenograft tumor model was constructed to confirm the function of FOXP4-AS1 in NSCLC progression. The results showed that FOXP4-AS1 was upregulated and miR-3184-5p was downregulated in NSCLC tissues and cell lines. Downregulation of FOXP4-AS1 significantly reduced cell proliferation and induced apoptosis of NSCLC cells in vitro. FOXP4-AS1 could regulated the expression of EIF5A by binding to miR-3184-5p. Rescue experiments showed that downregulation of miR-3184-5p or overexpression of EIF5A obviously attenuated the inhibitory effects of si-FOXP4-AS1 on cell proliferation, as well as the stimulating effects on cell apoptosis. Moreover, knockdown of FOXP4-AS1 could efficiently inhibited tumor development of NSCLC in vivo. Downregulation of FOXP4-AS1 attenuated the progression of NSCLC by regulating miR-3184-5p and EIF5A.

Toxic effects of ammonia and thermal stress on the intestinal microbiota and transcriptomic and metabolomic responses of Litopenaeus vannamei.

Ammonia and thermal stress frequently have harmful effects on aquatic animals. The intestine is an important barrier allowing the body to defend against stress. In this study, we investigated the intestinal microbiota and transcriptomic and metabolomic responses of Litopenaeus vannamei subjected to individual and combined ammonia and thermal stress. The results showed that obvious variation in the intestinal microbiota was observed after stress exposure, with increased levels of Firmicutes and decreased levels of Bacteroidetes and Planctomycetes. Several genera of putatively beneficial bacteria (Demequina, Weissella and Bacteroides) were abundant, while Formosa, Kriegella, Ruegeria, Rhodopirellula and Lutimonas were decreased; pathogenic bacteria of the genus Vibrio were increased under individual stress but decreased under combined stress. The intestinal transcriptome revealed several immune-related differentially expressed genes associated with the peritrophic membrane and antimicrobial processes in contrasting accessions. Haemolymph metabolomic analysis showed that stress exposure disturbed the metabolic processes of the shrimp, especially amino acid metabolism. This study provides insight into the underlying mechanisms associated with the intestinal microbiota, immunity and metabolism of L.vannamei in response to ammonia and thermal stress; ten stress-related metabolite markers were identified, including L-lactic acid, gulonic acid, docosahexaenoic acid, l-lysine, gamma-aminobutyric acid, methylmalonic acid, trans-cinnamate, N-acetylserotonin, adenine, and dihydrouracil.

Toxicological effects of microplastics in Litopenaeus vannamei as indicated by an integrated microbiome, proteomic and metabolomic approach.

Microplastics (MPs) are a hazardous pollutant of world concern that threaten aquatic organisms and ecosystems. In this study, we chose the worldwide-distributed shrimp Litopenaeus vannamei as a model and investigated the toxicological effects of five types of MPs on L. vannamei using several omics approaches. After 14 days of exposure to MPs, obvious intestinal microbiota variation was observed, such as increased abundances of Bacteroidetes and Proteobacteria and a decreased abundance of Firmicutes. Specifically, MPs induced several putative opportunistic pathogens and reduced lactic acid- and short-chain fatty acid-producing bacteria. Alternatively, MPs altered haemolymph proteome profiles, but the five types of MPs had different effects on the enriched pathways and the expression of immune-related proteins. Furthermore, MPs also caused haemolymph metabolite variation, especially in amino acid and alpha-linolenic acid metabolism, and 28 differential metabolites were altered in the five MP-treated groups. Changes in intestinal bacteria were correlated with the haemolymph proteins and metabolites of the shrimp. Overall, these results reveal the toxicological effects of MPs on the intestinal microbiota and the host's immunity and metabolism in shrimp.

Effects of Microcystis aeruginosa and microcystin-LR on intestinal histology, immune response, and microbial community in Litopenaeus vannamei.

