Role of exhaled hydrogen sulfide in the diagnosis of colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is often accompanied by increased excretion of hydrogen sulfide (H2S). This study aimed to explore the value of exhaled H2S in the diagnosis of CRC. METHODS: A total of 80 people with normal colonoscopy results and 57 patients with CRC were enrolled into the present observational cohort study. Exhaled oral and nasal H2S were detected by Nanocoulomb breath analyser. Results were compared between the two groups. Receiver operating characteristic (ROC) curves were analysed and area under the curves (AUCs) were calculated to assess the diagnostic value of exhaled H2S. Meanwhile, the clinicopathological features, including gender, lesion location and tumour staging of patients with CRC, were also collected and analysed. RESULTS: The amount of exhaled H2S from patients with CRC was significantly higher than that of those with normal colonoscopy results. The ROC curve showed an AUC value of 0.73 and 0.71 based on oral and nasal H2S detection, respectively. The exhaled H2S in patients with CRC was correlated with gender, lesion location and tumour progression, including depth of invasion, lymphatic metastasis and TNM (Tumor, Lymph Nodes, Metastasis) staging. CONCLUSION: Exhaled H2S analysis is a convenient and non-invasive detection method for diagnosing CRC, suggesting a potential role in population screening for CRC.

Establishment of a large-scale patient-derived high-risk colorectal adenoma organoid biobank for high-throughput and high-content drug screening.

BACKGROUND: Colorectal adenoma (CA), especially high-risk CA (HRCA), is a precancerous lesion with high prevalence and recurrence rate and accounts for about 90% incidence of sporadic colorectal cancer cases worldwide. Currently, recurrent CA can only be treated with repeated invasive polypectomies, while safe and promising pharmaceutical invention strategies are still missing due to the lack of reliable in vitro model for CA-related drug screening. METHODS: We have established a large-scale patient-derived high-risk colorectal adenoma organoid (HRCA-PDO) biobank containing 37 PDO lines derived from 33 patients and then conducted a series of high-throughput and high-content HRCA drug screening. RESULTS: We established the primary culture system with the non-WNT3a medium which highly improved the purity while maintained the viability of HRCA-PDOs. We also proved that the HRCA-PDOs replicated the histological features, cellular diversity, genetic mutations, and molecular characteristics of the primary adenomas. Especially, we identified the dysregulated stem genes including LGR5, c-Myc, and OLFM4 as the markers of adenoma, which are well preserved in HRCA-PDOs. Based on the HRCA-PDO biobank, a customized 139 compound library was applied for drug screening. Four drugs including metformin, BMS754807, panobinostat and AT9283 were screened out as potential hits with generally consistent inhibitory efficacy on HRCA-PDOs. As a representative, metformin was discovered to hinder HRCA-PDO growth in vitro and in vivo by restricting the stemness maintenance. CONCLUSIONS: This study established a promising HRCA-PDO biobank and conducted the first high-throughput and high-content HRCA drug screening in order to shed light on the prevention of colorectal cancer.

Generational effects and abnormalities in craniofacial chondrogenesis in zebrafish (Danio rerio) embryos upon maternal exposure to estrogen endocrine disrupting chemicals.

Bisphenol A (BPA) and diethyl phthalate (DEP) are estrogenic endocrine disrupting chemicals (EEDCs). The present study reconfirmed that the angle of the ceratohyal cartilage (CH) in embryos were larger from maternal BPA and E2, but smaller from DEP compared to the control. However, it is still unknown whether both the BPA and DEP chemicals disrupted the action of E2 and thereby influence the estrogen signaling pathways. Additionally, it remains unclear whether they also disrupted certain related genes in the migratory pathways of neural crest cells (NCCs) in their offspring. The present data showed that nuclear estrogen receptors and membrane estrogen receptors have different disrupted profiles among female zebrafish exposed to BPA (F-BPA), and DEP (F-DEP), and external E2 (F-E2). However, certain related genes in the migratory pathways of NCCs in embryos from F-BPA and F-E2 such as the sox10, chm1, and tgfbr1a mRNA expressions showed a positive relationship compared with CH angles; the gene expressions of sox9a, smad3, and col2a1a and the CH angles of embryos exhibited an opposite relationship upon F-DEP treatments. Thus, we suggested that the genes involved in NCCs migration were potentially induced by the residual maternal DEP contents. Two sets of genes, chm1/tgfb3 and chm1/gper1, exhibited an identical profile in the ovary and its offspring at 2 h of post fertilization upon F-E2 and F-BPA treatments, respectively. We suggested that the maternal mRNA from female to embryos were transferred before the maternal-to-zygotic transition stage.

