Regulation of RAS palmitoyltransferases by accessory proteins and palmitoylation.

Palmitoylation of cysteine residues at the C-terminal hypervariable regions in human HRAS and NRAS, which is necessary for RAS signaling, is catalyzed by the acyltransferase DHHC9 in complex with its accessory protein GCP16. The molecular basis for the acyltransferase activity and the regulation of DHHC9 by GCP16 is not clear. Here we report the cryo-electron microscopy structures of the human DHHC9-GCP16 complex and its yeast counterpart-the Erf2-Erf4 complex, demonstrating that GCP16 and Erf4 are not directly involved in the catalytic process but stabilize the architecture of DHHC9 and Erf2, respectively. We found that a phospholipid binding to an arginine-rich region of DHHC9 and palmitoylation on three residues (C24, C25 and C288) were essential for the catalytic activity of the DHHC9-GCP16 complex. Moreover, we showed that GCP16 also formed complexes with DHHC14 and DHHC18 to catalyze RAS palmitoylation. These findings provide insights into the regulatory mechanism of RAS palmitoyltransferases.

Electroacupuncture-modulated extracellular ATP levels in prefrontal cortex ameliorated depressive-like behavior of maternal separation rats.

Maternal separation (MS) is a type of early-life stress that has been linked to neuropsychiatric disorders, especially depression. Increasing evidence indicates that the adenosine triphosphate (ATP) level in the prefrontal cortex (PFC) is involved in the pathophysiology of depression. To investigate the potential relationship between ATP in PFC and antidepressant effects of electroacupuncture (EA) treatment, we assessed genes involved in ATP biosynthesis as well as the extracellular ATP levels in a rat model exposed to neonatal MS. Our results demonstrated that reduced expression of ABCG2 (an ATP-binding cassette protein) and ATP levels in the PFC of depressive-like rats exposed to MS can be attenuated by EA stimulus at the Baihui (GV20) and Yintang (GV29) acupoints. Moreover, the antidepressant effect of EA treatment was blocked by administration of suramin, a broad purinergic P2 receptor antagonist. Together, these results suggested that electroacupuncture may be able to modulate extracellular ATP levels in the PFC of depressive-like MS rats, potentially contributing to its antidepressant effects.

Predictive Value of the Neutrophil-to-Lymphocyte Ratio, Platelet-to-Lymphocyte Ratio, and Monocyte-to-Lymphocyte Ratio Esophageal Stricture After Endoscopic Submucosal Dissection of Early Esophageal Cancer.

BACKGROUND Endoscopic submucosal dissection (ESD) has become a preferred treatment method for patients with early esophageal cancer (EEC), but it can easily be complicated by esophageal stricture. In this study, we aimed to analyze the predictive value of neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and monocyte-to-lymphocyte ratio (MLR) for post-ESD esophageal stricture of EEC. MATERIAL AND METHODS A retrospective study of 285 patients with EEC who underwent ESD was conducted. Patients were divided into 2 groups according to whether there were complications of esophageal stricture: the stricture group (n=72) and the non-stricture group (n=213). A t test or chi-squared test was used to compare the clinical differences in different subgroups. Receiver operating characteristic (ROC) curves were plotted to determine and compare the predictive value of NLR, PLR, and MLR in post-ESD esophageal stricture. Spearman correlation was used to detect the relationship between NLR, PLR, and MLR and the severity of esophageal stricture. RESULTS The NLR, PLR, and MLR values in the stricture group were higher than those in the non-stricture group, and there was a positive correlation between NLR and MLR and the severity of stricture according to the Spearman correlation test (P<0.05). ROC analysis showed that the area under the ROC curve value of the combination of NLR, PLR, and MLR (0.850) was higher than the NLR (0.792), PLR (0.774), and MLR (0.768). CONCLUSIONS The combination of NLR, PLR, and MLR could help clinicians to predict post-ESD esophageal stricture in the early stage.

[Analysis of clinical features and genetic variant in a Chinese pedigree affected with familial adenomatous polyposis].

OBJECTIVE: To analyze the clinical features and genetic basis for a Chinese pedigree affected with familial adenomatous polyposis (FAP). METHODS: Clinical information of the patient was collected. Genomic DNA was extracted from peripheral blood sample of the patient and subjected to whole exome sequencing (WES). Candidate variant was verified by Sanger sequencing. RESULTS: The proband, a 33-year-old female, was found to have multiple adenomatous polyps in the intestine. WES revealed that she has harbored a heterozygous variant of the APC gene, namely c.1922dupA (p.N641fs*10), which was unreported previously. Based on the guidelines of the American College of Medical Genetics and Genomics, the variant was predicted to be likely pathogenic. CONCLUSION: The c.1922dupA (p.N641fs*10) variant of the APC gene probably underlay the FAP in this pedigree. Above finding has enabled genetic counseling for this family.

