Direct Measurement of pH Evolution in Aerosol Microdroplets Undergoing Ammonium Depletion: A Surface-Enhanced Raman Spectroscopy Approach.

Accurately measuring the pH of atmospheric aerosols is a prerequisite for understanding the multiphase chemistry that profoundly affects the environment and climate systems. Despite the advancements of experimental techniques for in situ pH measurements in aerosols, current studies are limited to measuring the static pH of aerosol microdroplets with an unperturbed composition. This steady-state scenario, however, deviates from the real-world aerosols undergoing atmospheric aging reactions, specifically, those characterized with a spontaneous displacement of strong bases (or acids) with high volatility. Here, we introduce a continuous and in situ measurement of aerosol pH by using a 4-mercaptopyridine-functionalized silver nanoparticle probe and surface-enhanced Raman spectroscopy. We find that the ammonium depletion─a spontaneous displacement of ammonium by dicarboxylic acid salts─continuously acidifies aerosol water over time. The decaying trends of pH in the aerosols under various humidity conditions can be unified with a universal exponential function. Such an exponentially decaying function further indicates that the ammonium depletion reaction is a self-limiting process. Our technique can be applied to study the dynamic change of aerosol acidity during the complex atmospheric aging processes, toward elucidating their implications on atmospheric chloride, nitrate, and ammonium cycles.

Clinical and prognostic implications of pretreatment albumin to C-reactive protein ratio in patients with hepatocellular carcinoma.

BACKGROUND: Despite recent advances in the treatments of hepatocellular carcinoma (HCC), the prognosis of HCC patients remains controversial. The purpose of this study was to investigate the prognostic performance of pretreatment albumin to C-reactive protein ratio (ACR) in patients with HCC. METHODS: This study included 409 initially diagnosed HCC patients retrospectively. The optimal cut-off points for distinguishing high and low ACR value was determined by the X-tile software. The chi-squared test was used for comparing the baseline clinicopathologic parameters in different groups and subgroups. The Cox regression with log-rank tests was used to analyze OS and DFS, and Kaplan-Meier curves was used to estimate the prognosis of HCC patients. RESULTS: Patients with lower ACR were significantly correlated with advanced clinical parameters, using a cut-off points of 5.4 (high ACR, n = 236 vs. low ACR, n = 173). Multivariate analysis demonstrated that ACR was associated with OS (HR = 0.544, 95% CI: 0.385-0.769, p = 0.001), with DFS (HR = 0.550, 95% CI: 0.392-0.772, p = 0.001). Treatment exposure (HR = 2.191; 95% CI: 1.533-3.132; p <  0.001), tumor size (HR = 1.973; 95% CI: 1.230-3.164; p = 0.005), serum AFP level (HR = 1.752; 95% CI: 1.277-2.403; p = 0.001), and TNM stage (HR = 0.470; 95% CI: 0.319-2.504; p <  0.001), were independent factors for OS in HCC patients. Treatment exposure (HR = 2.244; 95% CI: 1.590-3.166; p <  0.001), TNM stage (HR = 2.075; 95% CI: 1.436-3.000; p <  0.001), serum AFP level (HR = 1.819; 95% CI: 1.340-2.469; p = 0.001), tumor size (HR = 1.730; 95% CI: 1.113-2.689; p = 0.015), and ACR (HR = 0.550; 95% CI: 0.392-0.772; p = 0.001) were independent factors for DFS in HCC patients. CONCLUSIONS: Pretreatment ACR is a convenient and useful parameter for HCC patients predicting OS and DFS. Lower ACR was associated with advanced TNM stage, larger tumor size, and a high concentration of AFP. These results may help to design strategies to personalize management approaches among HCC patients.

Mesothelin-targeting chimeric antigen receptor-modified T cells by piggyBac transposon system suppress the growth of bile duct carcinoma.

