Molecular docking and molecular dynamics simulation of wheat gluten-derived antioxidant peptides acting through the Keap1-Nrf2 pathway.

BACKGROUND: In our previous study, we successfully identified five peptides from wheat gluten: Ala-Pro-Ser-Tyr (APSY), Leu-Tyr (LY), Pro-Tyr (PY), Arg-Gly-Gly-Tyr (RGGY) and Tyr-Gln (YQ). Molecular docking and molecular dynamics simulation methods were employed to investigate the interaction between these antioxidant peptides and the Kelch-like ECH-associated protein 1 (Keap1 protein), revealing the molecular mechanism of their non-competitive binding. In addition, the total antioxidant capacity of the five peptides was determined using the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) method. RESULTS: The affinities of APSY, LY, PY, RGGY and YQ were -8.9, -8.3, -8.5, -9.1 and - 7.9 kcal mol-1, respectively. The five peptides effectively bound to Keap1 protein through hydrogen, π-σ, π-alkyl and alkyl interactions. Significant roles were observed for the P1 pocket residue ARG-415 and the P3 pocket residue ALA-556 in the interactions of the Keap1-peptide complexes. Molecular dynamics simulations further elucidated the dynamic process of peptide binding to the Keap1 protein. All five peptides formed stable complexes with Keap1 protein, with van der Waals forces playing crucial roles in these complex systems, indicative of the peptides' strong binding ability to Keap1 protein. The van der Waals forces were -178.74, -123.11, -134.36, -132.59, and -121.44 kJ mol-1 for the Keap1-APSY, Keap1-LY, Keap1-PY, Keap1-RGGY and Keap1-YQ complexes, respectively. These peptides exhibited excellent antioxidant effects. Among them, the YQ peptide exhibited the highest total antioxidant capacity, with an activity value of 1.18 ± 0.06 mmol Trolox equivalent (TE) L-1 at a concentration of 0.10 mg mL-1. The RGGY, PY, LY and APSY peptides followed in descending order, with activity values of 0.91 ± 0.05, 0.72 ± 0.06, 0.62 ± 0.04 and 0.60 ± 0.05 mmol TE L-1, respectively. CONCLUSION: These results unveiled the molecular mechanism by which the five antioxidant peptides act on active pockets through the Keap1-Nrf2 signaling pathway, providing a theoretical basis for the development of antioxidants. © 2024 Society of Chemical Industry.

Structural identification and combination mechanism of iron (II)-chelating Atlantic salmon (Salmo salar L.) skin active peptides.

In order to utilize salmon skin for high value, and investigate the structural identification and combination mechanism of iron (II)-chelating peptides systemically, Atlantic salmon (Salmo salar L.) skin, a by-product of Atlantic salmon processing, was treated by two-step enzymatic hydrolysis to obtain salmon skin active peptides (SSAP). Then they reacted with iron (II) to obtain iron (II)-chelating salmon skin active peptides (SSAP-Fe) with a high iron (II) chelating ability of 98.84%. The results of Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD) spectroscopy, 8-anilino-1-naphthalenesulfonic acid ammonium salt hydrate (ANS) combined fluorescence measurement, isothermal titration calorimetry (ITC) and full wavelength ultraviolet (UV) scanning showed that the structural characteristics of SSAP changed before and after chelating iron (II). Reverse phase high performance liquid chromatography (RP-HPLC) and mass spectrometry were used to identify and quantify the peptides in SSAP-Fe. Four peptide sequences (STEGGG, GIIKYGDDFMH, PGQPGIGYDGPAGPPGPPGPPGAP and QNQRESWTTCRSQSSLPDG) were identified. The content of PGQPGIGYDGPAGPPGPPGPPGAP was the highest, at 25.17 µg/mg. The pharmacokinetic and pharmacodynamic properties of these four peptides were also investigated, and the results indicated that they have satisfactory predicted ADMET properties. Molecular docking technology was used to analyze the binding sites between iron (II) and SSAP, and it was found that PGQPGIGYDGPAGPPGPPGPPGAP had the lowest predicted binding energy with iron (II) and the most stable predicted binding energy with iron (II). This results showed that the stability of SSAP-Fe were closely related to the number of covalent bonds and the types of amino acids. This study revealed the structure and combination mechanism of SSAP-Fe, and indicated that SSAP-Fe prepared by chelation may be used as a Fe supplement that can be applied in functional foods or ingredients.

