Increased yield of 2-O-α-d-glucopyranosyl-l-ascorbic acid synthesis by α-glucosidase using rational design that regulating the ground state of enzyme and substrate complex.

BACKGROUND: α-Glucosidase (AG) is a bifunctional enzyme, it has a capacity to synthesize 2-O-α-d-glucopyranosyl-l-ascorbic acid (AA-2G) from l-ascorbic acid (L-AA) and low-cost maltose under mild conditions, but it can also hydrolyze AA-2G, which leads to low synthesis efficiency of AA-2G. MAIN METHODS AND MAJOR RESULTS: This study introduces a rational molecular design strategy to regulate enzymatic reactions based on inhibiting the formation of ground state of enzyme-substrate complex. Y215 was analyzed as the key amino acid site affecting the affinity of AG to AA-2G and L-AA. For the purpose of reducing the hydrolysis efficiency of AA-2G, the mutant Y215W was obtained by analyzing the molecular docking binding energy and hydrogen bond formation between AG and the substrates. Compared with the wild-type, isothermal titration calorimetry (ITC) results showed that the equilibrium dissociation constant (KD ) of the mutant for AA-2G was doubled; the Michaelis constant (Km ) for AA-2G was reduced by 1.15 times; and the yield of synthetic AA-2G was increased by 39%. CONCLUSIONS AND IMPLICATIONS: Our work also provides a new reference strategy for the molecular modification of multifunctional enzymes and other enzymes in cascade reactions system.

MACC1 and Gasdermin-E (GSDME) regulate the resistance of colorectal cancer cells to irinotecan.

Metastasis-associated in colon cancer 1 (MACC1) is an oncogene associated with the progression and metastasis of many solid cancer entities. High expression of MACC1 is found in colorectal cancer (CRC) tissues. So far, the role of MACC1 in CRC cell pyroptosis and resistance to irinotecan is unclear. The cleavage of Gasdermin-E (GSDME) is the main executors of activated pyroptosis. We found that GSDME enhanced CRC cell pyroptosis and reduced their resistance to irinotecan, while MACC1 inhibited the cleavage of GSDME and CRC cell pyroptosis, promoted CRC cell proliferation, and enhanced the resistance of CRC cells to irinotecan. Therefore, CRC cells with high MACC1 expression and low GSDME expression had higher resistance to irinotecan, while CRC cells with low MACC1 expression and high GSDME expression had lower resistance to irinotecan. Consistently, by analyzing CRC patients who received FOLFIRI (Fluorouracil + Irinotecan + Leucovorin) in combination with chemotherapy in the GEO database, we found that CRC patients with low MACC1 expression and high GSDME expression had higher survival rate. Our study suggests that the expression of MACC1 and GSDME can be used as detection markers to divide CRC patients into irinotecan resistant and sensitive groups, helping to determine the treatment strategy of patients.

Liver infiltration of multiple immune cells during the process of acute liver injury and repair.

BACKGROUND: Immune cells, including neutrophils, natural killer (NK) cells, T cells, NKT cells and macrophages, participate in the progression of acute liver injury and hepatic recovery. To date, there has been no systematic study on the quantitative changes in these different immune cells from initial injury to subsequent recovery. AIM: To investigate the infiltration changes of various immune cells in acute liver injury models over time, and to study the relationship between the changes in leukocyte cell-derived chemotaxin 2 (LECT2) and the infiltration of several immune cells. METHODS: Carbon tetrachloride- and concanavalin A-induced acute liver injury models were employed to mimic toxin-induced and autoimmune-mediated liver injury respectively. The quantitative changes in various immune cells were monitored at different time points. Serum samples were collected, and liver tissues were harvested. Ly6G, CD161, CD4, CD8 and F4/80 staining were used to indicate neutrophils, NK/NKT cells, CD4+ T cells, CD8+ T cells and macrophages, respectively. Lect2-KO mice were used to detect the function of LECT2. RESULTS: During the injury and repair process, different types of immune cells began to increase, reached their peaks and fell into decline at different time points. Furthermore, when the serum alanine transaminase (ALT) and aspartate transaminase (AST) indices reverted to normal levels 7 d after the injury, the infiltration of immune cells still existed even 14 d after the injury, showing an obvious lag effect. We found that the expression of LECT2 was upregulated in acute liver injury mouse models, and the liver injuries of Lect2-KO mice were less severe than those of wild-type mice. Compared with wild-type mice, Lect2-KO mice had different immune cell infiltration. CONCLUSION: The recovery time of immune cells was far behind that of serum ALT and AST during the process of liver repair. LECT2 could regulate monocyte/macrophage chemotaxis and might be used as a therapeutic target for acute liver injury.

