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Carbohydrate Active EnZymes (CAZymes) are significantly important for microbial communities to thrive in carbohydrate rich environments such as animal guts, agricultural soils, forest floors, and ocean sediments. Since 2017, microbiome sequencing and assembly have produced numerous metagenome assembled genomes (MAGs). We have updated our dbCAN-seq database (https://bcb.unl.edu/dbCAN_seq) to include the following new data and features: (i) ∼498 000 CAZymes and ∼169 000 CAZyme gene clusters (CGCs) from 9421 MAGs of four ecological (human gut, human oral, cow rumen, and marine) environments; (ii) Glycan substrates for 41 447 (24.54%) CGCs inferred by two novel approaches (dbCAN-PUL homology search and eCAMI subfamily majority voting) (the two approaches agreed on 4183 CGCs for substrate assignments); (iii) A redesigned CGC page to include the graphical display of CGC gene compositions, the alignment of query CGC and subject PUL (polysaccharide utilization loci) of dbCAN-PUL, and the eCAMI subfamily table to support the predicted substrates; (iv) A statistics page to organize all the data for easy CGC access according to substrates and taxonomic phyla; and (v) A batch download page. In summary, this updated dbCAN-seq database highlights glycan substrates predicted for CGCs from microbiomes. Future work will implement the substrate prediction function in our dbCAN2 web server.

Carbohydrate active enzymes (CAZymes) are made by various organisms for complex carbohydrate metabolism. Genome mining of CAZymes has become a routine data analysis in (meta-)genome projects, owing to the importance of CAZymes in bioenergy, microbiome, nutrition, agriculture, and global carbon recycling. In 2012, dbCAN was provided as an online web server for automated CAZyme annotation. dbCAN2 (https://bcb.unl.edu/dbCAN2) was further developed in 2018 as a meta server to combine multiple tools for improved CAZyme annotation. dbCAN2 also included CGC-Finder, a tool for identifying CAZyme gene clusters (CGCs) in (meta-)genomes. We have updated the meta server to dbCAN3 with the following new functions and components: (i) dbCAN-sub as a profile Hidden Markov Model database (HMMdb) for substrate prediction at the CAZyme subfamily level; (ii) searching against experimentally characterized polysaccharide utilization loci (PULs) with known glycan substates of the dbCAN-PUL database for substrate prediction at the CGC level; (iii) a majority voting method to consider all CAZymes with substrate predicted from dbCAN-sub for substrate prediction at the CGC level; (iv) improved data browsing and visualization of substrate prediction results on the website. In summary, dbCAN3 not only inherits all the functions of dbCAN2, but also integrates three new methods for glycan substrate prediction.

Supplementary Figures and Legends

Primers used for validation the gene expression level

The interplay between gut microbiota and the host immune system is emerging as a factor in the pathogenesis of colorectal cancer. Here, we set out to identify the effect of Akkermansia muciniphila (A. muciniphila) on colorectal cancer pathogenesis. A. muciniphila abundance was significantly reduced in patients with colorectal cancer from two independent clinical cohorts and the GMrepo dataset. Supplementation with A. muciniphila suppressed colonic tumorigenesis in ApcMin/+ mice and the growth of implanted HCT116 or CT26 tumors in nude mice. Mechanistically, A. muciniphila facilitated enrichment of M1-like macrophages in an NLRP3-dependent manner in vivo and in vitro. As a consequence, NLRP3 deficiency in macrophages attenuated the tumor-suppressive effect of A. muciniphila. In addition, we revealed that TLR2 was essential for the activation of the NF-κB/NLRP3 pathway and A. muciniphila induced M1-like macrophage response. We observed positive correlations between M1-like macrophages, NLRP3/TLR2 and A. muciniphila in patients with colorectal cancer, which corroborated these findings. In summary, A. muciniphila–induced M1-like macrophages provide a therapeutic target in the colorectal cancer tumor microenvironment.