Effect of the preoperative prognostic nutritional index on the long-term prognosis in patients with borderline resectable pancreatic cancer after pancreaticoduodenectomy.

Background: The preoperative prognostic nutritional index (PNI) is an indicator of systemic immune-nutritional condition and is a well-known prognostic biomarker in cancer patients. This study aims to reflect the correlation between the preoperative PNI and prognosis in patients with borderline resectable pancreatic cancer (BRPC) after pancreaticoduodenectomy (PD). Methods: Medical records of patients with BRPC after PD between Jan 2011 and Dec 2021 in our hospital were retrospectively analyzed. The preoperative PNI was calculated, and the receiver operating characteristic curve was obtained based on the preoperative PNI and the 1-year survival rate. Patients were divided into two groups (High-PNI and Low-PNI) following the best cut-off value of the preoperative PNI, and demographic and pathologic findings were compared between the two groups. Univariate and multivariate analysis were performed to identify risk factors in recurrence and long-term survival. Results: The best cut-off value for the preoperative PNI was 44.6 (sensitivity: 62.46%; specificity: 83.33%; area under the curve: 0.724). Patients in the low-PNI group had significantly shorter recurrence-free survival (P=0.008) and overall survival (P=0.009). The preoperative PNI (P=0.009) and lymph node metastasis (P=0.04) were independent risk factors for tumor recurrence. The preoperative PNI (P=0.001), lymph node metastasis (P=0.04), neoadjuvant chemotherapy (P=0.04) were independent risk factors for long-term survival in patients. Conclusion: The preoperative PNI, lymph node metastasis, neoadjuvant chemotherapy were independent risk factors for recurrence and long-term survival in patients with BRPC. The preoperative PNI might be an indicator that can predict BRPC patients' recurrence and survival. Patients with high-PNI would benefit from neoadjuvant chemotherapy.

Food-derived bioactive oligopeptide iron complexes ameliorate iron deficiency anemia and offspring development in pregnant rats.

This study aimed to investigate anemia treatment and other potential effects of two food-derived bioactive oligopeptide iron complexes on pregnant rats with iron deficiency anemia (IDA) and their offspring. Rats with IDA were established with a low iron diet and then mated. There were one control group and seven randomly assigned groups of pregnant rats with IDA: Control group [Control, 40 ppm ferrous sulfate (FeSO4)]; IDA model group (ID, 4 ppm FeSO4), three high-iron groups (H-FeSO4, 400 ppm FeSO4; MCOP-Fe, 400 ppm marine fish oligopeptide iron complex; WCOP-Fe, 400 ppm whey protein oligopeptide iron complex) and three low-iron groups (L-FeSO4, 40 ppm FeSO4; MOP-Fe, 40 ppm marine fish oligopeptide iron complex; WOP-Fe, 40 ppm whey protein oligopeptide iron complex). Rats in each group were fed the corresponding special diet during pregnancy until the day of delivery. After different doses of iron supplement, serum hemoglobin, iron, and ferritin levels in rats with IDA were significantly increased to normal levels (P < 0.05). Serum iron levels were significantly lower in two food-derived bioactive oligopeptide low-iron complex groups than in the low FeSO4 group (P<0.05). Liver malondialdehyde levels were significantly increased in the three high-iron groups compared with the other five groups (P < 0.05), and hemosiderin deposition was observed in liver tissue, indicating that the iron dose was overloaded and aggravated the peroxidative damage in pregnant rats. Liver inflammation was reduced in the three low-iron groups. Tumor necrosis factor α secretion was significantly decreased in all groups with supplemented oligopeptide (P < 0.05), with the concentration of tumor necrosis factor α declining to normal levels in the two whey protein oligopeptide iron complex groups. In the marine fish oligopeptide iron complex groups, body length, tail length, and weight of offspring were significantly increased (P < 0.05) and reached normal levels. Therefore, food-derived bioactive oligopeptide (derived from marine fish skin and milk) iron complexes may be an effective type of iron supplement for pregnancy to improve anemia, as well as reduce the side effects of iron overload, and improve the growth and nutritional status of offspring.

