Crystal structure and catalytic mechanism of the MbnBC holoenzyme required for methanobactin biosynthesis.

Methanobactins (Mbns) are a family of copper-binding peptides involved in copper uptake by methanotrophs, and are potential therapeutic agents for treating diseases characterized by disordered copper accumulation. Mbns are produced via modification of MbnA precursor peptides at cysteine residues catalyzed by the core biosynthetic machinery containing MbnB, an iron-dependent enzyme, and MbnC. However, mechanistic details underlying the catalysis of the MbnBC holoenzyme remain unclear. Here, we present crystal structures of MbnABC complexes from two distinct species, revealing that the leader peptide of the substrate MbnA binds MbnC for recruitment of the MbnBC holoenzyme, while the core peptide of MbnA resides in the catalytic cavity created by the MbnB-MbnC interaction which harbors a unique tri-iron cluster. Ligation of the substrate sulfhydryl group to the tri-iron center achieves a dioxygen-dependent reaction for oxazolone-thioamide installation. Structural analysis of the MbnABC complexes together with functional investigation of MbnB variants identified a conserved catalytic aspartate residue as a general base required for MbnBC-mediated MbnA modification. Together, our study reveals the similar architecture and function of MbnBC complexes from different species, demonstrating an evolutionarily conserved catalytic mechanism of the MbnBC holoenzymes.

Sequential administration of anti-PD-1 and anti-Tim-3 combined with an SA-GM-CSF-anchored vaccine overcomes adaptive immune resistance to reject established bladder cancer.

Program death receptor-1 (PD-1) and T-cell immunoglobulin and mucin domain-containing protein-3 (Tim-3) play an important role in tumor immune evasion. PD-1 blockade could produce an effective anti-tumor effect but the response rate was low due to lacking of tumor infiltrating lymphocytes (TILs) and existing of other negative regulatory pathways. Streptavidin(SA)-GM-CSF surface-anchored tumor cells vaccine could induce specific anti-tumor immune response. However, this vaccine failed to induce regression of established tumor because it also up-regulated PD-1 expression on tumor cells dependent on IFNγ and up-regulated PD-1/Tim-3 expression on CD8+ TILs. Subsets of CD8+ TILs assay showed that PD-1 expression was closely associated with CD8+ TILs exhaustion, and Tim-3 expression was closely correlated with secretion function but not proliferation of CD8+ TILs. Sequential administration of anti-PD-1 and anti-Tim-3 could further improve the efficacy of SA-GM-CSF-anchored vaccine therapy, and tumor regression was noted in over 50%. This triple therapy improves the specific cytotoxic activity and decreased the apoptosis of CD8+ TILs. These findings indicated that this triple therapy could induce a more robust anti-tumor immune response.

Increased Tim-3 expression on TILs during treatment with the Anchored GM-CSF vaccine and anti-PD-1 antibodies is inversely correlated with response in prostate cancer.

Programmed death receptor-1 (PD-1) and T cell immunoglobulin and mucin domain-containing protein-3 (Tim-3) play important roles in tumor immune evasion. PD-1 blockade could produce an effective antitumor effect in many solid tumors except prostate cancer (PCa) because of rare programmed death ligand-1 (PD-L1) expression on PCa cells. Streptavidin (SA)-GM-CSF surface-anchored tumor cell (Anchored GM-CSF) vaccines could increase the number of tumor-infiltrating lymphocytes (TILs) and induce specific antitumor immune responses. The Anchored-GM-CSF vaccine and anti-PD-1 antibodies exerted synergistic effects in mouse models of PCa metastasis. However, the response rate was low due to the presence of other negative regulatory pathways. Tim-3 expression could be upregulated at resistance to combination therapy with anti-PD-1 antibodies and the Anchored GM-CSF vaccine. Sequential administration of anti-PD-1 and anti-Tim-3 antibodies could further improve the efficacy of the Anchored GM-CSF vaccine therapy, and tumor regression was noted in over 60% of animals. This triple therapy improved the specific cytotoxic activity, proliferation and secretion of CD8+ TILs and reduced the production of tumor-promoting cytokines. These findings indicated that this triple therapy could induce a robust antitumor immune response in mouse models of PCa.

LncARSR sponges miR-129-5p to promote proliferation and metastasis of bladder cancer cells through increasing SOX4 expression.