The Effects of 6 Common Antidiabetic Drugs on Anti-PD1 Immune Checkpoint Inhibitor in Tumor Treatment.

Diabetes and cancer are common diseases and are frequently diagnosed in the same individual. These patients need to take antidiabetic drugs while receiving antitumor drugs therapy. Recently, immunotherapy offers significant advances for cancer treatment. However, it is unclear whether antidiabetic drugs affect immunotherapy. Here, by employing syngeneic mouse colon cancer model and melanoma model, we studied the effects of 6 common antidiabetic drugs on anti-PD1 immune checkpoint inhibitor in tumor treatment, including acarbose, sitagliptin, metformin, glimepiride, pioglitazone, and insulin. We found that acarbose and sitagliptin enhanced the tumor inhibition of anti-PD1, and metformin had no effect on the tumor inhibition of anti-PD1, whereas glimepiride, pioglitazone, and insulin weakened the tumor inhibition of anti-PD1. Our study suggests that cancer patients receiving anti-PD1 antibody therapy need serious consideration when choosing antidiabetic drugs. In particular, acarbose significantly inhibited tumor growth and further enhanced the therapeutic effect of anti-PD1, which can be widely used in tumor therapy. Based on this study, further clinical trials are expected.

LECT2: A pleiotropic and promising hepatokine, from bench to bedside.

LECT2 (leucocyte cell-derived chemotaxin 2) is a 16-kDa protein mainly produced by hepatocytes. It was first isolated in PHA-activated human T-cell leukaemia SKW-3 cells and originally identified as a novel neutrophil chemotactic factor. However, many lines of studies suggested that LECT2 was a pleiotropic protein, it not only functioned as a cytokine to exhibit chemotactic property, but also played multifunctional roles in some physiological conditions and pathological abnormalities, involving liver regeneration, neuronal development, HSC(haematopoietic stem cells) homeostasis, liver injury, liver fibrosis, hepatocellular carcinoma, metabolic disorders, inflammatory arthritides, systemic sepsis and systemic amyloidosis. Among the above studies, it was discovered that LECT2 could be a promising molecular biomarker and therapeutic target. This review summarizes LECT2-related receptors and pathways, basic and clinical researches, primarily in mice and human, for a better comprehension and management of these diseases in the future.

Association between urine microscopy and severe acute kidney injury in critically ill patients following non-cardiac surgery: a prospective cohort study.

BACKGROUND: Acute kidney injury (AKI) is a common and adverse complication following non-cardiac surgery. Evidence have shown urine microscopy could help early detection, differentiating the causes and predicting the progression of AKI. However, little evidence is available on AKI after non-cardiac surgery. Thus, we investigated the association between urine microscopy and severe AKI in critically ill patients after non-cardiac surgery. METHODS: This was a single-center prospective cohort study. The primary outcome was severe AKI, defined as stage 2 or 3 according to maximal KDIGO criteria within 7 days following non-cardiac surgery. Urine microscopy immediately, 6 and 12 hours after surgical intensive care unit (SICU) admission were examined and graded by a urine microscopy score (UMS) based on the observed quantification of renal tubular cells and casts in the sediment. Then, multivariate Logistic regression models were used to analyze the associations between UMS and postoperative severe AKI. RESULTS: From May 20, 2019 to November 24, 2020, 661 patients were enrolled with 147 patients (22.2%) developing postoperative severe AKI. Multivariate Logistic regression model showed elevated UMS (≥1) 6 and 12 hours after SICU admission were independently associated with postoperative severe AKI (OR 2.200, 95% CI: 1.182-4.095, P=0.013 and OR 2.949, 95% CI: 1.657-5.248, P<0.001, respectively). Furthermore, higher UMS 6 hours after SICU admission demonstrated correlation with greater risk of severe AKI with OR 3.887 (95% CI: 1.430-10.563) for UMS ≥3 and OR 2.429 (95% CI: 1.237-4.770) for UMS =1-2. The specificity and sensitivity of UMS ≥1 for severe AKI was 93.8% (95% CI: 91.7-95.9%) and 15.6% (95% CI: 9.7-21.5%), respectively. While the negative and positive predictive value was 79.5% (95% CI: 76.3-82.7%) and 41.8% (95% CI: 28.8-54.8%), respectively. In addition, patients with higher UMS (≥3, 1-2 and 0) had significantly more postoperative complications and longer SICU stay; and they also showed a trend toward other adverse postoperative outcomes. CONCLUSIONS: Early urine microscopy was independently associated with severe AKI in critically ill patients following non-cardiac surgery with higher UMS related to greater risk. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT03880110.