To identify candidate bacteria associated with aging, we performed fecal microbiota sequencing in young, middle-aged and older adults, and found lower Bifidobacterium adolescentis abundance in older individuals aged ≥60 years. Dietary supplementation of B. adolescentis improved osteoporosis and neurodegeneration in a mouse model of premature aging (Terc−/−) and increased healthspan and lifespan in Drosophila melanogaster and Caenorhabditis elegans. B. adolescentis supplementation increased the activity of the catalase (CAT) enzyme in skeletal muscle and brain tissue from Terc−/− mice, and suppressed cellular senescence in mouse embryonic fibroblasts. Transgenic deletion of catalase (ctl-2) in C. elegans abolished the effects of B. adolescentis on the lifespan and healthspan. B. adolescentis feeding also led to changes in oxidative stress-associated metabolites in Terc−/− mouse feces. These results suggest a role for B. adolescentis in improving the healthspan and lifespan through the regulation of CAT activity and host metabolism. This study shows that dietary supplementation with B. adolescentis increases the activity of the catalase enzyme and improves the healthspan and lifespan in multiple animal models. Deletion of the catalase enzyme in worms abolished the beneficial effects of the bacterium.

The interplay between gut microbiota and the host immune system is emerging as a factor in the pathogenesis of colorectal cancer. Here, we set out to identify the effect of Akkermansia muciniphila (A. muciniphila) on colorectal cancer pathogenesis. A. muciniphila abundance was significantly reduced in patients with colorectal cancer from two independent clinical cohorts and the GMrepo dataset. Supplementation with A. muciniphila suppressed colonic tumorigenesis in ApcMin/+ mice and the growth of implanted HCT116 or CT26 tumors in nude mice. Mechanistically, A. muciniphila facilitated enrichment of M1-like macrophages in an NLRP3-dependent manner in vivo and in vitro. As a consequence, NLRP3 deficiency in macrophages attenuated the tumor-suppressive effect of A. muciniphila. In addition, we revealed that TLR2 was essential for the activation of the NF-κB/NLRP3 pathway and A. muciniphila induced M1-like macrophage response. We observed positive correlations between M1-like macrophages, NLRP3/TLR2 and A. muciniphila in patients with colorectal cancer, which corroborated these findings. In summary, A. muciniphila–induced M1-like macrophages provide a therapeutic target in the colorectal cancer tumor microenvironment.

Long noncoding RNAs (lncRNAs) are emerging as a new class of important regulators of signal transduction in tissue homeostasis and cancer development. Liquid-liquid phase separation (LLPS) occurs in a wide range of biological processes, while its role in signal transduction remains largely undeciphered. In this study, we uncovered a lipid-associated lncRNA, small nucleolar RNA host gene 9 (SNHG9) as a tumor-promoting lncRNA driving liquid droplet formation of Large Tumor Suppressor Kinase 1 (LATS1) and inhibiting the Hippo pathway. Mechanistically, SNHG9 and its associated phosphatidic acids (PA) interact with the C-terminal domain of LATS1, promoting LATS1 phase separation and inhibiting LATS1-mediated YAP phosphorylation. Loss of SNHG9 suppresses xenograft breast tumor growth. Clinically, expression of SNHG9 positively correlates with YAP activity and breast cancer progression. Taken together, our results uncover a novel regulatory role of a tumor-promoting lncRNA (i.e., SNHG9) in signal transduction and cancer development by facilitating the LLPS of a signaling kinase (i.e., LATS1).

Background and aims To compare the relationships of five obesity-related routine anthropometric indicators (body mass index (BMI), body adiposity index (BAI), waist circumference (WC), waist-to-hip ratio (WHR), waist-to-height ratio (WHtR)) for hypertension in both sexes and among different age groups of the Chinese population. Methods and results A total of 12,064 adult participants (5638 males and 6426 females) were included. Odds ratios (OR) and 95% confidence intervals were used with binary logistic regression models to estimate the risk of hypertension for each obesity index. For the males, WHtR had the highest OR value in all age groups. The degrees of correlation between hypertension and the obesity indices for different age groups were different among the females. WC, BMI, and WHtR were the highest in the 18–44, 45–59, and ≥60 years age groups, respectively. Furthermore, we compared the area under the ROC curve (AUC) of each obesity index for the criterion of hypertension under the influence of risk factors. For the males, the AUC of WHtR was the largest (0.814, 0.710, and 0.662). WC (AUC = 0.820), BMI (AUC = 0.765), and WHtR (AUC = 0.668) tended to be the best criteria for hypertension among females in the 18–44, 45–59, and ≥60 years age groups respectively. In addition, BAI, as an obesity indicator proposed in recent years, has a positive association with hypertension except in 18–44 years women, which was not stronger than other obesity indicators. Conclusions For males, WHtR appears to be the best obesity index related with hypertension. For young, middle-aged, and elderly women, the best obesity indices related with hypertension are WC, BMI, and WHtR, respectively.