Microcystis aeruginosa (MA) is a primary hazardous cyanobacteria species in aquatic ecosystems that can produce microcystin-LR (MC-LR), which harms aquatic animals. The intestine is an important target tissue for MA and MC-LR. In this study, we investigated the effects of MA and MC-LR exposure on the intestinal microbiota variation and immune responses of Litopenaeus vannamei. Shrimp were experimentally exposed to MA and MC-LR for 72 h. The results showed that both MA and MC-LR exposure caused marked histological variation and apoptosis characteristics and increased oxidative stress in the intestine. Furthermore, the relative expression levels of antimicrobial peptide genes (ALF, Crus, Pen-3) decreased, while those of pro-inflammatory cytokines (MyD88, Rel, TNF-a), a pattern-recognition receptor (TLR4) and a mediator of apoptosis (Casp-3) increased. MA and MC-LR exposure also caused intestinal microbiota variation, including decreasing microbial diversity and disturbing microbial composition. Specifically, the relative abundance of Proteobacteria decreased in the two stress groups; that of Bacteroidetes decreased in the MA group but increased in the MC-LR group, while Tenericutes varied inversely with Bacteroidetes. Our results indicate that MA and MC-LR exposure causes intestinal histopathological and microbiota variations and induces oxidative stress and immune responses in L. vannamei. In conclusion, this study reveals the negative effects of MA and MC-LR on the intestinal health of shrimp, which should be considered in aquaculture.

RNA-seq revealed the signatures of immunity and metabolism in the Litopenaeus vannamei intestine in response to dietary succinate.

The intestine is important for nutrition, metabolism and immunity. Succinate (SA) plays a vital role in the physiological homeostasis of animal intestines. However, the effects of dietary SA on the intestinal immunity and metabolism in shrimp are not clear. In this study, we investigated the immune and metabolic responses in the intestine of Litopenaeus vannamei that were fed diets consisting of different levels of SA: 0 g/kg (Con) and 10 g/kg (SA) for 56 days. The results from a RNA-seq analysis identified 6005 differentially expressed genes (DEGs), including 2728 upregulated genes and 3277 downregulated genes, which were grouped into 312 pathways. The DEGs were most enriched in pathways related to protein synthesis and amino acid metabolism, including "ribosome", "aminoacyl-tRNA biosynthesis", "pyrimidine metabolism", and "arginine and proline metabolism"; additionally, carbohydrate and lipid metabolism pathways were also activated. A large number of immune-related genes were associated with mucus barrier modification, antimicrobial activity, pathogen attachment and recognition, antioxidant activity, and apoptosis. The expression patterns of several candidate genes involved in the immune response and nutrition metabolism were detected by qPCR. This study provides insight into the transcriptomic modulating mechanisms associated with intestinal immunity and the metabolism of L. vannamei in response to the intake of dietary SA.

Changes in the intestine microbial, digestion and immunity of Litopenaeus vannamei in response to dietary resistant starch.

Resistant starch (RS) is a constituent of dietary fibre that has beneficial effects on the intestine physiological function of animals. However, the roles of RS on shrimp intestine health is unknown. In this study, we investigated the the effects of dietary RS on the microbial composition, and digestive and immune-related indices in the intestine of Litopenaeus vannamei. The shrimp were fed with diets containing different levels of RS: 0 g/kg (Control), 10 g/kg (RS1), 30 g/kg (RS2) and 50 g/kg (RS3) for 56 days. The results showed that dietary RS improved the morphology of the intestine mucosa. RS also increased the activity of digestive enzymes (AMS, LPS, Tryp, and Pep) and immune enzymes (PO, T-AOC, T-NOS, and NO), and the expression levels of immune-related genes (proPO, ALF, Lys, HSP70, Trx, Muc-1, Muc-2, Muc-5AC, Muc-5B, and Muc-19). A microbiome analysis indicated that dietary RS increased the short-chain fatty acids (SCFAs) contents and altered the composition of the intestine microbial. Specifically, RS increased the abundances of Proteobacteria and decreased the abundance of Bacteroidetes. At the genus level, the beneficial bacteria (Lutimonas, Ruegeria, Shimia, Mesoflavibacter, and Mameliella) were enriched, which might be involved in degrading toxins and producing beneficial metabolites; while potential pathogens (Formosa and Pseudoalteromonas) were decreased in response to dietary RS. Our results revealed that dietary RS could improve the intestine health of L. vannamei, probably via modulating the intestine microbial composition and SCFAs contents, and enhancing the digestion and immunity of the shrimp.