Mitigating gas emissions from poultry litter composting with waste vinegar residue.

The composting process is important in the recycling of organic wastes produced in agriculture, food, and municipal waste management. This study explored the suitability of using waste vinegar residue (WVR) as an amendment in poultry litter (PL) composting. Four treatments, including poultry litter (CK), poultry litter+vinegar residue (VR), poultry litter+vinegar residue+lime (VR_Ca) and poultry litter+vinegar residue+biochar (VR_B), were conducted. During a 42-day composting period, the dynamics of carbon dioxide (CO2), ammonia (NH3), nitrous oxide (N2O) and methane (CH4) emissions, as well as the physicochemical properties and abundances of the bacteria and fungi of the feedstock were tracked to examine the potential barriers in the co-composting of WVR and PL. Compared to those of the CK, using a WVR amendment lowered the pH, increased the electrical conductivity significantly at the early stage, resulted in a strong inhibition of bacterial and fungal growth and delayed the thermophilic period of poultry litter composting while significantly reducing NH3 and N2O and GHG (CO2-e) emissions. A preadjustment of the WVR with alkaline biochar or lime lengthened the thermophilic period and increased the germination index (GI) by alleviating the inhibitory effect of the WVR on bacterial and fungal growth during composting. However, such preadjustment might reduce the mitigation effect on NH3. In conclusion, WVR can be recycled through co-composting with poultry litter, and the additional mitigation of N losses and N conservation can be achieved without halting compost quality.

Network Pharmacology-Based Study on the Mechanism of Gegen Qinlian Decoction against Colorectal Cancer.

PURPOSE: Gegen Qinlian decoction (GQD) has been used to treat gastrointestinal diseases, such as diarrhea and ulcerative colitis (UC). A recent study demonstrated that GQD enhanced the effect of PD-1 blockade in colorectal cancer (CRC). This study used network pharmacology analysis to investigate the mechanisms of GQD as a potential therapeutic approach against CRC. MATERIALS AND METHODS: Bioactive chemical ingredients (BCIs) of GQD were collected from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. CRC-specific genes were obtained using the gene expression profile GSE110224 from the Gene Expression Omnibus (GEO) database. Target genes related to BCIs of GQD were then screened out. The GQD-CRC ingredient-target pharmacology network was constructed and visualized using Cytoscape software. A protein-protein interaction (PPI) network was subsequently constructed and analyzed with BisoGenet and CytoNCA plug-in in Cytoscape. Gene Ontology (GO) functional and the Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analysis for target genes were then performed using the R package of clusterProfiler. RESULTS: One hundred and eighteen BCIs were determined to be effective on CRC, including quercetin, wogonin, and baicalein. Twenty corresponding target genes were screened out including PTGS2, CCNB1, and SPP1. Among these genes, CCNB1 and SPP1 were identified as crucial to the PPI network. A total of 212 GO terms and 6 KEGG pathways were enriched for target genes. Functional analysis indicated that these targets were closely related to pathophysiological processes and pathways such as biosynthetic and metabolic processes of prostaglandins and prostanoids, cytokine and chemokine activities, and the IL-17, TNF, Toll-like receptor, and nuclear factor-kappa B (NF-κB) signaling pathways. CONCLUSION: The study elucidated the "multiingredient, multitarget, and multipathway" mechanisms of GQD against CRC from a systemic perspective, indicating GQD to be a candidate therapy for CRC treatment.

miR­205 suppresses cell migration, invasion and EMT of colon cancer by targeting mouse double minute 4.