An endorectal ultrasound-based radiomics signature for preoperative prediction of lymphovascular invasion of rectal cancer.

OBJECTIVE: To investigate whether radiomics based on ultrasound images can predict lymphovascular invasion (LVI) of rectal cancer (RC) before surgery. METHODS: A total of 203 patients with RC were enrolled retrospectively, and they were divided into a training set (143 patients) and a validation set (60 patients). We extracted the radiomic features from the largest gray ultrasound image of the RC lesion. The intraclass correlation coefficient (ICC) was applied to test the repeatability of the radiomic features. The least absolute shrinkage and selection operator (LASSO) was used to reduce the data dimension and select significant features. Logistic regression (LR) analysis was applied to establish the radiomics model. The receiver operating characteristic (ROC) curve, calibration curve, and decision curve analysis (DCA) were used to evaluate the comprehensive performance of the model. RESULTS: Among the 203 patients, 33 (16.7%) were LVI positive and 170 (83.7%) were LVI negative. A total of 5350 (90.1%) radiomic features with ICC values of ≥ 0.75 were reported, which were subsequently subjected to hypothesis testing and LASSO regression dimension reduction analysis. Finally, 15 selected features were used to construct the radiomics model. The area under the curve (AUC) of the training set was 0.849, and the AUC of the validation set was 0.781. The calibration curve indicated that the radiomics model had good calibration, and DCA demonstrated that the model had clinical benefits. CONCLUSION: The proposed endorectal ultrasound-based radiomics model has the potential to predict LVI preoperatively in RC.

A strategy of vascular-targeted therapy for liver fibrosis.

BACKGROUND AND AIMS: No effective treatments are available for liver fibrosis. Angiogenesis is deeply involved in liver fibrogenesis. However, current controversial results suggest it is difficult to treat liver fibrosis through vascular targeting. There are three different microvessels in liver: portal vessels, liver sinusoids, and central vessels. The changes and roles for each of the three different vessels during liver fibrogenesis are unclear. We propose that they play different roles during liver fibrogenesis, and a single vascular endothelial cell (EC) regulator is not enough to fully regulate these three vessels to treat liver fibrosis. Therefore, a combined regulation of multiple different EC regulatory signaling pathway may provide new strategies for the liver fibrosis therapy. Herein, we present a proof-of-concept strategy by combining the regulation of leukocyte cell-derived chemotaxin 2 (LECT2)/tyrosine kinase with immunoglobulin-like and epidermal growth factor-like domains 1 signaling with that of vascular endothelial growth factor (VEGF)/recombinant VEGF (rVEGF) signaling. APPROACH AND RESULTS: The CCl4 -induced mouse liver fibrosis model and NASH model were both used. During fibrogenesis, vascular changes occurred at very early stage, and different liver vessels showed different changes and played different roles: decreased portal vessels, increased sinusoid capillarization and the increased central vessels the increase of portal vessels alleviates liver fibrosis, the increase of central vessels aggravates liver fibrosis, and the increase of sinusoid capillarization aggravates liver fibrosis. The combinational treatment of adeno-associated viral vector serotype 9 (AAV9)-LECT2-short hairpin RNA (shRNA) and rVEGF showed improved therapeutic effects, but it led to serious side effects. The combination of AAV9-LECT2-shRNA and bevacizumab showed both improved therapeutic effects and decreased side effects. CONCLUSIONS: Liver vascular changes occurred at very early stage of fibrogenesis. Different vessels play different roles in liver fibrosis. The combinational treatment of AAV9-LECT2-shRNA and bevacizumab could significantly improve the therapeutic effects on liver fibrosis.