Changes in the intestine barrier function of Litopenaeus vannamei in response to pH stress.

pH of water environment affects the survival of aquatic animals. Intestine barrier function influences the health of animals, which is related to its mucosa structure, immune components, and microbial communities. In this study, we investigated the histological structure, digestive and metabolic capacity, immune responses, and microbial composition in the intestine of Litopenaeus vanmei under three different conditions: control (pH 8.3), low pH stress (pH 6.9), and high pH stress (pH 9.7) for 72 h. The results showed both low and high pH stress disrupted the intestine morphological structure, and induced variations in the activities of digestive (AMS, LPS, Tryp, and Pep) and metabolic (HK, PK, CCO, and LDH) enzymes. Low and high pH stress also increased oxidative stress (MDA, LPO, PC, and ·O2- generation), and decreased the antioxidant enzyme activities (T-AOC, SOD, and GST); shrimp enhanced CAT activity and HSP70, Trx, MT and Fer gene transcripts as defense mechanism. Additionally, Immune confusion was also found in the shrimp intestine in response to low and high pH stress, including the antibacterial ability (T-NOS, PO, proPO, ALF, and Lys), pathogen recognition (TLR and Lec), apoptosis (Casp, IAP and p53), and mucus homeostasis (Muc-1, Muc-2, Muc-5AC, Muc-5B, and Muc-19). pH exposure also decreased the diversity of the intestine bacterial, disturbed the composition of microbiota, and decreased the microbial metabolite SCFA contents. Our results indicated that acute pH stress can impair the intestine barrier function of white shrimp, probably via destroying mucosa structure, confusing digestion and metabolism, inducing oxidative stress, disordering immunity, and disrupting the microbial composition.

Transcriptomic and microbiota response on Litopenaeus vannamei intestine subjected to acute sulfide exposure.

Harmful effects of water pollutants are myriad. Sulfide from water bodies affects the aquatic animals. Intestine barrier function serves as the front-line of animals defense. Our previous study confirmed the toxic effect of sulfide on intestine immune response of Litopenaeus vannamei, but the underlying mechanisms remained elusive. Therefore, in this study, we investigated the transcriptomic and microbiota responses of the L. vannamei intestine subjected to acute sulfide exposure. Sulfide decreased bacterial richness and altered the intestine microbiota composition. Specifically, sulfide increased the abundances of Bacteroidetes and Actinobacteria, but decreased the abundance of Proteobacteria. At the genus level, sulfide increased typical cellulolytic characteristics bacteria, such as Formosa, Sphingomonas, and Demequina. RNA-seq analysis identified differential expression of 1799 genes (701 up-regulated and 1098 down-regulated) were grouped into 267 pathways. The most enriched pathway 'amoebiasis' was related to the intestine mucus homeostasis. A number of immune-related genes associated with antimicrobial, antioxidant, pathogen attachment and recognition, and apoptosis processes in contrasting accessions; they were correlated with the abundance of intestine bacterial at the phylum level. This study provides an insight into the mechanisms associated with molecular and microbiota response and processes involved in adaptation strategies towards sulfide stress.

Changes in the Intestine Microbial, Digestive, and Immune-Related Genes of Litopenaeus vannamei in Response to Dietary Probiotic Clostridium butyricum Supplementation.

The intestine barrier serves as the front-line defense in shrimp. Clostridium butyricum (CB) can produce butyric acid that provides energy for the intestine epithelial cells of the host. However, the effects of dietary CB on the intestine microbiome and the digestion and immunity of the host is not clear. In this study, we therefore investigated the composition and metabolic activity of the intestine microbiome, and digestive and immune-related gene expression in Litopenaeus vannamei fed with diets containing different levels of CB: basal diet (control), 2.5 × 109 CFU kg-1 diet (CB1), 5.0 × 109 CFU kg-1 diet (CB2), and 1.0 × 1010 CFU kg-1 diet (CB3) for 56 days. Dietary CB altered the composition of the intestine microbiome. Specifically, the dominant bacterial phylum Proteobacteria was enriched in the CB3 group and weakened in the CB1 and CB2 groups. The Bacteroidetes was enriched in the CB1 and CB2 groups and weakened in the CB3 group. The Firmicutes was enriched in all three CB groups. At the genus level, the potential pathogen (Desulfovibrio and Desulfobulbus) were weakened, and beneficial bacteria (Bacillus, Clostridium, Lachmoclostridium, Lachnospiraceae, and Lactobacillus) were enriched in response to dietary CB; these might contribute to the expression of the host digestive genes (α-amylase, lipase, trypsin, fatty acid-binding protein, and fatty acid synthase) and immune-related genes (prophenoloxidase, lipopolysaccharide and ß-1,3-glucan binding protein, lysozyme, crustin, and superoxide dismutase). Additionally, CB enhanced the bacterial metabolism, especially that of carbohydrates, polymers, amino acids, carboxylic acids, and amines. These results revealed that dietary CB had a beneficial effect on the intestine health of L. vannamei by modulating the composition of the intestine microbiome, enhancing the microbial metabolism activity, and promoting the digestion and immunity of the host. The optimal dietary supplementation dosage was found to be 5.0 × 109 CFU kg-1 in the diet.