MiR-1301-3p Inhibits Epithelial-Mesenchymal Transition via Targeting RhoA in Pancreatic Cancer.

Micro(mi)RNAs play an essential role in the epithelial-mesenchymal transition (EMT) process in human cancers. This study aimed to uncover the regulatory mechanism of miR-1301-3p on EMT in pancreatic cancer (PC). The miRNA profilings from Gene Expression Omnibus data sets (GSE31568, GSE41372, and GSE32688) demonstrated the downregulation of miR-1301-3p in PC tissues, which was validated with 72 paired PC tissue samples through qRT-PCR detection. The low level of miR-1301-3p was associated with a poor prognosis for PC patients from the PC cohort of The Cancer Genome Atlas and the validation cohort. Gene Ontology analyses indicated that the target genes of miR-1301-3p were involved in cell cycle and adherent junction regulation. In vitro assays revealed that miR-1301-3p suppressed the proliferation and migration abilities of PC cells. Western blotting and luciferase reporter assays suggested that miR-1301-3p inhibited RhoA expression by targeting its 3'-untranslated region; RhoA upregulated N-cadherin and vimentin levels; however, it downregulated the E-cadherin level. In conclusion, our study showed that miR-1301-3p could serve as a prognostic biomarker for PC and suppress PC cell malignancy by targeting the RhoA-induced EMT process.

Ratio of CA19-9 Level to Total Tumor Volume as a Prognostic Predictor of Pancreatic Carcinoma After Curative Resection.

Background: Pancreatic ductal adenocarcinoma (PDAC) is known to have a poor prognosis, early local invasion, and distant metastasis. Surgical resection is the most effective treatment, and tumor recurrence can be the key factor affecting the surgical outcome. Serum carbohydrate antigen 19-9 (CA19-9) is a tumor marker with high sensitivity to pancreatic cancer; elevated CA19-9 levels often indicate poor biological behavior. Tumor size is also a crucial factor that affects the prognosis. Therefore, we developed a program to evaluate the effect of the ratio of CA19-9 to total tumor volume (CA19-9/TTV) as a prognostic marker on tumor recurrence and long-term survival in patients with PDAC following pancreaticoduodenectomy (PD). Methods: Data from 200 patients who underwent PD for PDAC were retrospectively analyzed. CA19-9/TTV was calculated according to preoperative CA19-9 and TTV, and patients were divided into two groups according to the optimal cut-off value. Univariate and multivariate analyses were performed on the clinicopathological data to screen the risk factors affecting postoperative recurrence and long-term prognosis of patients with PDAC undergoing PD. Results: The receiver operating characteristic curve showed that the best cut-off value was 5.62 (area under curve [AUC], 0.633; 95% CI: 0.548-0.718). Multivariate analysis showed that tumor differentiation and CA19-9/TTV were independent risk factors for the long-term prognosis of PDAC (P = 0.004, P = 0.007), as well as for tumor recurrence (P = 0.008, P = 0.008). Conclusion: CA19-9/TTV is an independent risk factor for the prognosis of PDAC and may be a new marker for lower survival benefits.

Browsing makes you feel less bad: An ecological momentary assessment of passive Qzone use and young women's negative emotions.

Passively browsing other's content on social networking sites (SNS) is popular among young females. It remains unclear how passive use relates to female's negative emotions, and especially whether short-term associations are confined by individual differences. This study used ecological momentary assessment over the course of one week to examine the associations between passive use of SNS and negative emotions, and investigated whether individual differences (i.e., self-esteem and life satisfaction) would moderate this relationship. A sample of N = 99 Chinese female undergraduates (Mage = 18.81, SDage = 0.84) completed a baseline online survey on self-esteem and life satisfaction, and received a daily online survey about their Qzone use as well as their negative emotions for seven days. Multilevel modeling revealed that passively browsing Qzone on a given day was associated with less negative emotions. However, the within-person association was independent of self-esteem and life satisfaction. Implications of passive SNS use are discussed especially regarding their potential to reduce negative feelings among Chinese young female undergraduate students, at least in the short term.