Background The interplay between gut microbiota and tumor microenvironment (TME) in the pathogenesis of colorectal cancer (CRC) is largely unknown. Here, we elucidated the functional role of B. adolescentis (B.a) and its possible mechanism on the manipulation of cancer-associated fibroblasts (CAFs) in CRC. Methods The relative abundance of B.a in the patients with CRC or the healthy individuals was analyzed. The role of B.a was evaluated in the CRC animal models. The single cell-RNA sequencing (scRNA-seq) was used to investigate the cell subsets in the TME. The expression level of Wnt signaling and its downstream GAS1 in CAFs were explored by western blot and qRT-PCR. Overexpression of GAS1 in NIH/3T3 was performed to investigate its effect on the subcutaneous tumor formation. Multi-immunofluorescence assay examined the expression level of CD143 and GAS1 on the tissue array. Results We found that B. a abundance was significantly reduced in CRC patients from two independent cohorts and the bacteria database of GMrepo. Supplementation with B. a suppressed ApcMin/+ spontaneous or AOM/DSS-induced tumorigenesis in mice. scRNA-seq revealed that B. a facilitated a new subset CD143+CAFs to highly express GAS1 and GAS1+CAFs existed a suppressive effect. Mechanistically, GAS1 were activated by the Wnt/β-catenin signaling in CD143+CAFs. B. a abundance was correlated with the expression level of CD143 and GAS1. The level of CD143+CAFs predicted the more satisfied survival in CRC patients. Conclusions These results highlight that B. a induced a new subset CD143+CAFs and provides a novel therapeutic target for probiotic-based modulation of TME in CRC.

The enrichment of Fusobacterium nucleatum (Fn) has been identified in CRC patients and associated with worse outcomes. However, whether Fn was involved in the metastasis of CRC was not well determined. Here, we found that the abundance of Fn was significantly increased in CRC patients with lymph nodes metastasis. To further clarify the role of Fn in CRC metastasis, we performed transwell and wound healing assays after incubating CRC cell lines with or without Fn and injected Fn-treated or untreated CRC cells into nude mice via tail vein. The results indicated that Fn infection promoted CRC cells migration in vitro, as well as lung metastasis in vivo. Interestingly, colonization of Fn was detected in metastatic lung lesions of nude mice by fluorescence in situ hybridization. Mechanistically, RNA sequencing and validation study revealed that Fn significantly upregulated the expression of long non-coding RNA Keratin7-antisense (KRT7-AS) and Keratin7 (KRT7) in CRC cells. Importantly, Fn-induced CRC lung metastasis was attenuated by the depletion of KRT7-AS. In addition, KRT7-AS facilitated CRC cells migration by upregulating KRT7. Subsequently, we found that NF-κB signaling pathway was involved in the upregulation of KRT7-AS upon Fn infection. In conclusion, Fn infection upregulated KRT7-AS/KRT7 by activating NF-κB pathway, which promoted CRC cell migration in vitro and metastasis in vivo.