Physiological and immune response in the gills of Litopenaeus vannamei exposed to acute sulfide stress.

Sulfide is a harmful environmental pollutant that affects the survival and immunity of shrimps. The gill is important for shrimp respiratory and osmotic adjustment, the physiological and immune homeostasis of the organ can be influenced by sulfide. In this study, we investigated the acute toxicity of sulfide (5 mg/L) on the morphology, physiological and immune response in the gills of Litopenaeus vannamei. H&E stain showed that sulfide stress damaged the gills histological structure. Specifically, osmoregulation capacity including of Na+/K+-ATPase and Ca2+/Mg2+-ATPase activity was both increased at 6 h and 12 h, and decreased at 72 h; the contents of free amino acid including of Gly, Pro, Ser, Thr and Ala were decreased at 72 h. Respiratory metabolic enzymes, such as cytochrome c oxidase and succcinate dehydrogenase activity was decreased at 12 h-72 h, while fumarate reductase and lactate dehydrogenase activity kept a higher level at 12 h-72 h. Significant variations in the activities of immune enzymes (acid phosphatase, alkaline phosphatase, total antioxidant capacity and lysozyme). The expression of immune-related genes (heat shock protein 70, thioredoxin and caspase-3) was increased at first and then decreased, while hypoxia inducible factor 1α kept a higher level at 6 h-72 h. These results revealed that sulfide stress influenced the L. vannamei gills physiological and immune function by damaging histological structure, and confusing osmoregulation, respiratory metabolic and immune capacity.

Impairment of the intestine barrier function in Litopenaeus vannamei exposed to ammonia and nitrite stress.

Intestine barrier serves as the front-line of shrimp defense, which rely on its structural integrity, microbial composition, and mucus immune compounds. Mucins are the major organic components of the intestine mucus layer that contribute to the immunity of intestine mucus. In this study, we examined the histological structure, microbial composition, and mucin genes expression in the intestines of Litopenaeus vanmei under three different conditions: control, ammonia stress, and nitrite stress for 72 h. H&E stain showed that ammonia and nitrite stress exposure both damaged the intestine mucosal tissue. High-throughput 16S rDNA sequencing revealed that two stresses exposure decreased the bacterial diversity, and altered the composition of intestine microbial. Specifically, the dominant bacterial phyla Bacteroidetes abundance was increased, while Proteobacteria and Planctomycetes were decreased; at the genus level, Formosa abundance was increased and Photobacterium was decreased, opportunistic pathogens including Nautella and Pseudoalteromonas was also increased. Intestine mucus immune genes including mucin-2 and mucin-19 were up-regulated, while mucin-1, mucin-5AC, and mucin-5B were down-regulated in two stress exposure groups. These results revealed that ammonia and nitrite stress harmed the intestine barrier function of L. vannamei by damaging the mucosal tissue, disrupting the composition of intestine microbial, and suppressing the immune function.

Nitrite stress disrupts the structural integrity and induces oxidative stress response in the intestines of Pacific white shrimp Litopenaeus vannamei.

The acute toxicity of nitrite on the mucosal morphology and oxidative stress response was investigated in the intestines of Litopenaeus vannamei after the exposure of 20 mg/L nitrite for 72 hr. The duration of nitrite stress influenced the shrimp survival, and the cumulative mortality rate was 36.7% at 72 hr. Hematoxylin and eosin (HE) stain showed that nitrite stress damaged the intestine connective and epithelium tissue. Specifically, nitrite stress increased the content of reactive oxygen species (ROS) production (O2- generation capacity), lipid peroxidation (LPO), and malondialdehyde (MDA). Meanwhile, the activities of antioxidant enzymes including superoxide dismutase (SOD) increased first, then catalase (CAT) and glutathione peroxidase (GPx) increased to the highest at 24 and 12 hr, respectively. The relative expression level of antioxidant genes including heat shock protein 70 (HSP70), ferritin, and thioredoxin (Trx) increased to the highest at 6, 12, and 48 hr respectively. After exposure to nitrite stress for 72 hr, the levels of antioxidant enzymes activity and genes expression all decreased. These results revealed that nitrite stress harmed the intestine barrier of L. vannamei by damaging the mucosal structure, inducing the oxidative stress, and confusing the antioxidant status.