Emerging evidences have indicated that long non-coding RNAs (lncRNAs) are potential biomarkers, playing important roles in the development of cancer. LncRNA Activated in RCC with Sunitinib Resistance (lncARSR) is a novel lncRNA that functions as a potential biomarker and is involved in the progression of cancers. However, the clinical significance and molecular mechanism of lncARSR in bladder cancer (Bca) remains unknow. In this study, we discovered that lncARSR was significantly up-regulated in bladder cancer. In addition, increased expression of lncARSR was positively correlated with higher histological grade and larger tumor size. Further experiments demonstrated that suppression of lncARSR attenuated the proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) process of Bca cells. Mechanistically, lncARSR was mainly located in the cytoplasm and acted as a miRNA sponge to positively modulate the expression of Sex-determining region Y-related high-mobility-group box transcription factor 4 (SOX4) via sponging miR-129-5p and subsequently promoted the proliferation and metastasis of Bca cells, thus playing an oncogenic role in Bca pathogenesis. In conclusion, our study indicated that lncARSR plays a critical regulatory role in Bca cells and lncARSR may serve as a potential diagnostic biomarker and therapeutic target for bladder cancer.

The prognostic value of the site of invasion in T3aN0M0 clear cell renal cell carcinoma.

BACKGROUND: The 7th Tumor-Node-Metastasis system for clear cell renal cell carcinoma (ccRCC) classified renal sinus fat invasion (SFI), perirenal fat invasion (PFI), or renal vein invasion (RVI) as stage pT3a. However, their close interactions and prognostic value of them remain controversial. The goal of this study is to further analyze their prognostic values for patients with T3aN0M0 ccRCC. METHODS: The data of 1,869 pT3aN0M0 ccRCC patients receiving the radical nephrectomy surgery were collected from the National Cancer Institute Surveillance, Epidemiology, and End Results database of United states from 2010 to 2014. These Patients were grouped as SFI, PFI, SFI + RVI, SFI + PFI, PFI + RVI, and SFI + PFI + RVI according to their corresponding manifestations. Cancer-specific survival (CSS) was determined using the Kaplan-Meier method. Univariate and Multivariate cox proportional-hazards regression methods were used to evaluate the impacts of clinical pathologic parameters on CSS. RESULTS: Patients with SFI or PFI alone had the similar CSS (P = 0.286) and patients with SFI + PFI + RVI had the worst outcomes. Moreover, significantly more patients with SFI + PFI + RVI had tumor diameter ≥7cm than patients with PFI + RVI, SFI + PFI (68.80% vs. 65.32%, 58.77%, and 55.04%, P = 0.026), respectively. Multivariable analysis showed that RVI + PFI (P = 0.013) and PFI + SFI + RVI (P = 0.011) were the independent factors of CSS. CONCLUSIONS: The results suggest that invasion location can help distinguish patients with T3aN0M0 ccRCC with increased risk of cancer-related mortality.

Combination immunotherapy with interleukin-2 surface-modified tumor cell vaccine and programmed death receptor-1 blockade against renal cell carcinoma.

Immunotherapy may be an effective way to prevent postoperative recurrence of renal cell carcinoma. Streptavidin-interleukin-2 (SA-IL-2) surface-modified tumor cell vaccine developed through our protein-anchor technology could induce specific antitumor T-cell responses, but this immunotherapy cannot completely eradicate the tumor. These effector T cells highly expressed programmed death receptor-1 (PD-1), and the expression of programmed death ligand-1 (PD-L1) in the tumor environment also was upregulated after SA-IL-2-modified vaccine therapy. PD-1/PD-L1 interaction promotes tumor immune evasion. Adding PD-1 blockade to SA-IL-2-modified vaccine therapy increased the number of CD4+ , CD8+ and CD8+ interferon-γ+ but not CD4+ Foxp3+ T cells. PD-1 blockade could rescue the activity of tumor-specific T lymphocytes induced by the SA-IL-2-modified vaccine. Combination therapy delayed tumor growth and protected mice against a second Renca cells but not melanoma cells challenge. Taken together, PD-1 blockade could reverse immune evasion in the treatment with SA-IL-2-modified vaccine, and eventually induce a stronger specific antitumor immune response against renal cell carcinoma.

PD-1 Blockade Overcomes Adaptive Immune Resistance in Treatment with Anchored-GM-CSF Bladder Cancer Cells Vaccine.