Colon cancer is one of the most frequent malignant tumors, and microRNA (miR)­205 is involved in the tumor progression. The present study aimed to explore the effects of miR­205 on human colon cancer and its targeting mechanism. The levels of miR­205 and mouse double minute 4 (MDM4) were determined via reverse transcription­quantitative (RT­q)PCR and western blot analysis. A luciferase activity assay was performed to analyze the association between miR­205 and MDM4. Cell viability, migration and invasion were determined via Cell Counting Kit­8, wound healing and Transwell assays, respectively. The levels of epithelial­mesenchymal transition (EMT)­associated factors were determined by RT­qPCR and western blot analysis. It was identified that MDM4 was overexpressed in colon cancer tissues and cells, and that there was a negative correlation between miR­205 and MDM4 expression in colon cancer. Similarly, miR­205 inhibited MDM4 expression by binding to its 3'untranslated region. in addition, miR­205 directly targeted MDM4, accompanied by suppressed proliferation, migration and invasion of HCT116 cells. EMT processes were suppressed in miR­205­overexpressed cells; upregulation of E­cadherin, and downregulation of N­cadherin, vimentin, matrix metalloproteinase (MMP)2 and MMP9 were observed. Collectively, miR­205 conspicuously depressed the viability, migration, invasion and EMT process of human colon cancer cells via targeting MDM4. miR­205 could be potentially used in the treatment of human colon cancer.

MicroRNA-214 promotes the proliferation, migration and invasion of gastric cancer MKN28 cells by suppressing the expression of Dact2.

The present study examined the expression of Dapper, antagonist of ß-catenin 2 (Dact2) and microRNA (miR)-214 in gastric cancer at tissue and cellular levels, and to understand their biological roles. A total of 42 gastric cancer patients were enrolled in the present study. Bioinformatics tool was used to predict the miR molecule that potentially regulates Dact2 expression. To measure the expression of miR-214 and Dact2, reverse transcription-quantitative polymerase chain reaction was employed. Mixed gastric adenocarcinoma type MKN28 cells were transfected with negative control (NC), miR-214 mimics or inhibitor. The CCK-8 assay was used to investigate the proliferation of mixed gastric adenocarcinoma type MKN28 cells. To study migration and invasion abilities of mixed gastric adenocarcinoma type MKN28 cells, the Transwell assay was performed. To determine the expression of Dact2 protein, western blotting was conducted and the rescue assay was utilized to study the biological roles of miR-214 and Dact2 in mixed gastric adenocarcinoma type MKN28 cells. To test whether Dact2 is a direct target of miR-214, the dual luciferase reporter assay was performed. Results indicated that the expression of miR-214 was elevated, but expression of Dact2 mRNA was decreased in gastric cancer tissues, which was closely correlated with the invasion, metastasis, occurrence and development of gastric cancer. Notably, miR-214 promoted the proliferation of mixed gastric adenocarcinoma type MKN28 cells in vitro, whereas but Dact2 inhibited the proliferation of these cells. Downregulation of miR-214 expression or upregulation of Dact2 expression inhibited the migration and invasion of mixed gastric adenocarcinoma type MKN28 cells. Furthermore, miR-214 regulated the expression of Dact2 protein and its downstream ß-catenin protein in mixed gastric adenocarcinoma type MKN28 cells. Dact2 reversed the effect of miR-214 on the proliferation, migration and invasion of mixed gastric adenocarcinoma type MKN28 cells. In addition, miR-214 directly targeted the 3'-UTR seeding region of Dact2 mRNA to regulate its expression. The present study demonstrated that expression of miR-214 was upregulated in gastric cancer tissues, and positively correlated with lymphatic metastasis and clinical staging. In addition, expression of Dact2 was downregulated in gastric cancer tissues and negatively correlated with lymphatic metastasis and clinical staging. Notably, the present findings suggest that miR-214 promoted the proliferation, migration and invasion of mixed gastric adenocarcinoma type MKN28 cells by suppressing the expression of Dact2.

Bortezomib protects against dextran sulfate sodium­induced ulcerative colitis in mice.

Bortezomib, a first­in­class proteasome inhibitor, is a standard method of treatment in multiple myeloma. In the present study, the therapeutic effect of bortezomib was evaluated in an ulcerative colitis model induced by dextran sulfate sodium (DSS) in mice, and the mechanism of action was also investigated. Mice were administered with 3% DSS drinking water for 7 consecutive days and then they were intraperitoneally treated with bortezomib (0.2, 0.6 or 1 mg/kg) for 1, 3 or 7 days. Mice in the control group and the DSS group were provided the same volume of PBS, respectively. Body weight, stool characteristics and hematochezia were observed. Serum levels of tumor necrosis factor­α (TNF­α), C­reactive protein (CRP), albumin (ALB) and colonic activity of superoxide dismutase (SOD) were evaluated using specific kits. The expression of the transcription factor nuclear factor­κB (NF­κB) p65 gene and the DNA­binding activity of NF­κB protein were also evaluated. Administration of bortezomib attenuates colonic inflammation in mice. After 3 or 7 days of treatment, Disease Activity Index (DAI) as well as histological scores and NF­κB p65 protein expression were significantly reduced in mice treated with bortezomib at a dose of 0.6 or 1 mg/kg/day. Furthermore, it was also revealed that bortezomib was able to reduce serum levels of CRP and TNF­α caused by DSS and increase the level of ALB in serum and the activity of SOD in colonic tissues. These results demonstrated that bortezomib exerts a protective effect on DSS­induced colitis, and its underlying mechanisms are associated with the NF­κB gene inhibition that mitigates colon inflammatory responses in intestinal epithelial cells.