Gastric cancer (GC) patients with metastasis had limited treatment options and dismal outcome. We have previously reported the aberrant expression of Zic family member 1 (Zic1) in GC. However, the functional roles and underlying mechanism of Zic1 in GC metastasis remain unknown. Here, we demonstrate that lower expression of Zic1 was correlated with more lymph node metastasis and poor outcome of GC patients. Ectopic expression of Zic1 suppressed both lung metastasis and peritoneal tumor dissemination of GC in mice. The metastatic suppressing ability of Zic1 was mediated by regulating the process of cell invasion, adhesion and epithelial-mesenchymal transition (EMT). Mechanistically, Zic1 could downregulate Wnt targets including c-Myc and Cyclin D1 by inhibiting LEF transcriptional activity in GC cells. Notably, Zic1 was inversely related to the expression of Cyclin D1 in GC tissues tested. In addition, Zic1 could physically interact with β-catenin/transcription factor 4 (TCF4) and disrupt their complex formation, while not affecting β-catenin nuclear localization. Collectively, our study indicated that Zic1 suppressed GC metastasis through attenuating Wnt/β-catenin signaling and the EMT process. Our work may provide novel therapeutic strategies for the metastasis of GC.

Eukaryotic cells contain subcellular organelles with spatiotemporal regulation to coordinate various biochemical reactions. The various organelles perform their essential biological functions by employing specific biomolecules, including nucleic acids. Recent studies have revealed that noncoding RNAs (ncRNAs) are highly compartmentalized in cells and that their spatial distribution is intimately related to their functions. Dysregulation of subcellular ncRNAs can disrupt cellular homeostasis and cause human diseases. Mitochondria are responsible for energy generation to fuel cell growth and proliferation. Therefore, identifying mitochondria-associated ncRNAs helps to reveal new regulatory mechanisms and physiological functions of mitochondria. In this review, we summarize the latest advances in subcellular ncRNAs derived from either the nuclear or mitochondrial genome. We also discuss available biological approaches for investigating organelle-specific ncRNAs. Exploring the distribution and function of subcellular ncRNAs may facilitate the understanding of endomembrane dynamics and provide potential strategies for clinical transformation. This article is categorized under: RNA Export and Localizationgt; RNA Localization Regulatory RNAs/RNAi/Riboswitchesgt; Regulatory RNAs RNA Methodsgt; RNA Analyses in Cells.

Emerging evidence has shown that open reading frames inside long noncoding RNAs (lncRNAs) could encode micropeptides. However, their roles in cellular energy metabolism and tumor progression remain largely unknown. Here, we identified a 94 amino acid–length micropeptide encoded by lncRNA LINC00467 in colorectal cancer. We also characterized its conservation across higher mammals, localization to mitochondria, and the concerted local functions. This peptide enhanced the ATP synthase construction by interacting with the subunits α and γ (ATP5A and ATP5C), increased ATP synthase activity and mitochondrial oxygen consumption rate, and thereby promoted colorectal cancer cell proliferation. Hence, this micropeptide was termed ATP synthase–associated peptide (ASAP). Furthermore, loss of ASAP suppressed patient-derived xenograft growth with attenuated ATP synthase activity and mitochondrial ATP production. Clinically, high expression of ASAP and LINC00467 predicted poor prognosis of colorectal cancer patients. Taken together, our findings revealed a colorectal cancer–associated micropeptide as a vital player in mitochondrial metabolism and provided a therapeutic target for colorectal cancer.

Background Obesity is associated with type 2 diabetes mellitus (T2DM). However, the obesity index that is most closely related to type 2 diabetes remains controversial. Therefore, the aim of this study was to compare the associations of five anthropometric indices (body mass index [BMI], body adiposity index, waist circumference [WC], waist-to-hip ratio, and waist-to-height ratio [WHtR]) with T2DM among Chinese adults divided into four groups according to sex and age. Methods A total of 4007 adult participants (1669 men and 2338 women) were included in the study. Odds ratios (ORs) and 95% confidence intervals were used with binary logistic regression models to estimate the risk of T2DM for each obesity index. Furthermore, we compared the area under the receiver operating characteristic curve (AUC) of each obesity index for the criterion of T2DM under the influence of risk factors. Results WC had the highest OR (3.211 and 1.452) and AUC (0.783 and 0.614) in both age groups of men. However, WHtR (OR = 2.366, AUC = 0.771) and BMI (OR = 1.596, AUC = 0.647) were the optimal criteria for predicting T2DM among females in the 18–59 and ≥ 60 years age groups, respectively. Conclusions This study suggests that there is a positive association between obesity-related anthropometric indices and T2DM in different sex and age groups. WC appears to be the optimal anthropometric index for predicting T2DM in men. The optimal obesity indices related to T2DM were WHtR and BMI for women aged 18–59 and ≥ 60 years, respectively.