Chimeric antigen receptor modified T cell-based immunotherapy is revolutionizing the field of cancer treatment. However, its potential in treating bile duct carcinoma has not been fully explored. Herein, we developed the second-generation mesothelin-targeting chimeric antigen receptor-modified T cells with the 4-1BB co-stimulatory module by the piggyBac transposon system. Mesothelin-targeting chimeric antigen receptor was expressed by 66.0% of mesothelin-targeting chimeric antigen receptor-modified T cells post electrophoretic transfection and stimulation with K562-meso cells; the expressions of activation markers were tested by flow cytometry assay and showed greater activation of mesothelin-targeting chimeric antigen receptor-modified T cells than control T cells (CD107α: 71.9% vs 48.6%; CD27: 92.1% vs 61.8%; CD137: 55.5% vs 8.4%; CD28: 98.0% vs 82.1%; CD134: 37.5% vs 10.4%). Furthermore, mesothelin-targeting chimeric antigen receptor-modified T cells exerted cytotoxicity toward mesothelin-expressing EH-CA1b and EH-CA1a cells in an effector-to-target ratio-dependent manner, while leaving mesothelin-negative GSC-SD and EH-GB1 cells and normal liver L02 cells almost unharmed. Mesothelin-targeting chimeric antigen receptor-modified T cells secreted cytokines at higher levels when co-cultured with mesothelin-positive EH-CA1a and EH-CA1b cells than with mesothelin-negative GSC-SD and EH-GB1 cells. Enhanced cytotoxicity and cytokine secretion of mesothelin-targeting chimeric antigen receptor-modified T cells compared to control T cells were also observed when co-cultured with 293-meso cells (interferon γ: 85.1% ± 1.47% vs 8.3% ± 2.50%, p = 0.000; tumor necrosis factor α: 90.9% ± 4.67% vs 18.5% ± 3.62%, p = 0.0004; interleukin 2: 60.8% ± 2.00% vs 15.6% ± 2.06%, p = 0.002; interleukin 6: 6.4% ± 2.95% vs 1.7% ± 0.63%, p = 0.055). In addition, mesothelin-targeting chimeric antigen receptor-modified T cells showed greater inhibitory and proliferative capability than control T cells within EH-CA1a cell xenografts. This study shows the potential of mesothelin-targeting chimeric antigen receptor-modified T cells in treating bile duct carcinoma.

Synergistic antitumor activity of triple-regulated oncolytic adenovirus with VSTM1 and daunorubicin in leukemic cells.

V-set and transmembrane domain-containing 1 (VSTM1), which is downregulated in bone marrow cells from leukemia patients, may provide a diagnostic and treatment target. Here, a triple-regulated oncolytic adenovirus was constructed to carry a VSTM1 gene expression cassette, SG611-VSTM1, and contained the E1a gene with a 24-nucleotide deletion within the CR2 region under control of the human telomerase reverse transcriptase promoter, E1b gene directed by the hypoxia response element, and VSTM1 gene controlled by the cytomegalovirus promoter. Real-time quantitative PCR and Western blot analyses showed that SG611-VSTM1 expressed VSTM1 highly efficiently in the human leukemic cell line K562 compared with SG611. In Cell Counting Kit-8 and flow cytometric assays, SG611-VSTM1 exhibited more potent anti-proliferative and pro-apoptotic effects in leukemic cells compared with SG611 and exerted synergistic cytotoxicity with low-dose daunorubicin (DNR) in vitro. In xenograft models, SG611-VSTM1 intratumorally injected at a dose of 1 × 10(9) plaque forming units combined with intraperitoneally injected low-dose DNR displayed significantly stronger antitumor effects than either treatment alone. Histopathologic examination revealed that SG611-VSTM1 induced apoptosis of leukemic cells. These results implicate an important role for VSTM1 in the pathogenesis of leukemia, and SG611-VSTM1 may be a promising agent for enhancing chemosensitivity in leukemia therapy.

Elevated Preoperative Serum Alanine Aminotransferase/Aspartate Aminotransferase (ALT/AST) Ratio Is Associated with Better Prognosis in Patients Undergoing Curative Treatment for Gastric Adenocarcinoma.

The level of anine aminotransferase/aspartate aminotransferase (ALT/AST) ratio in the serum was often used to assess liver injury. Whether the ALT/AST ratio (LSR) was associated with prognosis for gastric adenocarcinoma (GA) has not been reported in the literature. Our aim was to investigate the prognostic value of the preoperative LSR in patients with GA. A retrospective study was performed in 231 patients with GA undergoing curative resection. The medical records collected include clinical information and laboratory results. We investigated the correlations between the preoperative LSR and overall survival (OS). Survival analysis was conducted with the Kaplan-Meier method, and Cox regression analysis was used to determine significant independent prognostic factors for predicting survival. A p value of <0.05 was considered to be statistically significant. A total of 231 patients were finally enrolled. The median overall survival was 47 months. Multivariate analysis indicated that preoperative LSR was an independent prognostic factor in GA. Patients with LSR ≤ 0.80 had a greater risk of death than those with LSR > 0.80. The LSR was independently associated with OS in patients with GA (hazard ratio: 0.610; 95% confidence interval: 0.388-0.958; p = 0.032), along with tumor stages (hazard ratio: 3.118; 95% confidence interval: 2.044-4.756; p < 0.001) and distant metastases (hazard ratio: 1.957; 95% confidence interval: 1.119-3.422; p = 0.019). Our study first established a connection between the preoperative LSR and patients undergoing curative resection for GA, suggesting that LSR was a simple, inexpensive, and easily measurable marker as a prognostic factor, and may help to identify high-risk patients for treatment decisions.