Plasmacytoid dendritic cells mediate the tolerogenic effect of CD8+regulatory T cells in a rat tolerant liver transplantation model.

BACKGROUND: Tolerance is more easily induced in liver transplant models than in other organs; CD8+CD45RClowregulatory T cells (Tregs) have been shown to induce tolerance in heart allografts. Whether CD8+CD45RClowTregs could induce tolerance in a liver transplant model and how dendritic cells (DCs) mediate the CD8+CD45RClowTregs effect remains to be investigated. METHODS: A rat liver transplantation model was established and used to test tolerance and acute rejection compared to control groups. Liver function and histopathological changes of allograft were examined by enzyme-linked immunosorbent assay (ELISA) and haematoxylin and eosin (H&E) staining, respectively. The distribution and proportion of CD8+CD45RClowTregs and plasmacytoid dendritic cells (pDCs) in the allografts and spleen were determined using flow cytometry. Cytokine secretion levels were determined using ELISA and real-time quantitative PCR (qRT-PCR). RESULTS: The rat liver transplantation model was well established, with a success rate of 93.3% (28/30). The mean survival time of the tolerant and acute-rejection rats were 156 and 14 days, respectively. The proportions of CD8+CD45RClowTegs were higher in the allografts of tolerant rats than in those of acute-rejection rats (33.1 ± 4.3 and 12.4 ± 4.6, respectively; P = 0.04). Significant accumulation of pDCs was observed in tolerant liver graft rats compared to that in acute-rejection rats (1.46 ± 0.23 and 0.80 ± 0.20, respectively; P = 0.02). Importantly, CD8+CD45RClowTregs were positively associated with the frequency of pDCs (P = 0.001, r2 = 0.775). The protein and mRNA expression of IL-10 and TGF-ß in the allograft group were increased, possibly being responsible for tolerance induction. CONCLUSION: CD8+CD45RClowT cells interact with pDCs through the induction of IL-10 and TGF-ß expression and are responsible for inducing immune tolerance in rat liver transplantation.

Prognostic Value of Preoperative NLR and Vascular Reconstructive Technology in Patients With Pancreatic Cancer of Portal System Invasion: A Real World Study.

The purpose was aimed to establish a simple computational model to predict tumor prognosis by combining neutrophil to lymphocyte Ratio (NLR) and biomarkers of oncological characteristics in patients undergoing vascular reconstructive radical resection of PDAC. The enrolled patients was divided into high or low NLR group with the cutoff value determined by the receiver operator characteristic (ROC) curve. Different vascular anastomoses were selected according to the Chaoyang classification of PDAC. Survival rates were calculated using the Kaplan-Meier and evaluated with the log-rank test. Cox risk regression model was used to analyze the independent risk factors for prognostic survival. The optimal cut-off value of NRL was correlated with the differentiation, tumor size, TNM stage and distant metastasis of advanced PDAC. A curative resection with vascular reconstructive of advanced PDAC according to Chaoyang classification can obviously improve the survival benefits. Cox proportional hazards demonstrated higher evaluated NLR, incisal margin R1 and lymphatic metastasis were the independent risk predictor for prognosis with the HR > 2, meanwhile, age beyond 55, TNM stage of III-IV or Tumor size > 4cm were also the obvious independent risk predictor for prognosis with the HR ≤ 2. The advanced PADC patients marked of RS group (3 < RS ≤ 6) showed no more than 24 months of survival time according to RS model based on the six independent risk predictors. Vascular reconstruction in radical resection of advanced PDAC improved survival, higher elevated NLR (>2.90) was a negative predictor of DFS and OS in those patients accompanying portal system invasion.