Senescent microenvironments play an important role in tumor progression. Here, we report that doxorubicin (DOX)‐pretreated or replicative senescent stromal cells (WI‐38 and HUVEC) promote colorectal cancer (CRC) cell growth and invasion in vitro and in vivo. These pro‐tumorigenic effects were attenuated by exogenous administration of Klotho, an anti‐aging factor. We subsequently identified several senescence‐associated secretory phenotype (SASP)‐associated genes, including CCL2, which were significantly upregulated in both types of senescent stromal cells during replication and DNA damage‐induced senescence. Importantly, we found that the secretion of CCL2 by senescent stromal cells was significantly higher than that seen in nonsenescent cells or in senescent cells pretreated with Klotho. Notably, CCL2 was found to accelerate CRC cell proliferation and invasion, while this effect could be blocked by administration of a specific CCR2 antagonist. We further show that Klotho can suppress NF‐κB activation during DOX‐induced senescence and thus block CCL2 transcription. Low expression of Klotho, or high expression of CCL2 in patient tumor tissues, correlated with poor overall survival of CRC patients. Collectively, our findings suggest that senescent stromal cells are linked to progression of CRC. Klotho can suppress the senescent stromal cell‐associated triggering of CRC progression by inhibiting the expression of SASP factors including CCL2. The identification of key SASP factors such as CCL2 may provide potential therapeutic targets for improving CRC therapy.

Background: Obesity is associated with type 2 diabetes mellitus (T2DM), but the obesity index most closely related to type 2 diabetes is still controversial. Therefore, the aim of this study was to compare the associations of five anthropometric indices (body mass index (BMI), body adiposity index (BAI), waist circumference (WC), waist-to-hip ratio (WHR), and waist-to-height ratio (WHtR)) with Type 2 diabetes mellitus (T2DM) among Chinese adults divided into four groups according to sex and age.Methods: A total of 4,007 adult participants (1,669 males and 2,338 females) were included. Odds ratios (OR) and 95% confidence intervals were used with binary logistic regression models to estimate the risk of T2DM for each obesity index. Furthermore, we compared the area under the ROC curve (AUC) of each obesity index for the criterion of hypertension under the influence of risk factors. Results: For the males, WC had the highest value of OR (3.211 and 1.452) and AUC (0.783 and 0.614) in both age groups. However, WHtR (OR=2.366, AUC=0.771) and BMI (OR=1.596, AUC=0.647) tended to be the best criteria for T2DM among females in the 18-59 and ≥60 years age groups, respectively.Conclusions: This study suggests that there is a positive association of obesity-related anthropometric indices with T2DM in different sex and age groups. For males, WC appears to be the best anthropometric index for predicting T2DM. For 18-59 and ≥60 age women, the best obesity indices related with T2DM are WHtR and BMI, respectively.

Microbiota-host interaction was involved in aging, while the specific bacterium was undetermined. To identify candidate bacterium with aging, we performed fecal microbiota sequencing. Less richness of gut microbial community, and a reduction of B.adolescentis abundance was observed in elderly individuals. B. adolescentis supplement improved osteoporosis and neurodegeneration in telomerase RNA component deletion (Terc−/−) aged mice. B.adolescentis induced prolongevity and healthspan improvement in Drosophila melanogaster and C. elegans. Transgenic deletion of ctl-2 in C. elegans abolished the effect on lifespan and healthspan by B. adolescentis. The catalase activity was decreased in skeletal muscle and brain tissues of Terc−/− mice, as well as cellular senescence in mouse embryonic fibroblasts. B. adolescentis alleviated ROS accumulation by regulation of oxidative stress-associated metabolites. These results suggest a role for B. adolescentis in improving lifespan and healthspan by regulating catalase activity and host metabolism. Supplement with commensal bacteria is a promising strategy against age related diseases.