Purpose: To investigate whether PD-L1 mediated adaptive resistance could occur in treatment with Anchored-GM-CSF vaccine and whether PD-1 blockade combined with Anchored-GM-CSF vaccine could induce a greater anti-tumor immune response than either immunotherapy alone. Materials and Methods: After establishing long-established subcutaneous metastasis bladder cancer models, mice were treated with Anchored-GM-CSF vaccine and/or anti-PD-1 antibody. T-lymphocyte-cytotoxicity, flow cytometric analysis, immunohistochemical, immunofluorescence staining, CD8+ -T cell apoptosis and enzyme-linked immunosorbent assays were performed to evaluate the efficacy of combination therapy with anchored-GM-CSF vaccine and PD-1 blockade and to explore the related mechanism. Results: Anchored-GM-CSF vaccine could significantly increase the number of mature DCs and up-regulate PD-L1 expression dependent on IFN-γ released from CD8+ T cells. Anchored-GM-CSF vaccine combined with anti-PD-1 antibody could effectively inhibit tumor growth and even cause regression of the established tumor. More CD4+ and CD8+ T cells appeared at tumor sites and in peripheral blood in the combination therapy group than in the control groups. Splenocytes from mice of the combination therapy group exhibited the most potent cytotoxicity to MB49 cells. Apoptotic assays showed that PD-1 blockade could significantly reduce CD8+ T cells apoptosis. Conclusions: Anchored-GM-CSF vaccines and anti-PD-1 antibodies have synergistic effects in metastatic bladder cancer treatment. PD-1 blockade can overcome immune resistance in treatment with the Anchored-GM-CSF vaccine, while Anchored-GM-CSF vaccine can enhance the efficacy of PD-1 blockade therapy.

LncRNA-SNHG7 regulates proliferation, apoptosis and invasion of bladder cancer cells assurance guidelines.

PURPOSE: The purpose of this study was to investigate the expression of long-chain non-coding RNA (lncRNA) SNHG7 in bladder cancer tissues and cell lines, and to explore its impact on bladder cancer cell proliferation, apoptosis and invasion processes. METHODS: Bladder cancer tissues and near-cancer tissues were collected. The expression of lncRNA-SNHG7 in tissues and cell lines was detected by real-time PCR (RT-PCR). The expression of lncRNA-SNHG7 was downregulated by RNA interference (siRNA) as detected by RT-PCR that was used to detect the interference efficiency. CCK-8, flow cytometry and Transwell assays were used to evaluate the effect of lncRNASNHG7 on the proliferation, apoptosis and invasion capability of bladder cancer cells. The downregulation effect of lncRNA-SNHG7 on Epithelial-Mesenchymal Transition (EMT) related markers was tested by westernblot. RESULTS: lncRNA-SNHG7 was upregulated in bladder cancer cell lines. After the expression of lncRNA-SNHG7 was downregulated, the proliferation of bladder cancer cells was decreased, the apoptosis was increased, and the invasion ability of cells was decreased. The expression of E-cadherin was increased, but the expression of N-cadherin, vimentin and snail were decreased. Increased expression of lncRNASNHG7 in cancer tissues was significantly related to tumor size, metastasis and stage. CONCLUSIONS: This study showed that lncRNA -SNHG7 is overexpressed in bladder cancer tissues and cells. Downregulation of lncRNA-SNHG7 can inhibit the proliferation of bladder cancer cells and promote apoptosis, as well as inhibit cell invasion, which provides a potential molecular target for future tumor targeted therapy.

The cGas-Sting Signaling Pathway Is Required for the Innate Immune Response Against Ectromelia Virus.

Activation of the DNA-dependent innate immune pathway plays a pivotal role in the host defense against poxvirus. Cyclic GMP-AMP synthase (cGAS) is a key cytosolic DNA sensor that produces the cyclic dinucleotide cGMP-AMP (cGAMP) upon activation, which triggers stimulator of interferon genes (STING), leading to type I Interferons (IFNs) production and an antiviral response. Ectromelia virus (ECTV) has emerged as a valuable model for investigating the host-Orthopoxvirus relationship. However, the role of cGas-Sting pathway in response to ECTV is not clearly understood. Here, we showed that murine cells (L929 and RAW264.7) mount type I IFN responses to ECTV that are dependent upon cGas, Sting, TANK binding kinase 1 (Tbk1), and interferon regulatory factor 3 (Irf3) signaling. Disruption of cGas or Sting expression in mouse macrophages blocked the type I IFN production and facilitated ECTV replication. Consistently, mice deficient in cGas or Sting exhibited lower type I IFN levels and higher viral loads, and are more susceptible to mousepox. Collectively, our study indicates that the cGas-Sting pathway is critical for sensing of ECTV infection, inducing the type I IFN production, and controlling ECTV replication.

Ileal versus sigmoid neobladder as bladder substitute after radical cystectomy for bladder cancer: A meta-analysis.