MiR-374b-5p inhibits KDM5B-induced epithelial-mesenchymal transition in pancreatic cancer.

Micro(mi)RNAs play a critical regulatory role in the progression and metastasis of pancreatic cancer (PC). In this study, we aimed to reveal the mechanisms of miR-374b-5p in regulating epithelial-mesenchymal transition (EMT) in PC. Gene Expression Omnibus datasets (GSE24279 and GSE71533) and the pancreatic ductal adenocarcinoma (PDAC) cohort of The Cancer Genome Atlas were employed to screen for potential prognostic miRNAs. The expression of miR-374b-5p was measured by quantitative real-time polymerase chain reaction (qRT-PCR) in 78 paired PDAC tissue samples. The biological effects of miR-374b-5p were investigated using in vitro and in vivo assays. Luciferase reporter assays and immunohistochemical tests were conducted to verify the interaction between miR-374b-5p and its predicted direct target, KDM5B. MiR-374b-5p was downregulated in PC tissues, and a low level of miR-374b-5p was associated with poor overall survival, greater tumor size, and more lymph node metastasis in PC. In vitro assays indicated that overexpression of miR-374b-5p suppressed the proliferation, migration, and invasion of PC cells. Mechanistically, miR-374b-5p suppressed the expression of KDM5B, which inhibited E-cadherin expression but promoted N-cadherin and vimentin expression. Finally, in vivo assays demonstrated that miR-374b-5p overexpression suppressed tumor growth and lung metastasis in PANC-1 cells. Thus, our findings indicate that miR-374b-5p could be a potential prognostic biomarker and therapeutic target for KDM5B-induced EMT in PC.

Single-cell transcriptomics reveals heterogeneous progression and EGFR activation in pancreatic adenosquamous carcinoma.

Pancreatic adenosquamous carcinoma (PASC) - a rare pathological pancreatic cancer (PC) type - has a poor prognosis due to high malignancy. To examine the heterogeneity of PASC, we performed single-cell RNA sequencing (scRNA-seq) profiling with sample tissues from a healthy donor pancreas, an intraductal papillary mucinous neoplasm, and a patient with PASC. Of 9,887 individual cells, ten cell subpopulations were identified, including myeloid, immune, ductal, fibroblast, acinar, stellate, endothelial, and cancer cells. Cancer cells were divided into five clusters. Notably, cluster 1 exhibited stem-like phenotypes expressing UBE2C, ASPM, and TOP2A. We found that S100A2 is a potential biomarker for cancer cells. LGALS1, NPM1, RACK1, and PERP were upregulated from ductal to cancer cells. Furthermore, the copy number variations in ductal and cancer cells were greater than in the reference cells. The expression of EREG, FCGR2A, CCL4L2, and CTSC increased in myeloid cells from the normal pancreas to PASC. The gene sets expressed by cancer-associated fibroblasts were enriched in the immunosuppressive pathways. We demonstrate that EGFR-associated ligand-receptor pairs are activated in ductal-stromal cell communications. Hence, this study revealed the heterogeneous variations of ductal and stromal cells, defined cancer-associated signaling pathways, and deciphered intercellular interactions following PASC progression.

Rapamycin treated tol-dendritic cells derived from BM-MSCs reversed graft rejection in a rat liver transplantation model by inducing CD8+CD45RC-Treg.