A precise molecular subtyping of ulcerative colitis reveals the immune heterogeneity and predicts clinical drug responses.

BACKGROUND AND AIMS: We sought to identify novel molecular subtypes of ulcerative colitis (UC) based on large-scale cohorts and establish a clinically applicable subtyping system for the precision treatment of the disease. METHODS: Eight microarray profiles containing colon samples from 357 patients were utilized. Expression heterogeneity was screened out and stable subtypes were identified among UC patients. Immune infiltration pattern and biological agent response were compared among subtypes to assess the value in guiding treatment. The relationship between PRLR and TNFSF13B genes with the highest predictive value was further validated by functional experiments. RESULTS: Three stable molecular subtypes were successfully identified. Immune cell infiltration analysis defined three subtypes as innate immune activated UC (IIA), whole immune activated UC (WIA), and immune homeostasis like UC (IHL). Notably, the response rate towards biological agents (infliximab/vedolizumab) in WIA patients was the lowest (less than 10%), while the response rate in IHL patients was the highest, ranging from 42 to 60%. Among the featured genes of subtypes, the ratio of PRLR to TNFSF13B could effectively screen for IHL UC subtype suitable for biological agent therapies (Area under curve: 0.961-0.986). Furthermore, we demonstrated that PRLR expressed in epithelial cells could inhibit the expression of TNFSF13B in monocyte-derived macrophages through the CXCL1-NF-κB pathway. CONCLUSIONS: We identified three stable UC subtypes with a heterogeneous immune pattern and different response rates towards biological agents for the first time. We also established a precise molecular subtyping system and classifier to predict clinical drug response and provide individualized treatment strategies for UC patients.

Six immune-related promising biomarkers may promote hepatocellular carcinoma prognosis: a bioinformatics analysis and experimental validation.

BACKGROUND: Abnormal miRNA and mRNA expression and dysregulated immune microenvironment have been found to frequently induce the progression of hepatocellular carcinoma (HCC) in recent reports. In particular, the immune-related competing endogenous RNAs (ceRNA) mechanism plays a crucial role in HCC progression. However, the underlying mechanisms remain unclear. METHODS: Differentially expressed immune-related genes were obtained from the Immport, GEO, and TCGA databases. The mRNA and protein expression levels in HCC tissues and adjacent normal tissues were confirmed, and we further investigated the methylation levels of these biomarkers to explore their function. Then, the TIMER and TISCH databases were used to assess the relationship between immune infiltration and hub genes. Survival analysis and univariate and multivariate Cox models were used to evaluate the association between hub genes and HCC diagnosis. Hub gene expression was experimentally validated in six HCC cell lines and 15 HCC samples using qRT-PCR and immunohistochemistry. The hub genes were uploaded to DSigDB for drug prediction enrichment analysis. RESULTS: We identified that patients with abnormal miRNAs (hsa-miR-125b-5p and hsa-miR-21-5p) and their targeted genes (NTF3, PSMD14, CD320, and SORT1) had a worse prognosis. Methylation analysis of miRNA-targeted genes suggested that alteration of methylation levels is also a factor in the induction of tumorigenesis. We also found that the development of HCC progression caused by miRNA-mRNA interactions may be closely correlated with the infiltration of immunocytes. Moreover, the GSEA, GO, and KEGG analysis suggested that several common immune-related biological processes and pathways were related to miRNA-targeted genes. The results of qRT-PCR, immunohistochemistry, and western blotting were consistent with our bioinformatics results, suggesting that abnormal miRNAs and their targeted genes may affect HCC progression. CONCLUSIONS: Briefly, our study systematically describes the mechanisms of miRNA-mRNA interactions in HCC and predicts promising biomarkers that are associated with immune filtration for HCC progression.