Dipetidyl peptidase-4 and transferrin receptor serve as prognostic biomarkers for acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is the most common hematological malignancy in adult patients. Ferroptosis-related signatures have been shown to act as regulators of the progression of multiple cancer types, but the role of ferroptosis in AML remains to be elucidated. We performed the present study to preliminarily investigate the roles of ferroptosis-related genes (FRGs) in AML. METHODS: The transcriptome data of AML patients was downloaded from The Cancer Genome Atlas (TCGA) and the transcriptome data of normal samples was obtained from the Genotype-Tissue Expression (GTEx) database. FRGs were selected via public articles. Expression levels of FRGs between AML and normal samples were analyzed. The prognostic model based on FRGs was constructed via lasso regression. The expression levels and prognostic role of FRGs were identified from the risk model. We also performed validation experiments to verify the expression levels of the final selected genes via immunohistochemistry, polymerase chain reaction (PCR), and RNA-seq. Finally, we explored the associations between immune infiltration, drug sensitivity, and the selected FRGs. RESULTS: The transcriptome data of 151 AML samples were retrieved from TCGA and 70 bone marrow normal samples were retrieved from the GTEx database. Additionally, 23 FRGs were collected from the published articles. There were 22 differentially expressed FRGs, and among them, dipetidyl peptidase-4 (DPP4) (P= 0.011, HR =1.504), GPX4 (P=0.055, HR =1.569), LPCAT3 (P<0.001, HR =2.243), SLC7A11 (P=0.012, HR =2.243), and transferrin receptor (TFRC) (P=0.029, 0.774) had a significant influence on the prognosis of AML patients via lasso regression. The area under the curve (AUC) values of the 1-, 3-, and 5-year receiver operating characteristic (ROC) curves of the FRG signatures indicated that this model is novel and effective method for predicting the prognosis of AML patients. DPP4 (P<0.001) was overexpressed while LPCAT3 (P<0.001), TFRC (P<0.001), GPX4 (P<0.001), and SLC7A11 (P<0.001) were downregulated, further validation experiment results indicated that DPP4 was significantly downregulated but TFRC was upregulated in AML samples. Dysregulation of DPP4 and TFRC influence numbers of chemotherapy regimens sensitivity. CONCLUSIONS: DPP4 and TFRC act as biomarkers for predicting and diagnosing AML, and their expression levels also have significant correlations with drug resistance in AML.

Key immune-related gene ITGB2 as a prognostic signature for acute myeloid leukemia.

BACKGROUND: The tumor microenvironment (TME) has an essential role in tumorigenesis, progression, and therapeutic response in many cancers. Currently, the role of TME in acute myeloid leukemia (AML) is unclear. This study investigated the correlation between immune-related genes and prognosis in AML patients. METHODS: Transcriptome RNA-Seq data for 151 AML samples were downloaded from TCGA database (https://portal.gdc.cancer.gov/), and the immune related genes (irgs) were selected from Immport database. Bioinformatics screening was used to identify irgs for AML, and genes with a critical role in the prognosis of AML were selected for further analysis. To confirm the prognostic role of irgs in AML, we undertook protein-protein interaction (PPI) network analysis of the top 30 interacting genes. We then investigated associations between immune cell infiltration and prognosis in AML patients. Immunohistochemistry was used to validate protein expression levels between AML and normal bone marrow samples. Analysis of the drug sensitivity of the selected gene was then performed. RESULTS: The integrin lymphocyte function-associated antigen 1 (CD11A/CD18; ITGAL/ITGB2) was identified as the key immune-related gene that significantly influenced prognosis in AML patients. Overexpression of ITGB2 indicated poor prognosis in AML patients (P=0.007). Risk modeling indicated that a high-risk score led to poor outcomes (P=3.076e-08) in AML patients. The risk model showed accuracy for predicting prognosis in AML patients, with area under curve (AUC) at 1 year, 0.816; AUC at 3 years, 0.82; and AUC at 5 years, 0.875. In addition, we found that ITGB2 had a powerful influence on immune cell infiltration into AML TME. The results of immunohistochemistry showed that AML patients had significantly higher ITGB2 protein expression than normal samples. The AML patients were divided into 2 groups based on ITGB2 risk scores. Drug sensitivity test results indicated that the high-risk group was sensitive to cytarabine, axitinib, bosutinib, and docetaxel, but resistant to cisplatin and bortezomib. CONCLUSIONS: In the present study, we found that ITGB2 may be able to serve as a biomarker for assessing prognosis and drug sensitivity in AML patients.

siRNAs Targeting Mouse-Specific lncRNA AA388235 Induce Human Tumor Cell Pyroptosis/Apoptosis.