Argonaute 2 promotes angiogenesis via the PTEN/VEGF signaling pathway in human hepatocellular carcinoma.

AIM: Argonaute2 (AGO2) protein is the active part of RNA-induced silencing complex, cleaving the target mRNA strand complementary to their bound siRNA. An increasing number of miRNAs has been identified as essential to angiogenesis of hepatocellular carcinoma (HCC). In this study we investigated how AGO2 affected HCC angiogenesis. METHODS: Human HCC cell lines HepG2, Hep3B, Huh7, SMMC-7721, Bel-7404, MHCC97-H and LM-3, and human umbilical vein endothelial cells (HUVEC) were tested. The expression of AGO2 in HCC cells was knocked down with siRNA and restored using recombinant adenovirus expressing Ago2. The levels of relevant mRNAs and proteins were examined using RT-PCR, Western blot and EILSA. Nude mice were implanted with Huh7 or SMMC-7721 cells, and tumor volumes were measured. After the mice were euthanized, the xenograft tumors were used for immunohistological analysis. RESULTS: In 6 HCC cell lines, AGO2 protein expression was significantly correlated with VEGF expression (r=+0.79), and with VEGF secretion (r=+0.852). Knockdown of Ago2 in Huh7 cells and SMMC-7721 cells substantially decreased VEGF expression, whereas the restoration of AGO2 reversed both VEGF expression and secretion. Furthermore, knockdown of Ago2 significantly up-regulated the expression of PTEN (a tumor suppressor involved in the inhibition of HCC angiogenesis), and vice versa. Moreover, the specific PTEN inhibitor bisperoxovanadate (7, 14, 28 nmol/L) dose-dependently restored the expression of VEGF and the capacity of HCC cells to induce HUVECs to form capillary tubule structures. In the xenograft nude mice, knockdown of Ago2 markedly suppressed the tumor growth and decreased PTEN expression and CD31-positive microvascular in the xenograft tumors. CONCLUSION: A direct relationship exists between the miRNA processing machinery AGO2 and HCC angiogenesis that is mediated by the AGO2/PTEN/VEGF signaling pathway. The results suggest the high value of Ago2 knockdown in anti-angiogenesis therapy for HCC.

Traditional Chinese medicine bufalin inhibits infectious hematopoietic necrosis virus infection in vitro and in vivo.

Infectious hematopoietic necrosis virus (IHNV) causes infectious hematopoietic necrosis and severe economic losses to salmon and trout aquaculture worldwide. Currently, the only commercial vaccine against IHNV is a DNA vaccine with some biosafety concerns. Hence, more effective vaccines and antiviral drugs are needed to prevent IHNV infection. In this study, 1,483 compounds were screened from a traditional Chinese medicine monomer library, and bufalin showed potential antiviral activity against IHNV. The 50% cytotoxic concentration of bufalin was >20 µM, and the 50% inhibitory concentration was 0.1223 µΜ against IHNV. Bufalin showed the inhibition of diverse IHNV strains in vitro, which confirmed that it had an inhibitory effect against all IHNV strains, rather than random activity against a single strain. The bufalin-mediated block of IHNV infection occurred at the viral attachment and RNA replication stages, but not internalization. Bufalin also inhibited IHNV infection in vivo and significantly increased the survival of rainbow trout compared with the mock drug-treated group, and this was confirmed by in vivo viral load monitoring. Our data showed that the anti-IHNV activity of bufalin was proportional to extracellular Na+ concentration and inversely proportional to extracellular K+ concentration, and bufalin may inhibit IHNV infection by targeting Na+/K+-ATPase. The in vitro and in vivo studies showed that bufalin significantly inhibited IHNV infection and may be a promising candidate drug against the disease in rainbow trout. IMPORTANCE: Infectious hematopoietic necrosis virus (IHNV) is the pathogen of infectious hematopoietic necrosis (IHN) which outbreak often causes huge economic losses and hampers the healthy development of salmon and trout farming. Currently, there is only one approved DNA vaccine for IHN worldwide, but it faces some biosafety problems. Hence, more effective vaccines and antiviral drugs are needed to prevent IHNV infection. In this study, we report that bufalin, a traditional Chinese medicine, shows potential antiviral activity against IHNV both in vitro and in vivo. The bufalin-mediated block of IHNV infection occurred at the viral attachment and RNA replication stages, but not internalization, and bufalin inhibited IHNV infection by targeting Na+/K+-ATPase. The in vitro and in vivo studies showed that bufalin significantly inhibited IHNV infection and may be a promising candidate drug against the disease in rainbow trout.