Reproductive and Developmental Alterations on Zebrafish (Danio rerio) Upon Long-Term Exposure of Di-ethyl phthalate (DEP), Di-isononyl phthalate (DINP) and Di (2-ethylhexyl) phthalate (DEHP).

Di (2-ethylhexyl) phthalate (DEHP), di-ethyl phthalate (DEP) and di-isononyl phthalate (DINP) are all endocrine disrupting chemicals (EDCs) for organisms. However, little research has been done on the effects of long-term EDC exposure. The present study found that the zebrafish barely grew during the 7 months of DINP exposure. The fecundity rate (%) of female spawning was lower in the DEHP treatment by 4 months compared to other exposure groups. Zebrafish treated with 12.5-25.0 ppm of DEP for 4 months presented no spawning. Gonadal-somatic index (GSI) levels significantly decreased, and there were more oocytes in the atresia and peri-nucleus stage compared to the control group. In addition, the hatching rate of embryos were 71.02%, 56.92%, and 21.70% for females treated with DINP, DEHP and DEP, respectively. There were also abnormal craniofacial chondrogenesis development on 72 hpf embryos upon females treated with the three EDCs. In conclusion, long term exposure of DEHP, DINP, and DEP did not only affect the reproductive capacity of female zebrafish, but the 3 plasticizers also influence craniofacial cartilage development of its offspring.

Effect of different oesophagojejunostomy methods on the quality of life of gastric cancer patients after totally laparoscopic total gastrectomy with self-pulling and latter transected technique: study protocol for a randomised trial.

INTRODUCTION: Gastric cancer is the fifth most common cancer worldwide and the detection rate of proximal gastric cancer has been increasing. Currently, surgical resection using gastrectomy and proper perigastric lymphadenectomy is the only treatment option to enhance the survival rate of patients with gastric cancer. Laparoscopic total gastrectomy (LTG) is increasingly performed for adenocarcinoma of the oesophagogastric junction. However, totally LTG (TLTG) is only performed by a few surgeons due to difficulty associated with oesophagojejunostomy (OJ), in which there is no consensus on a standardised anastomosis technique. We propose a randomised trial to compare functional end-to-end anastomosis (FETE) and side-to-side anastomosis (Overlap) for OJ. METHODS AND ANALYSIS: A prospective, randomised, open-label, single-centre, interventional trial has been designed to evaluate the quality of life (QoL) outcomes and safety of FETE and Overlap, with a 1-year follow-up as the primary endpoint. The trial began in 2020 and is scheduled to enrol 96 patients according to a previous sample size calculation. Patients were randomly allocated to the FETE or Overlap groups with a follow-up of 1 year to assess QoL after the procedure. All relevant clinical data including biological markers were collected. The primary indicator is the D-value between the postoperative and preoperative QoL. Student's t-tests will be used to compare continuous variables, while χ2 tests or Fisher's exact tests will be used to compare categorical variables. Statistical analysis will be performed with SPSS V.23.0 statistical software. A p<0.05 will be considered statistically significant. ETHICS AND DISSEMINATION: This study has been approved by the Hospital Institutional Review Board of Huashan Hospital, Fudan University (2020-1055). The results will be submitted for publication in peer-reviewed journals. TRIAL REGISTRATION NUMBER: ChiCTR2000035583.

Gut Microbiota and Immune Checkpoint Inhibitors-Based Immunotherapy.

Application of immune checkpoint inhibitors (ICIs) is a major breakthrough in the field of cancer therapy, which has displayed tremendous potential in various types of malignancies. However, their response rates range widely in different cancer types and a significant number of patients experience immune-related adverse effects (irAEs) induced by these drugs, limiting the proportion of patients who can truly benefit from ICIs. Gut microbiota has gained increasing attention due to its emerging role in regulating the immune system. In recent years, numerous studies have shown that gut microbiota can modulate antitumor response, as well as decrease the risk of colitis due to ICIs in patients receiving immunotherapy. The present review analyzed recent progress of relevant basic and clinical studies in this area and explored new perspectives to enhance the efficacy of ICIs and alleviate associated irAEs via manipulation of the gut microbiota.