Species-specific lncRNAs significantly determine species-specific functions through various ways, such as epigenetic regulation. However, there has been no study focusing on the role of species-specific lncRNAs in other species yet. Here, we found that siRNAs targeting mouse-specific lncRNA AA388235 could significantly induce death of human tumor cells, although it has no effect on mouse tumor cells and normal human cells. The mechanism studies showed that these siRNAs could activate the response of human tumor cells to exogenous nucleic acids, induce pyroptosis and apoptosis in the presence of GSDME, but induce apoptosis in the absence of GSDME. They also significantly inhibited the growth of human tumor cells in vivo. 17 siRNAs were designed for seven more mouse-specific lncRNAs selected randomly, among which 12 siRNAs targeting five lncRNAs induced death in human tumor cell. Our study not only demonstrates that the siRNAs designed for knocking down mouse-specific lncRNA AA388235 can be potential tumor therapeutic drugs, but also suggests that non-human species-specific lncRNAs are a huge potential library that can be used to design siRNAs for tumor treatment. Large-scale screening based on this is promising.

Long-term Survival after Combined Epidural-General Anesthesia or General Anesthesia Alone: Follow-up of a Randomized Trial.

BACKGROUND: Experimental and observational research suggests that combined epidural-general anesthesia may improve long-term survival after cancer surgery by reducing anesthetic and opioid consumption and by blunting surgery-related inflammation. This study therefore tested the primary hypothesis that combined epidural-general anesthesia improves long-term survival in elderly patients. METHODS: This article presents a long-term follow-up of patients enrolled in a previous trial conducted at five hospitals. Patients aged 60 to 90 yr and scheduled for major noncardiac thoracic and abdominal surgeries were randomly assigned to either combined epidural-general anesthesia with postoperative epidural analgesia or general anesthesia alone with postoperative intravenous analgesia. The primary outcome was overall postoperative survival. Secondary outcomes included cancer-specific, recurrence-free, and event-free survival. RESULTS: Among 1,802 patients who were enrolled and randomized in the underlying trial, 1,712 were included in the long-term analysis; 92% had surgery for cancer. The median follow-up duration was 66 months (interquartile range, 61 to 80). Among patients assigned to combined epidural-general anesthesia, 355 of 853 (42%) died compared with 326 of 859 (38%) deaths in patients assigned to general anesthesia alone: adjusted hazard ratio, 1.07; 95% CI, 0.92 to 1.24; P = 0.408. Cancer-specific survival was similar with combined epidural-general anesthesia (327 of 853 [38%]) and general anesthesia alone (292 of 859 [34%]): adjusted hazard ratio, 1.09; 95% CI, 0.93 to 1.28; P = 0.290. Recurrence-free survival was 401 of 853 [47%] for patients who had combined epidural-general anesthesia versus 389 of 859 [45%] with general anesthesia alone: adjusted hazard ratio, 0.97; 95% CI, 0.84 to 1.12; P = 0.692. Event-free survival was 466 of 853 [55%] in patients who had combined epidural-general anesthesia versus 450 of 859 [52%] for general anesthesia alone: adjusted hazard ratio, 0.99; 95% CI, 0.86 to 1.12; P = 0.815. CONCLUSIONS: In elderly patients having major thoracic and abdominal surgery, combined epidural-general anesthesia with epidural analgesia did not improve overall or cancer-specific long-term mortality. Nor did epidural analgesia improve recurrence-free survival. Either approach can therefore reasonably be selected based on patient and clinician preference.

Immune-related long non-coding RNAs can serve as prognostic biomarkers for clear cell renal cell carcinoma.