OBJECTIVE: To investigate the influence of tolerance dendritic cells (tolDCs), generated from Bone marrow mesenchymal stem cells (BM-MSCs) treated with rapamycin (Rapa) on liver allograft survival in a rat acute liver transplantation model. METHODS: Different GM-CSF induction project was used to obtain immature DCs (imDCs), mature DCs (matDCs) or tolDCs from BM-MSCs. First, MLR was performed to analyze the activity of tolDCs on polyclonaly stimulated total T cells. Then, co-cultured imDCs, matDCs and tolDCs with CD8+T cells isolated by magnetic activated cell sorting to analyze the influence on its regulatory characteristic. Last, the established rat acute liver transplantation model were adoptive transfused with imDCs, matDCs or tolDCs isolated by anti-CD11c immunomagnetic beads. The phenotype of DC cells and level of CD8+Treg in the culture system and in vivo, the expression of CD8 and CD45RC in the tissues were analyzed by flow cytometry and immunohistochemistry, respectively. RESULTS: The loGM-CSF plus IL-4 decreased the costimulatory molecules of CD80/86 and MHC class II of DCs comparison with hiGM-CSF from BM-MSCs no matter whether stimulation by LPS (P<0.05). Rapa treated not only reduced the expression of CD80/86 and MHC class II but also down-regulated the expression of CD11c after LPS stimulation which was more obviously in tolDCs by loGM-CSF project (P<0.05). Moreover, tolDCs displayed a rather higher level of IL-10 and low level of IL-12p70 than others (P<0.01), which shown a rather lower stimulative effect on the proliferation of T cells comparison with matDCs and imDCs. Co-cultured with CD8+Treg showed an improvement on induction of CD8+TCR+CD45RC-T cells (CD8+Treg) in ex vivo. The rats transfused with tolDCs has a delayed survival benefits with high level of CD8+Tregs (P<0.01) and high expression of CD45RC in liver tissue (P<0.01) and spleen when comparison with other groups. The infused tolDCs improved a mean survival time (MST) of 32 days comparison with a MTS of 9.5 days and 15.75 days displayed by rat that per-infused with matDCs and imDCs, respectively. CONCLUSION: Rapa modified tolDCs derived from BM-MSCs reversed graft rejection by improve tolerance characteristics of CD8+CD45RC-Treg in acute liver rat transplantation.

In-hospital mortality of liver transplantation and risk factors: a single-center experience.

BACKGROUND: Liver transplantation (LT) is a life-saving treatment for patients with end-stage liver disease and acute liver failure. However, in-hospital death cannot be avoided. We designed this study to analyze patients' in-hospital mortality rate after LT and the factors correlated with in-hospital death. METHODS: The data of patients who received LT in our hospital between January 11, 2015, and November 19, 2019, were obtained from the China Liver Transplant Registry and medical records. The in-hospital mortality rate was calculated, and factors related to mortality, cause of death, and factors related to cause of death were analyzed by reviewing patients' data. RESULTS: A total of 529 patients who underwent cadaveric LT were enrolled in this study. Modified piggyback orthotopic LT was performed for all patients. Seventy patients died in the hospital after LT, and the in-hospital mortality rate was 13.2%. Factors including model for end-stage liver disease (MELD) score, Child-Pugh grading, intraoperative blood loss, and anhepatic phase were correlated with in-hospital death. MELD score and intraoperative blood loss were determined as the two independent risk factors of in-hospital death. The first two causes of death were infection (34.3%) and primary non-function (15.7%). Pulmonary fungal infection was the main cause of infectious death. MELD score was the independent risk factor for infectious death, and both body mass index of donors and cold ischemic time were independent risk factors of primary non-function. CONCLUSIONS: In-hospital death poses a threat to certain patients undergoing LT. Our study suggests that the main cause of in-hospital death is an infection, followed by primary non-function.

Overexpression of fibrinogen-like protein 2 alleviates acute rejection in rat models of liver transplantation.

BACKGROUND: The role of cluster of differentiation (CD)8+ regulatory T cells (Tregs) has previously been elucidated in tolerance models. Fibrinogen-like protein 2 (FGL2), that is secreted by Treg cells, which exhibited immunosuppressive functions, may alleviate acute rejection (AR). However, the precise role of CD8+ Tregs and FGL2 in the AR of rat liver transplantation remains unknown. Our previous study found that CD8+CD45RClow Tregs played crucial roles in maintaining immune tolerance. Here, we elucidated the role of CD8+ CD45RClowTreg and FGL2 in AR of rat liver transplantation. METHODS: A rat non-materialized AR of liver transplantation model was established using donors infected with no-load adeno-associated virus and adeno-associated virus expressing FGL2. RESULTS: There was an accumulation of tolerogenic CD8+CD45RClow in allografts compared with blank groups. Moreover, the proportion of CD8+CD45RClow Tregs was increased with longer survival time. Furthermore, we detected higher levels of FGL2 in the allografts infected with AAV-FGL2 in rats with AR of liver transplantation. We found that FGL2 could alleviate AR, and the survival time was prolonged in the recipients of donors infected with AAV-FGL2. CONCLUSIONS: Our data suggest that CD8+CD45RClow Tregs was accumulated in allografts. The presence of FGL2 alleviated AR and prolonged survival time in the AR of liver transplantation rat model, suggesting that FGL2 and CD8+CD45RClow Tregs may serves as novel therapeutic targets for AR in liver transplantation.