Maternal effect and dietary supplementation of estradiol-17ß on female zebrafish (Danio rerio) affects the swimming behavior and stress-coping styles of its offspring.

Extrinsic estradiol-17ß (E2) is an environmental hormone. Female fish exposure to waterborne E2 might affect the development of craniofacial cartilage of its offspring. The present study investigates the effects of maternal E2 on larval craniofacial cartilage development by administering oral feed containing E2 (F-E2) to female zebrafish, and examines whether the swimming behavior and their stress coping style are influenced by maternal E2. The results showed that E2 contents responded to dosage and time in male fish after being fed with a diet containing E2. In addition, the E2 contents in female ovaries showed a significant increase after 250 mg of E2/kg treatment for 14 d. On the other hand, the fecundity rate of F-E2 group was lower around 2 folds than FC (female fed 0 mg of E2/kg) group. Craniofacial chondrogenesis on 72 hpf (hours of post fertilization) of F-E2 larvae were abnormalities, and a recovery to a normal developmental pattern was observed at the 96 hpf stage. The swimming speed was slower for F-E2 larvae compared to the FC larvae; and the F-E2 juvenile seems to be less responsive to cortisol (LRC) after cold stress. According to the results, we suggested that F-E2 larvae might have worse environmental adaptability than FC larvae.

Machine Learning Model in Predicting Sarcopenia in Crohn's Disease Based on Simple Clinical and Anthropometric Measures.

Sarcopenia is associated with increased morbidity and mortality in Crohn's disease. The present study is aimed at investigating the different diagnostic performance of different machine learning models in identifying sarcopenia in Crohn's disease. Patients diagnosed with Crohn's disease at our center provided clinical, anthropometric, and radiological data. The cross-sectional CT slice at L3 was used for segmentation and the calculation of body composition. The prevalence of sarcopenia was calculated, and the clinical parameters were compared. A total of 167 patients were included in the present study, of which 127 (76.0%) were male and 40 (24.0%) were female, with an average age of 36.1 ± 14.3 years old. Based on the previously defined cut-off value of sarcopenia, 118 (70.7%) patients had sarcopenia. Seven machine learning models were trained with the randomly allocated training cohort (80%) then evaluated on the validation cohort (20%). A comprehensive comparison showed that LightGBM was the most ideal diagnostic model, with an AUC of 0.933, AUCPR of 0.970, sensitivity of 72.7%, and specificity of 87.0%. The LightGBM model may facilitate a population management strategy with early identification of sarcopenia in Crohn's disease, while providing guidance for nutritional support and an alternative surveillance modality for long-term patient follow-up.

The Role of Exhaled Hydrogen Sulfide in the Diagnosis of Colorectal Adenoma.

PURPOSE: Exhaled determination can detect metabolite hydrogen sulfide in the intestine. We aim to analyze the predictive value of hydrogen sulfide in the diagnosis of colorectal adenoma. METHODS: We recruited seventy patients diagnosed with colorectal adenoma as the observation group and sixty-six healthy subjects as the control group. The colorectal adenoma was diagnosed by colonoscopy at the Endoscopy Center of Huashan Hospital affiliated to Fudan University from June 2018 to November 2019. Exhaled gas was collected through the nose and mouth, respectively, and hydrogen sulfide in exhaled gas was determined according to the manufacturer's instructions. RESULTS: Receiver operating characteristic (ROC) curve was analyzed based on the exhaled data of the observation group and the control group. The ROC curve showed an area under ROC curve (AUC) 0.724 for nasal exhaled H2S, which had a diagnostic value. When nasal exhaled H2S was >13.3 part per billion (ppb), the sensitivity and the specificity of predicting colorectal adenoma were 57% and 78%, respectively. The exhaled H2S of the observation group was significantly different from that of the control group. The AUC value was 0.716 as a prognostic factor of colorectal adenoma. As exhaled H2S was >28.8 ppb, the sensitivity and the specificity of predicting colorectal adenoma were 63% and 77%, respectively. CONCLUSION: Exhaled and nasal H2S determination has a predictive value for colorectal adenoma as a novel and noninvasive method. Therefore, it is worth conducting more research to analyze exhaled and nasal H2S.