Organelles entail specialized molecules to regulate their essential cellular processes. However, systematically elucidating the subcellular distribution of functional molecules such as long non-coding RNAs (lncRNAs) in tissue homeostasis and diseases has not been fully achieved. Here, we characterized the organelle-associated lncRNAs from mitochondria, lysosome, and endoplasmic reticulum (ER), respectively, and revealed the diverse and abundant distribution of lncRNAs. Among them, we identified mitochondrial lncRNA Growth-Arrest-Specific 5 (GAS5) as a tumor suppressor in maintaining cellular energy homeostasis. Mechanistically, energy stress-induced GAS5 modulated mitochondria TCA flux by declining metabolic tandem association of FH-MDH2-CS, the canonical members of the TCA cycle. Remarkably, the expression of GAS5 negatively related with levels of its associated mitochondrial metabolic enzymes and breast cancer development. Together with the detailed functional annotations, this subcellular lncRNA identification revealed the human cell’s inquisitively complex architecture, aiding in the development of new strategies for the clinical application of organelle-associated lncRNAs.

Background: MicroRNAs (miRNAs) play important roles in post-transcriptional regulation and may serve as targets for the diagnosis and treatment of cancers. Nevertheless, a comprehensive understanding of miRNAs profiles in gastric cancer (GC) progression is still lacking. Methods: Bioinformatics analyses, qRT-PCR, western blotting, and immunohistochemistry were applied for detecting miR-129-5p, HOXC10 and Cyclin D1 expression in GC tissues. Kaplan-Meier analysis with log-rank test was used for survival analyses. ChIP assay was performed for identifying potential gene regulated by HOXC10. Series in vitro and in vivo assays were used for exploring the function of miR-129-5p, HOXC10 and Cyclin D1 in the progression of GC. Findings: MiR-129-5p is downregulated in gastric cancer tissues and inhibits gastric cancer proliferation by inducing cycle cell arrest. MiR-129-5p directly targeted the 3’ untranslated regions (3’ UTR) of HOXC10 mRNA and downregulated its expression. Importantly, miR-129-5p could reverse the oncogenic effect induced by HOXC10. We systemically screened the downstream target of HOXC10 by ChIP-sequencing, and found that HOXC10 could transcriptionally regulate the expression of Cyclin D1 and facilitate G1/S cell cycle transition. Interpretation: MiR-129-5p/HOXC10/Cyclin D1 axis modulates cell cycle and gastric tumorigenesis, which might provide potential prognostic biomarkers and therapeutic targets for gastric cancer patients. Funding Statement: This work was supported by National Natural Science Foundation of China (31771540, 81802348, 81972276); Natural Science Foundation of Zhejiang Province (LY18H160011, LYY19H310011); Fundamental Research Funds for the Central Universities (2017QNA7005); Zhejiang Provincial Medical and Health Science and Technology Project (2018KY464). Declaration of Interests: The authors declare no potential conflicts of interest. Ethical Approval Statement: Animal experimental protocols were approved by the Committee of Animal Ethics of Zhejiang University. Informed written consent was obtained from all participants, and the study was approved by the ethics committee of Sir Run Run Shaw Hospital and The First Affiliated Hospital in Zhejiang University.