Comprehensive circular RNA expression profiling constructs a ceRNA network and identifies hsa_circ_0000673 as a novel oncogene in distal cholangiocarcinoma.

Circular RNAs (circRNAs) play an important role in cholangiocarcinoma (CCA) development; however, the expression and functions of circRNAs in distal CCA (dCCA) remain unknown. Herein, we explored the expression profile of circRNAs in six paired dCCA tumor and adjacent normal tissue samples using microarray. A total of 171 differentially expressed (DE) circRNAs were identified in dCCA tissues. Host genes of DE circRNAs were enriched in the cellular cytoskeleton and adheren junction. Bioinformatics analyses were used to establish a circRNA-microRNA-mRNA network for dCCA. Protein-protein interaction networks were constructed, and five hub genes were associated with the regulation of the cell cycle based on gene set enrichment analyses. Five DE circRNAs were validated with qRT-PCR in 40 pairs of dCCA tissues, and hsa_circ_0000673 showed promising diagnostic performance in distinguishing dCCA from normal tissues (AUC = 0.85, p < 0.01). Overexpression of hsa_circ_0000673 was associated with tumor invasion (p = 0.001), poor differentiation (p = 0.041), and residual tumor (p = 0.044). In vitro experiments indicated that inhibition of hsa_circ_0000673 suppressed the proliferation, migration, and invasion of CCA cells. This research provided a landscape of dysregulated circRNAs in dCCA and identified hsa_circ_0000673 as a potential biomarker and therapeutic target for dCCA.

Reduction of FoxP3+ Tregs by an immunosuppressive protocol of rapamycin plus Thymalfasin and Huaier extract predicts positive survival benefits in a rat model of hepatocellular carcinoma.

BACKGROUND: Investigate immunoregulation and anti-tumor immunity of FoxP3+Tregs after treatment with rapamycin (RAPA/SRL) plus thymalfasin (Zadaxin) and Huaier extract (PS-T) in a hepatocellular carcinoma (HCC) rat model simulating HCC relapse after liver transplant (LT). METHODS: We successfully established a rat model simulating HCC relapse after LT using an optimized chemical induction method with TACROLIMUS, methylprednisolone, and diethylnitrosamine as identified by visible liver nodules and hematoxylin-eosin staining. The model rats were then treated with RAPA, Zadaxin, and PS-T. Immune status changes were analyzed by flow cytometry, and protein expression of Akt and mTOR was determined by western blotting. Cytokines were measured by ELISAs. RESULTS: Combined therapy by RAPA plus Zadaxin and PS-T obviously alleviated hepatic pathological changes and significantly decreased the levels of FoxP3+Tregs in peripheral blood, the spleen, and the liver (P<0.05) and expression of mTOR protein (P<0.01) in the liver, obviously improved survival time (P=0.02). Moreover, the levels of CD8+T cells were increased significantly to almost normal levels (P<0.05) in comparison with no SRL monotherapy protocols. Inhibitory cytokines were also decreased in accordance with FoxP3+Tregs. Significant decreases of IL-10 and TGF-ß were observed after SRL-based therapy (P<0.01) in comparison with the other groups. Serum alpha fetoprotein (AFP) and vascular endothelial growth factor (VEGF) levels were also decreased significantly (P<0.05). FoxP3+Tregs showed a negative correlation with CD8+ and CD4+/CD8+T cells and a positive correlation with AFP, and VEGF (P<0.05). CONCLUSIONS: SRL-based therapy reduces FoxP3+Tregs to decrease secreted inhibitory cytokines which may enhancement the viability and number of CD8+T cells to exert anti-tumor effects that are mainly mediated through the AKT-mTOR signaling pathway.