CBX2 Expression in Colorectal Mucosa-adenoma-adenocarcinoma Sequence.

OBJECTIVE: To investigate the expression of chromobox 2 (CBX2) in colorectal adenoma (CRA) and colorectal cancer (CRC), and analyse its correlation with various clinicopathological parameters. STUDY DESIGN: Observational study. PLACE AND DURATION OF STUDY: Pathology Department, Huashan Hospital, Fudan University, from December 2019 to December 2020. METHODOLOGY: The mRNA level of CBX2 in colorectal mucosa, CRA and CRC was evaluated in gene expression profiling interactive analysis (GEPIA) and gene expression omnibus (GEO) dataset, then verified by quantitative real-time PCR (qRT-PCR). CBX2 expression by immunohistochemistry was determined in 122 samples, then its correlation was analysed with various clinicopathological parameters. Diagnostic value of CBX2 was estimated by the receiver operating characteristic curve (ROC). Prognostic value of CBX2 mRNA expression was evaluated via the Kaplan-Meier method in GEPIA. RESULTS: CBX2 expression rate in CRC (89.8%) was greater than adenoma (37.74%) and mucosa (20%). CBX2 protein levels were highest in adenocarcinoma and lowest in mucosa with intermediate level in adenoma. The area under curve (AUC) of CBX2 was 0.810 and 0.734, respectively, in distinguishing CRA from mucosa and CRC from CRA. CBX2 hyperexpression was not significantly correlated with clinicopathological variables, either in CRA or CRC. Kaplan-Meier survival analysis revealed that CBX2 mRNA hyperexpression was not associated with overall survival (OS) of CRC. Although the disease-free survival (DFS) of high CBX2 patients was shorter than those with low expression, but no obvious significance was found in colon adenocarcinoma (COAD, p=0.052) and rectal adenocarcinoma (READ, p=0.097). CONCLUSION: CBX2 expression progressively increased in the sequence of mucosa-adenoma-carcinoma, which may be used as a diagnostic biomarker and therapeutic target for CRA and CRC. Key Words: CBX2, Colorectal adenoma, Colorectal cancer, Biomarker.

Distinguishing colorectal adenoma from hyperplastic polyp by WNT2 expression.

BACKGROUND: Colorectal adenoma (CRA) is a classical premalignant lesion, with high incidence and mainly coexisting with hyperplastic polyp (HPP). Hence, this study aimed to distinguish CRA from HPP by molecular expression profiling and advance the prevention of CRA and its malignance. METHODS: CRA and paired HPP biopsies were collected by endoscopy. Through RNA-sequencing (RNA-seq), the differentially expressed genes (DEGs) were obtained. Functional enrichment analysis was performed based on the DEGs. The STRING database and Cytoscape were used to construct the protein-protein interaction (PPI) network and perform module analysis. Hub genes were validated by real-time quantitative PCR (RT-qPCR) and immunohistochemistry. The ROC curve was drawn to establish the specificity of the hub genes. RESULTS: 485 significant DEGs were identified including 133 up-regulated and 352 down-regulated. The top 10 up-regulated genes were DLX5, MMP10, TAC1, ACAN, TAS2R38, WNT2, PHYHIPL, DKK4, DUSP27, and ABCA12. The top 10 down-regulated genes were SFRP2, CHRDL1, KBTBD12, RERGL, DPP10, CLCA4, GREM2, TMIGD1, FEV, and OTOP3. Wnt signaling pathway and extracellular matrix (ECM) were up-regulated in CRA. Three hub genes including WNT2, WNT5A, and SFRP1 were filtered out via Cytoscape. Further RT-qPCR and immunohistochemistry confirmed that WNT2 was highly expressed in CRA. The area under the ROC curve (AUC) at 0.98 indicated the expression level of WNT2 as a candidate to differ CRA from HPP. CONCLUSION: Our study suggests Wnt signaling pathway and ECM are enriched in CRA, and WNT2 may be used as a novel biomarker for distinguishing CRA from HPP and preventing the malignance of CRA.