PURPOSE: To evaluate and compare the functional outcomes of ileal and sigmoid neobladders in patients underwent radical cystectomy. METHODS: Relevant studies were identified by searching PubMed, Embase, and Cochrane Library. The studies comparing the functional outcomes of sigmoid neobladder (SN) and ileal neobladder (IN) in patients underwent radical cystectomy were included. RESULTS: A total of 12 cohort studies were included in this meta-analysis. From our analysis, more early complications were observed in SN group than in IN group (RR = 1.37, 95% CI: 1.03-1.81). Both daytime and nighttime continence rates were significantly better in IN group than in SN group (RR = 0.87, 95%CI: 0.81-0.94) (RR = 0.73, 95%CI: 0.60-0.90). More patients could spontaneous voiding in SN group than in IN group (RR = 1.12, 95%CI: 1.00-1.26). According to the urodynamic study, ileal neobladder exhibited bigger capacity (WMD = -84.93, 95%CI: -160.36 to -9.50), lower pressure at capacity (WMD = 11.18, 95%CI: 4.29-18.06), better compliance (WMD = -25.55, 95%CI: -32.45 to -18.64), and greater post-void residual volume(WMD = -23.48, 95%CI: -36.75 to -10.21); There was no significant difference in the max voiding flow rate or void volume between the two groups (WMD = -1.00, 95%CI: -3.73-1.73) (WMD = -27.00, 95%CI: 70.05-16.06). No significant difference was found in the serum creatinine between the two groups (WMD = -0.05, 95%CI: -0.12-0.03). CONCLUSIONS: Ileal neobladder seems able to provide more favorable patient's satisfaction, while sigmoid neobladder may provide a better chance of spontaneous voiding. This meta-analysis may provide some useful evidences for urological surgeons to choose the ideal bladder substitute for patients underwent radical cystectomy.

The association of tea consumption with bladder cancer risk: a meta-analysis.

The association between tea consumption and bladder cancer has been confirmed in several animal studies, but one epidemiological study in 2001 showed no association between them. In order to provide an accurate assessment of this, we conducted a meta-analysis on tea consumption and bladder cancer risk. Studies were identified by a literature search in PubMed from January 1980 to March 2012 and the reference lists of relevant studies. Random effect models were used to calculate summary relative risk estimates (RR) and their corresponding 95% confidence intervals (CI) based on high contrast to low intake values. Twenty-four publications (6 cohort studies and 18 case-control studies) based on consumption of overall tea, black tea, and green tea to bladder cancer risk were included in this analysis. For overall tea, the summary RR indicated no association between tea consumption and bladder cancer (RR= 1.09, 95%CI: 0.85-1.40). In subgroup analyses, we found a moderate increase of bladder cancer risk in smoking group (RR= 1.77, 95%CI: 1.04-3.01). In the black tea group, no statistically significant association was observed (RR= 0.84, 95%CI: 0.70-1.01). Interestingly, in the subgroup of sex, a protective effect was observed between tea consumption and bladder cancer risk in female (RR= 0.61, 95%CI: 0.38- 0.98). For green tea group, there was no relationship associated with bladder cancer risk (RR= 1.03, 95%CI: 0.82- 1.31). In conclusion, our data suggest that high overall tea intake in smokers increased the risk of bladder cancer, and high black tea intake in female may reduce the risk of bladder cancer.

Serum cystatin C is an early biomarker for assessment of renal function in burn patients.

BACKGROUND: Early detection of renal dysfunction is important in burn patients. This study evaluated whether serum cystatin C (CysC) is a potentially accurate and sensitive marker for identification of reduced glomerular filtration rate (GFR) and the risk factors of impaired renal function in major burn patients. METHODS: A total of 48 adult patients with major burn injury were enrolled. Renal function was assessed using serum creatinine (sCr), 24-h urinary creatinine clearance (24-hCrCl), sCr-based formulae and CysC-based formulae on the second day and seventh day post-burn. RESULTS: There was a high prevalence (27.1%) of acute impaired renal function in major burn patients in the first week post-burn. CysC-based formulae for estimated GFR (eGFR) are more accurate and sensitive for detection of impaired renal function than sCr-based formulae. Multivariate logistic regression analysis demonstrated that age (OR, 2.08; 95% CI 1.26-4.77) and the percentage of burn area (OR, 3.41; 95% CI 1.64-6.95) were independent risk factors of impaired renal function. CONCLUSIONS: The results of this study suggest that CysC is a more accurate and sensitive marker for identification of acute impaired renal function in major burn patients than sCr. It is important to measure CysC and calculate eGFR to prevent acute renal failure and modify drug doses in burn patients, especially those of older age and with major burn areas.