BACKGROUND: The immune microenvironment is a critical regulator of clear cell renal cell carcinoma (ccRCC) progression. However, the underlying mechanisms the regulatory role of immune-related long non-coding RNAs (irlncRNAs) in the ccRCC tumor microenvironment (TME) are still obscure. Herein, we investigated prognostics role of irlncRNAs for ccRCC. METHODS: The raw data of patients with ccRCC were downloaded from The Cancer Genome Atlas (TCGA) database, and immune-related genes were obtained from the ImmPort database. First, we investigated the correlation between the immune-related genes and irlncRNAs. Then, we identified the differentially expressed irlncRNA pairs (ILRPs) between normal and cancer tissue samples, and prognostic model was constructed with the differentially expressed ILRPs. We further explored whether the signature risk scores of ILRPs had a considerable impact on immune cell infiltration. Finally, we performed a drug sensitivity analysis based on risk score. RESULTS: There were 13 upregulated and 40 downregulated irlncRNAs between the ccRCC and normal tissue samples. We further selected the irlncRNAs that significantly affect the prognosis of patients with ccRCC via univariate Cox, lasso regression, and multivariate regression analyses. Twelve ILRPs were used to construct a prognostic signature. The model showed the ILRPs model could be used to assess the prognosis of ccRCC patients. Study of the influence of risk score and clinical characteristics on the prognosis of patients with ccRCC showed risk score to be an independent factor affecting the outcome of ccRCC. We further performed the difference analysis of immune cell abundance between ccRCC and normal tissue samples. The results showed that patients with higher abundance of M0 macrophages, plasma cells, follicular helper T cells, and regulatory T cells (Tregs) had a poor outcome. Finally, we performed a drug sensitivity analysis based on risk score. The results showed that high-risk score patients are sensitive to orafenib, sunitinib, temsirolimus, cisplatin, and gemcitabine. CONCLUSIONS: Our study has developed a novel and reasonable ILPRs model for prognostic prediction, which does not require transcriptional levels to be detected.

ZBP1 is a significant pyroptosis regulator for systemic lupus erythematosus.

BACKGROUND: Systemic lupus erythematosus (SLE) is a common autoimmune disease that affects all organs. Recently, several studies have shown that pyroptosis playsa significant process in the occurrence and progression of SLE. However, no study has investigated the association between pyroptosis genes and SLE. We conducted this study to examine this association. METHODS: The GSE11090, GSE20864, and GSE112087 gene microarrays of normal and SLE patient samples were downloaded from the Gene Expression Omnibus database. A differentially expressed gene (DEG) analysis was performed using the LIMMA package in R software. Log2 fold change |logFC| >0.5 and a false discovery rate (FDR) <0.05 setting for DEGs' screening value. We also performed an enrichment function analysis of the DEGs. To explore the role of pyroptosis genes in SLE, we selected pyroptosis genes that intersected with the DEGs for further analysis, we also examined the expression levels of the selected genes, their association with immune cell infiltration, and conducted western blotting and polymerase chain reaction analyses to confirm the selected genes expression levels in the SLE and normal samples. RESULTS: A total of 3,398 identical genes were obtained from 3 datasets for the differential analysis. 84 upregulated genes and 52 downregulated genes were identified in SLE. The enrichment function analysis revealed that DEGs act as key regulators of nicotinamide adenine dinucleotide (NADH) dehydrogenase activity, phospholipid scramblase activity, double-stranded ribonucleic acid (RNA) binding, and the interferon signaling pathway. We identified the SLE-related pyroptosis gene, Z-DNA binding protein 1 (ZBP1), by intersecting the DEGs of SLE and 40 pyroptosis genes. The differential analysis indicated that ZBP1 was more highly expressed in SLE patients compared to normal samples (P<0.001). Additionally, the expression of ZBP1 was higher in females than males (P=0.008). The SLE samples had different immune cell infiltration than the normal samples, and ZBP1 was significantly correlated with immune cell infiltration in the SLE samples. Finally, the validation experiments results showed that ZBP1 expression levels were significantly more highly expressed in female and SLE patients, than male and normal patients. CONCLUSIONS: ZBP1 may indicate that females have a high incidence rate of SLE, and it plays a significant role in the occurrence and progression of SLE.

Deficiency of ß-arrestin2 exacerbates inflammatory arthritis by facilitating plasma cell formation.