Dummy molecularly imprinted microspheres prepared by Pickering emulsion polymerization for matrix solid-phase dispersion extraction of three azole fungicides from fish samples.

Dummy molecularly imprinted microspheres (DMIMs) were synthesized by Pickering emulsion polymerization and used as the matrix solid-phase dispersion extraction (MSPD) sorbent for sample pre-treatment of azole fungicides in fish samples. Alpha-(2,4-Dichlorophenyl)-1H-imidazole-1-ethanol (DCE) was used as the fragment dummy template for the imprinting of climbazole (CBZ), clotrimazole (CMZ) and miconazole (MNZ). The morphology of the microspheres were characterized by scanning electron microscopy (SEM) and nitrogen adsorption measurements, narrow diameter distribution (20-50 µm) with regular spherical shape and high surface area (SBET = 408.91 ± 6.72 m2 g-1) were achieved. Good class-selectivity of the DMIMs was found for CBZ, CMZ and MNZ by static adsorption experiments. The imprinted microspheres as MSPD sorbent was then evaluated for the extraction and purification of CBZ, CMZ and MNZ in fish samples. The extracted azole fungicides were detected by HPLC-DAD analysis at 225 nm. MSPD conditions including elution, mass ratio of sample/sorbent and washing were carefully evaluated. The optimized MSPD method have good recoveries (89.2-101.5%) and reproducibility (RSDs 1.6-4.8%, n = 5) for fish samples spiked at 0.5, 2.5 and 12.5 µg g-1. The limits of detection (LODs) were 0.045, 0.036 and 0.033 µg g-1 for CBZ, CMZ and MNZ, respectively. The results show that this method has a good application prospect for the pretreatment of azole fungicides in fish samples.

Overexpression of NNT-AS1 Activates TGF-ß Signaling to Decrease Tumor CD4 Lymphocyte Infiltration in Hepatocellular Carcinoma.

Nicotinamide nucleotide transhydrogenase-antisense RNA1 (NNT-AS1) is a long noncoding RNA (lncRNA) that has been shown to be overexpressed in hepatocellular carcinoma (HCC). However, the molecular mechanism involving NNT-AS1 in HCC remains to be extensively investigated. The activation of TGF-ß signaling inhibits tumor-infiltrating lymphocytes (TILs) and results in tumor immune evasion. We thus planned to explore the mechanism by which NNT-AS1 activates the TGF-ß signaling pathway and inhibits TILs in HCC. High levels of NNT-AS1 were detected in HCC tissues by both RNAscope and real-time quantitative PCR (RT-qPCR) assays. The levels of proteins involved in TGF-ß signaling and those of CD4 T lymphocytes were quantified by immunohistochemistry (IHC). HCC cell lines (HepG2 and Huh7) were used to explore the effects of NNT-AS1 on TGF-ß signaling activation. In these analyses, RNAscope detection demonstrated that NNT-AS1 levels were significantly increased in HCC cancer tissues (P = 0.0001). In addition, the elevated NNT-AS1 levels in cancer tissue were further confirmed by RT-qPCR analysis of HCC cancer tissues (n = 64) and normal tissues (n = 26) (P = 0.0003). Importantly, the overall survival time of HCC patients who exhibited higher levels of NNT-AS1 expression was significantly shorter than that of HCC patients who had lower levels of NNT-AS1 expression (P = 0.0402). Further mechanistic investigation indicated that NNT-AS1 inhibition significantly decreased the levels of TGF-ß, TGFBR1, and SMAD5 in HCC cells. In HCC tissues, IHC detection showed that relatively high NNT-AS1 levels were associated with a reduction in infiltrated CD4 lymphocyte numbers. In conclusion, this research identifies a novel mechanism by which NNT-AS1 impairs CD4 T cell infiltration via activation of the TGF-ß signaling pathway in HCC.