Effects of bisphenol A or diethyl phthalate on cartilage development and the swimming behavior of zebrafish (Danio rerio) through maternal exposure.

Waterborne bisphenol A (BPA) and diethyl phthalate (DEP) are endocrine disruptive chemicals that impact the reproductive system of fish. The present study checks the effectiveness of the reproductive capacity on zebrafish after BPA and DEP exposure, and consequently investigates its effect on their development and the swimming behavior of its offspring. The exposure of BPA and DEP to zebrafish reveals that the levels of ovarian 17ß-estradiol (E2) and relative mRNA expression (RRE) ratios (Treatment/Control) of hepatic vitellogenin (vtg1) could be induced and decreased. Liver RRE levels in estrogen receptors (ERs) are also affected. Among the ERs, esr2a significantly increased upon BPA exposure, and esr1 and esr2b decreased upon DEP exposure. In addition, the ceratohyal cartilage (CH) angle of larvae whose mothers were exposed to BPA (F-BPA) was significantly bigger, but the CH angle of larvae whose mothers were exposed to DEP (F-DEP) was significantly smaller than the control. The swimming performance of larvae from F-DEP was more compromised than the control, but the situation did not appear in the larvae from the F-BPA group. The success rate of larvae hatching from F-BPA and F-DEP was lower than control group. Moreover, the successful rate of female spawns was higher in the control group compared to the treatment groups exposed to BPA and DEP. We suggested that both maternal BPA and DEP disrupt E2 levels, and influence the CH development of larvae, resulting in a decrease in successful hatching. Only the swimming behavior of larvae from maternal DEP was disrupted.

Inhibition of SPATS2 Suppresses Proliferation and Invasion of Hepatocellular Carcinoma Cells through TRIM44-STAT3 Signaling Pathway.

Hepatocellular carcinoma (HCC) is a major global health burden and its treatment options are limited. Spermatogenesis associated serine rich 2(SPATS2), a recent defined oncogene, was found to be a prognostic biomarker in HCC. However, the explicit mechanism underlying SPATS2 was urged to be elucidated. In vitro, knockdown of SPATS2 hampered the proliferation, invasion and migration of HCC cells. Moreover, phosphorylation of signal transducer and activator of transcription 3 (STAT3) and its downstream oncogenes were dramatically suppressed by SPATS2 knockdown. In addition, tripartite motif containing 44 (TRIM44) was found to be positively associated with SPATS2 in TCGA and declined after SPATS2 knockdown in HCC cells. Overexpression of TRIM44 rescued the effect of SPATS2 silencing on p-STAT3 and its downstream oncogenes. In vivo, SPATS2 silencing was confirmed to impede HCC tumor development in nude mice. In our own cohort containing 112 HCC patients, high SPATS2 protein level is indicative of an unfavorable clinicopathological feature and poor prognosis and could serve as an independent risk factor. Collectively, the present study is the first to propose the mechanism of significance of SPATS2-TRIM44-p-STAT3 in HCC and provide a new theoretical basis for targeted therapy.

Asymptomatic Hyperamylasemia in Stevens-Johnson Syndrome Is Associated with Intestinal Barrier Dysfunction.

METHODS: A retrospective study on SJS patients was conducted at a tertiary medical center. All patients diagnosed as SJS, with available serum amylase index, were included. Clinical data of all subjects were retrospectively collected and analyzed. Colonic mucosal biopsies were obtained to measure tight junction protein expression. RESULTS: A total of nine patients were included in the present study for study analysis. The average serum amylase of the study cohort was 228.78 ± 204.18 U/L. Among which, five patients had a positive fecal occult blood test (FOBT). Colonic mucosal biopsies were obtained and stained with occludin and zonula occludens-1 (ZO-1). The expression of occludin and ZO-1 was significantly downregulated in SJS patients (p < 0.01), which was indicative of intestinal barrier dysfunction. CONCLUSION: Hyperamylasemia often extends beyond pancreatic diseases. Clinical awareness of asymptomatic hyperamylasemia secondary to other systemic diseases can help avoid unnecessary overexamination and overtreatment.