ß-arrestin2 (ß-arr2) is, a key protein that mediates desensitization and internalization of G protein-coupled receptors and participates in inflammatory and immune responses. Deficiency of ß-arr2 has been found to exacerbate collagen antibody-induced arthritis (CAIA) through unclear mechanisms. In this study we tried to elucidate the molecular mechanisms underlying ß-arr2 depletion-induced exacerbation of CAIA. CAIA was induced in ß-arr2-/- and wild-type (WT) mice by injection of collagen antibodies and LPS. The mice were sacrificed on d 13 after the injection, spleen, thymus and left ankle joints were collected for analysis. Arthritis index (AI) was evaluated every day or every 2 days. We showed that ß-arr2-/- mice with CAIA had a further increase in the percentage of plasma cells in spleen as compared with WT mice with CAIA, which was in accordance with elevated serum IgG1 and IgG2A expression and aggravating clinical performances, pathologic changes in joints and spleen, joint effusion, and joint blood flow. Both LPS stimulation of isolated B lymphocytes in vitro and TNP-LPS challenge in vivo led to significantly higher plasma cell formation and antibodies production in ß-arr2-/- mice as compared with WT mice. LPS treatment induced membrane distribution of toll-like receptor 4 (TLR4) on B lymphocytes, accordingly promoted the nuclear translocation of NF-κB and the transcription of Blimp1. Immunofluorescence analysis confirmed that more TLR4 colocalized with ß-arr2 in B lymphocytes in response to LPS stimulation. Depletion of ß-arr2 restrained TLR4 on B lymphocyte membrane after LPS treatment and further enhanced downstream NF-κB signaling leading to additional increment in plasma cell formation. In summary, ß-arr2 depletion exacerbates CAIA and further increases plasma cell differentiation and antibody production through inhibiting TLR4 endocytosis and aggravating NF-κB signaling.

Paeoniflorin-6'-O-benzene sulfonate down-regulates CXCR4-Gßγ-PI3K/AKT mediated migration in fibroblast-like synoviocytes of rheumatoid arthritis by inhibiting GRK2 translocation.

OBJECTIVE: This study aims to explore the effect of paeoniflorin-6'-O-benzene sulfonate (CP-25) on the migration of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) and the mechanism focused on CXCR4-Gßγ-PI3K/AKT signaling. METHODS: Human synovial tissues were collected from RA and osteoarthritis (OA) patients. Immunohistochemistry (IHC) and Western blot were used to detect the protein expression of CXCR4, GRK2, Gßγ, PI3K, p-PI3K, AKT and p-AKT. Transwell was adopted to analyse the migration of fibroblast-like synoviocytes (FLS). Co-immunoprecipitation (Co-IP) and laser scanning confocal microscopy (LSCM) were used to detect the combination of GRK2 and Gßγ, the combination of PI3K and Gßγ. RESULTS: The expression level of CXCR4, GRK2, Gßγ, p-p85 and p-AKT were increased in RA synovial tissue according to the results of IHC and Western blot. In vitro, the migration of FLS was increased after stimulation of CXCL12, inhibition of Gßγ suppressed the migration and phosphorylation of p85 and AKT induced by CXCL12 in FLS, and CP-25 had the same effect as inhibition of Gßγ. The membrane expression of GRK2, Gßγ, PI3K and the combination of GRK2 and Gßγ, the combination of PI3K and Gßγ in FLS were increased after the stimulation of CXCL12, and CP-25 had an ability in reducing the membrane expression and the combination of these proteins. CONCLUSION: Excessive migration of FLS in RA was associated with over-activation of PI3K/AKT signaling, and the activity of Gßγ was involved in the over-activation of PI3K/AKT. CP-25 down-regulated CXCR4-Gßγ-PI3K/AKT signals by inhibiting GRK2-Gßγ complex membrane translocation.

Engagement of Robo1 by Slit2 induces formation of a trimeric complex consisting of Src-Robo1-E-cadherin for E-cadherin phosphorylation and epithelial-mesenchymal transition.

Loss of E-cadherin elicits epithelial-mesenchymal transition (EMT). While both the Src family of membrane-associated non-receptor tyrosine kinases (SFKs) and Slit2 binding to Roundabout 1 (Robo1) have been shown to induce E-cadherin repression and EMT, whether these two signaling pathways are mechanistically coupled remains unknown in epithelial cells. Here we found that Slit2 and Robo1 overexpression activated Src kinases for tyrosine phosphorylation, degradation of E-cadherin and induction of EMT. Specific blockade of Slit2 binding to Robo1 inactivated Src, prevented E-cadherin phosphorylation and EMT induction. Biochemically, the cytoplasmic CC3 motif of Robo1 (CC3) bound directly to the SH2 and 3 domains of c-Src and the cytoplasmic domains of E-cadherin. Slit2 induced Robo1 association with endogenous c-Src and E-cadherin, whereas ectopic expression of CC3 dissociated this protein complex in colorectal epithelial cells. These results indicate that Slit2 not only induces Robo1 binding to Src, but also recruits Src to E-cadherin for tyrosine phosphorylation of E-cadherin, leading to E-cadherin degradation and EMT induction in colorectal epithelial cells.