Expression of Toll-like receptors in the mucosa of patients with ulcerative colitis.

Patients with ulcerative colitis (UC) have a high risk of developing colorectal cancer. The aim of the present study was to evaluate the expression pattern of Toll-like receptors (TLRs) in the colonic mucosa of patients with UC. Colonic mucosal biopsy specimens were collected during colonoscopy from 30 patients with UC and 30 patients with normal findings as controls. The protein and mRNA expression levels of TLRs 1-4 and TLR9 were measured by immunohistochemistry and reverse transcription-quantitative polymerase chain reaction analysis, respectively. The results showed that the mRNA and protein expression of TLR2, TLR4 and TLR9, but not TLR1 and TLR3, was significantly increased in the colonic mucosa of patients with UC compared with that in the normal controls. TLR (TLR2, TLR4 and TLR9) immunoreactivity was found in the cytoplasm of epithelial cells in the mucosa, and occasionally in the endothelium of small vessels of the stromal tissues. In conclusion, TLR2, TLR4 and TLR9 expression may be important in the biological pathogenesis of UC. TLR alterations in the innate response system may contribute to the pathogenesis of UC.

Functional mechanism of the enhancement of 5-fluorouracil sensitivity by TUSC4 in colon cancer cells.

5-Fluorouracil (5-FU) is the chemotherapeutic drug of choice for the treatment of metastatic colorectal cancer (CRC). Tumor suppressor candidate 4 (TUSC4), also referred to as nitrogen permease regulator-like 2 (NPRL2), is located at chromosome 3p21.3 and expressed in numerous normal tissues, including the heart, liver, skeletal muscle, kidney, and pancreas. The aim of the present study was to investigate the functional mechanism by which TUSC4 affects sensitivity to 5-FU and to determine its clinical significance in CRC. The results of the present study demonstrated that TUSC4 overexpression increases the sensitivity of HCT116 cells to 5-FU. The IC50 of 5-FU was reduced in cells transduced with TUSC4 compared with negative control (NC) cells, and the effect of TUSC4 on 5-FU sensitivity was time dependent. Following TUSC4 transduction in HCT116 cells, a proportion of the cells were arrested in the G1 phase of the cell cycle, and a reduction in the S phase population was observed. Flow cytometry analysis revealed that TUSC4 transduction and 5-FU treatment increased apoptosis compared with NC cells. The mechanism through which TUSC4 overexpression enhances 5-FU sensitivity involves the downregulation of the function of the PI3K/Akt/mTOR network. Furthermore, 5-FU upregulated caspase-3 and caspase-9, promoting apoptosis in TUSC4-overexpressing cells compared with cells that were transduced with TUSC4 or treated with 5-FU and NC cells. The findings of the present study indicate that TUSC4 has potential as a biomarker for the prediction of the response to 5-FU and prognosis in patients with colorectal cancer and other types of human cancer. TUSC4 may also act as a molecular therapeutic agent for enhancing the patient's response to 5-FU treatment.

Relationship between tumor and peripheral blood NPRL2 mRNA levels in patients with colorectal adenoma and colorectal cancer.

NPRL2 is a tumor suppressor gene involved in the progression of human cancer. The present study investigated whether NPRL2 expression correlates with colorectal cancer (CRC) progression. Colorectal tissue and peripheral blood samples were obtained from 62 patients with CRC, 38 patients with colorectal adenomas and 51 normal controls. NPRL2 mRNA levels in tissue samples and blood were measured using quantitative real-time PCR. NPRL2 protein expression was determined by immunohistochemistry. NPRL2 protein expression in CRCs was significantly lower than in the adenomas or normal colorectal tissue. NPRL2 mRNA expression was significantly decreased in adenomas compared with normal controls (P<0.0001) and it was further decreased in colorectal tumors compared with adenomas (P<0.0001). NPRL2 mRNA levels expression correlated with tumor stage. In addition, NPRL2 mRNA levels in the blood correlated with the levels detected in tumors. Furthermore, receiver operating characteristic (ROC) analysis showed that NPRL2 expression in blood could distinguish colorectal adenomas and CRCs from normal controls. NPRL2 mRNA expression in CRC tumor tissues and peripheral blood correlated with colorectal tumor progression. Based on our findings, we can conclude that NPRL2 mRNA blood levels could be a potentially useful marker for the detection of early stage adenomas and CRCs.