To evaluate potency and safety of 14-day bismuth–furazolidone quadruple regimens and to compare efficacies of five proton pump inhibitors (PPIs) for the initial eradication of Helicobacter pylori (H. pylori), 175 eligible patients were enrolled and randomly assigned to 14-day quadruple regimens consisting of bismuth (400 mg), amoxicillin (1 g), furazolidone (100 mg), and a PPI, twice a day. PPIs used were Group A (pantoprazole capsules, 40 mg), Group B (pantoprazole tablets, 40 mg), Group C (lansoprazole, 30 mg), Group D (esomeprazole, 20 mg), and Group E (rabeprazole, 10 mg). H. pylori status was reassessed by 13C urea breath test on day 56 as the primary outcome. Gastrointestinal symptoms, parenteral side effects, compliance, and stool type were recorded simultaneously. The total eradication rates were 86.9% (152/175 [95% CI 80.9–91.5%]) and 95.6% (152/159 [91.1–98.2%]) by intention-to-treat (ITT) and per-protocol (PP) analysis. The efficacies of Group A, B, C, D, and E by ITT analysis were 91.4% (32/35 [76.9–98.2%]), 85.7% (30/35 [69.7–95.2%]), 88.6% (31/35 [73.3–96.8%]), 85.7% (30/35 [69.7–95.2%]), and 82.9% (29/35 [66.4–93.4%]) (p > 0.05). In the PP analysis, the efficacies were 97.0% (32/33), 93.8% (30/32), 93.9% (31/33), 100% (30/30), and 93.5% (29/31) (p > 0.05). Gastrointestinal symptoms and stool type were improved significantly (p

Although immunotherapy such as immune checkpoint inhibitors has shown promising efficacy in cancer treatment, the responsiveness among patients is relatively limited. Activation of the cyclic guanosine monophosphate-adenosine monophosphate synthase/interferon gene stimulator (cGAS/STING) signaling pathway to upregulate innate immunity has become an emerging strategy for enhancing tumor immunotherapy. Herein, ZnS@BSA (bovine serum albumin) nanoclusters synthesized via a self-assembly approach are reported, where the released zinc ions under acidic tumor microenvironment significantly enhance cGAS/STING signals. Meanwhile, intracellular zinc ions can produce reactive oxygen species, which is further facilitated by the generated H2 S gas from ZnS@BSA via specifically inhibiting catalase in hepatocellular carcinoma cells. It is found that the nanoclusters activate the cGAS/STING signals in mice, which promotes the infiltration of CD8+ T cells at the tumor site and cross-presentation of dendritic cells, leading to an improved immunotherapy efficacy against hepatocellular carcinoma.

Background: The enrichment of Fusobacterium nucleatum (Fn) has been identified in CRC patients and associated with worse outcomes. However, whether Fn was involved in the metastasis of CRC was not well determined. Methods: CRC cell lines were incubated with Fn and the migration ability was analyzed by transwell and would healing assays. Fn-treated or untreated CRC cells were intravenously injected into nude mice to evaluate the lung metastasis. Fluorescence in situ hybridization was performed to detect the colonization of Fn. RNA sequencing and validation studies were performed to identify the mechanism by which Fn promote CRC metastasis. Findings::The abundance of Fn was significantly increased in CRC patients with lymph nodes metastasis.Fn infection promoted CRC cells migration in vitro, as well as lung metastasis in vivo. Colonization of Fn was detected in metastatic lung lesions of nude mice. RNA sequencing revealed that Fn significantly upregulated the expression of long non-coding RNA Keratin7-antisense (KRT7-AS) and Keratin7 (KRT7) in CRC cells. Fn-induced CRC lung metastasis was attenuated by the depletion of KRT7-AS. In addition, KRT7-AS facilitated CRC cells migration by upregulating KRT7. Fn upregulated KRT7-AS by activating NF-κB signaling pathway. Importantly, the expression of KRT7-AS was positively correlated with the abundance of Fn in CRC tissues. Interpretation: Fn infection activated NF-κB pathway, leading to increased expression of KRT7-AS/KRT7, which promoted CRC cell migration in vitro and metastasis in vivo. Funding Statement: This work was supported by National Key R&D Program of China(2016YFC0107003 and 2016YFC1303200) , the National Natural Science Foundation of China (81702308). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: All animal procedures were performed in compliance with ethical standards and approved by the Animal Care Committee of Zhejiang University.

Testing is the last step to check the correctness of a software system. However, due to its incompleteness, we still do not know if the implementation behavior matches the design behavior. In this paper, we provide a new solution for web applications. Since web application has special navigation character, we can get rich information in the log file after testing. From such log file, we extract the Petri net based behavior of the web application. The behavior is then checked by SPIN. Market Information System has been adopted as the case study.