LECT2, a Ligand for Tie1, Plays a Crucial Role in Liver Fibrogenesis.

Liver fibrosis is a very common condition seen in millions of patients with various liver diseases, and yet no effective treatments are available owing to poorly characterized molecular pathogenesis. Here, we show that leukocyte cell-derived chemotaxin 2 (LECT2) is a functional ligand of Tie1, a poorly characterized endothelial cell (EC)-specific orphan receptor. Upon binding to Tie1, LECT2 interrupts Tie1/Tie2 heterodimerization, facilitates Tie2/Tie2 homodimerization, activates PPAR signaling, and inhibits the migration and tube formations of EC. In vivo studies showed that LECT2 overexpression inhibits portal angiogenesis, promotes sinusoid capillarization, and worsens fibrosis, whereas these changes were reversed in Lect2-KO mice. Adeno-associated viral vector serotype 9 (AAV9)-LECT2 small hairpin RNA (shRNA) treatment significantly attenuates fibrosis. Upregulation of LECT2 is associated with advanced human liver fibrosis staging. We concluded that targeting LECT2/Tie1 signaling may represent a potential therapeutic target for liver fibrosis, and serum LECT2 level may be a potential biomarker for the screening and diagnosis of liver fibrosis.

HGF/R-spondin1 rescues liver dysfunction through the induction of Lgr5+ liver stem cells.

Induction of endogenous adult stem cells by administering soluble molecules provides an advantageous approach for tissue damage repair, which could be a clinically applicable and cost-effective alternative to transplantation of embryonic or pluripotent stem cell-derived tissues for the treatment of acute organ failures. Here, we show that HGF/Rspo1 induce liver stem cells and rescue liver dysfunction. Carbon tetrachloride treatment promotes both fibrosis and Lgr5+ liver stem cell proliferation, whereas Lgr5 knockdown worsens fibrosis. Injection of HGF in combination with Rspo1 increases the number of Lgr5+ liver stem cells and improves liver function by attenuating fibrosis. We observe Lgr5+ liver stem cells in human liver fibrosis tissues, and once they are isolated, these cells are able to form organoids, and treatment with HGF/Rspo1 promotes their expansion. We suggest that Lgr5+ liver stem cells represent a valuable target for liver damage treatment, and that HGF/Rspo1 can be used to promote liver stem cell expansion.

Angelica sinensis polysaccharides promotes apoptosis in human breast cancer cells via CREB-regulated caspase-3 activation.

Angelica sinensis polysaccharide (ASP) is purified from the fresh roots of Angelica sinensis (AS). This traditional Chinese medicine has been used for thousands of years for treating gynecological diseases and used in functional foods for the prevention and treatment of various diseases, such as inflammation and cancer. The antitumor activity of ASP is related to its biological activities, because it suppresses a variety of pro-proliferative or anti-apoptotic factors that are dramatically expressed in cancer cells of given types. In this study, we show that angelica sinensis polysaccharide induced apoptosis in breast cancer cells of T47D over-expressing the Cyclic AMP response element binding protein (CREB), inducing apoptosis-related signaling pathway activity. The result also found that ASP caused cell death was linked to caspase activity, accompanied by the loss of mitochondrial membrane potential, cytochrome c release, and Bax translocation from the cytosol to the mitochondria. We found that ASP significantly affected the poly-ADP-ribose polymerase (PARP), Bcl-2 Associated X Protein (Bax), Bcl-2, Bcl-xL and apoptotic protease activating facter-1 (Apaf1) protein expression in a dose- and time-dependent manner. DAPI staining and Flow cytometry were used to analyze apoptosis. The nude mice xenograft model was used to evaluate the antitumor effect of ASP in vivo. ASP has profound antitumor effect on T47D cells, probably by inducing apoptosis through CREB signaling pathway. Thus, these results suggest that ASP would be a promising therapeutic agent for breast cancer.