[Wnt/b-catenin signaling pathway affects the protein expressions of caspase-3, XIAP and Grp-78 in hepatocellular carcinoma].

To investigate the relationship and significance of Wnt/b-catenin signaling pathway with caspase-3, XIAP, HSP27and Grp-78. The HCC cell line HepG2 was transfected with small interfering RNA (siRNA) directed against b-catenin. After 72 and 96 h, protein was extracted and the protein expressions of b-catenin, caspase-3, XIAP, Grp-78 and HSP27 were detected by Western blot. b-catenin protein expression was inhibited at both time points and the expression at 96 h was higher than that at 72 h (F = 160.72, P is less than to 0.01). Interestingly, Caspase-3 protein expression was decreased at 72 h and increased to normal at 96 h (F = 136.10, P is less than to 0.01), while p-caspase-3 protein expression increased at 72 h and decreased to normal at 96 h (F = 98.65, P is less than to 0.01). XIAP protein expression decreased at 72 h (F = 37.29, P is less than to 0.01) and increased at 96 h. Grp-78 protein expression increased at 72 h and decreased to normal at 96 h ( F = 58.72, P is less than to 0.01). HSP27 protein expression showed no change following transfection ( F = 1.91, P is more than to 0.05). Wnt/b-catenin signaling pathway is related to the protein expressions of caspase-3, XIAP and Grp-78, but not related to HSP27 protein expression in HCC. Wnt/b-catenin signaling pathway may participate in the regulation of HCC apoptosis, proliferation and differentiation through affecting these factors.

STAT3 and beta-catenin signaling pathway may affect GSK-3beta expression in hepatocellular carcinoma.

BACKGROUND/AIMS: To study the correlation and significance of beta-catenin, STAT3 and GSK-3beta signaling pathway in hepatocellular carcinoma (HCC). METHODOLOGY: The HCC cell line HepG2 was transfected with small interfering RNA (siRNA) directed against 8-catenin or STAT3. After 72 and 96h, protein was extracted and the protein expression of beta-catenin, STAT3, and GSK-3beta was detected by Western blot analysis. RESULTS: After siRNA directed against beta-catenin was transfected into HepG2 cells, beta-catenin protein expression was decreased at 72 and 96h, GSK-3beta and p-GSK-3beta protein expression increased gradually at 72 and 96h, and STAT3 protein expression showed no change following transfection. After siRNA directed against STAT3 was transfected into HepG2 cells, STAT3 protein expression was decreased at 72 and 96h and beta-catenin, GSK-3beta and p-GSK-3beta protein expression all increased at 72h and decreased at 96 h after transfection. CONCLUSION: In HCC, the beta-catenin signaling pathway may regulate GSK-3beta protein expression and the STAT3 signaling pathway may regulate beta-catenin and GSK-3beta protein expression, thereby playing key roles during HCC genesis and development.

Trial of a novel endoscopic tattooing biopsy forceps on animal model.

AIM: To tattoo gastric mucosa with a novel medical device which could be used to monitor and follow-up gastric mucosal lesions. METHODS: Combining endoscopic biopsy with sclerotherapy injection, we designed a new device that could perform biopsy and injection simultaneously. We performed endoscopies on a pig by using a novel endoscope tattoo biopsy forceps for 15 mo. At the same time, we used two-step method combining sclerotherapy injection needle with endoscopic biopsy. The acuity, inflammation and duration of endoscopy were compared between two methods. RESULTS: Compared with the old two-step method, although the inflammation induced by our new device was similar, the duration of procedure was markedly decreased and the acuity of tattooing was better than the old two-step method. All characteristics of the novel device complied with national safety guidelines. Follow-up gastroscopy after 15 mo showed the stained site with injection of 1:100 0.5 mL of India ink was still markedly visible with little inflammatory reaction. CONCLUSION: Endoscopic tattooing biopsy forceps can be widely used in monitoring precancerous lesions. Its safety and effectiveness has been established in animals.