[Potential molecular mechanisms of QiZhenYuanDan in treatment of atherosclerosis based on network pharmacology].

Objective: By means of network pharmacology, potential targets and molecular pathways of QiZhenYuanDan in the treatment of atherosclerosis (AS) were studied. Methods: TCMSP database was used to obtain the main active components and target information of Astragali Radix, Fructus Ligustri Lucidi, Corydalis Rhizoma and Salvia Miltiorrhiza in QiZhenYuanDan. Disease targets were retrieved by OMIM and other databases. Molecular networks were constructed using Cytoscape. STRING database was searched and PPI network diagram was drawn to obtain the key targets of QiZhenYuanDan in the treatment of AS; and the targets were uploaded to Metascape data platform for GO and KEGG analysis. Results: There were 118 targets of intersection between QiZhenYuanDan and AS, which were used as the predicted targets of QiZhenYuanDan on AS. GO analysis showed that the biological functions of QiZhenYuanDan in the treatment of AS targets mainly involved biological processes, such as the cytokine-mediated signaling pathway, cytokine receptor binding. KEGG pathway was mainly enriched in 155 signaling pathways, including PI3K-Akt, HIF-1, NF-κB signal pathway and inflammatory bowel disease pathway. Conclusion: Based on the result of network pharmacology study, the mechanisms of Qizhenyuandan for AS treatment was preliminarily revealed. The active ingredients such as quercetin and kaempferol act on targets such as IL-6 and PI3K-Akt, and exert anti-AS effects by inhibiting apoptosis, oxidative stress, as well as inflammatory responses. Our result indicates that QiZhenYuanDan exhibits anti-AS effect via a multi-component, multi-target and multi-route synergistic process.

Correlations between hippocampal functional connectivity, structural changes, and clinical data in patients with relapsing-remitting multiple sclerosis: a case-control study using multimodal magnetic resonance imaging.

Multiple sclerosis is associated with structural and functional brain alterations leading to cognitive impairments across multiple domains including attention, memory, and the speed of information processing. The hippocampus, which is a brain important structure involved in memory, undergoes microstructural changes in the early stage of multiple sclerosis. In this study, we analyzed hippocampal function and structure in patients with relapsing-remitting multiple sclerosis and explored correlations between the functional connectivity of the hippocampus to the whole brain, changes in local brain function and microstructure, and cognitive function at rest. We retrospectively analyzed data from 20 relapsing-remitting multiple sclerosis patients admitted to the Department of Neurology at the China-Japan Union Hospital of Jilin University, China, from April 2015 to November 2019. Sixteen healthy volunteers were recruited as the healthy control group. All participants were evaluated using a scale of extended disability status and the Montreal cognitive assessment within 1 week before and after head diffusion tensor imaging and functional magnetic resonance imaging. Compared with the healthy control group, the patients with relapsing-remitting multiple sclerosis had lower Montreal cognitive assessment scores and regions of simultaneously enhanced and attenuated whole-brain functional connectivity and local functional connectivity in the bilateral hippocampus. Hippocampal diffusion tensor imaging data showed that, compared with the healthy control group, patients with relapsing-remitting multiple sclerosis had lower hippocampal fractional anisotropy values and higher mean diffusivity values, suggesting abnormal hippocampal structure. The left hippocampus whole-brain functional connectivity was negatively correlated with the Montreal cognitive assessment score (r = -0.698, P = 0.025), and whole-brain functional connectivity of the right hippocampus was negatively correlated with extended disability status scale score (r = -0.649, P = 0.042). The mean diffusivity value of the left hippocampus was negatively correlated with the Montreal cognitive assessment score (r = -0.729, P = 0.017) and positively correlated with the extended disability status scale score (r = 0.653, P = 0.041). The right hippocampal mean diffusivity value was positively correlated with the extended disability status scale score (r = 0.684, P = 0.029). These data suggest that the functional connectivity and presence of structural abnormalities in the hippocampus in patients with relapse-remission multiple sclerosis are correlated with the degree of cognitive function and extent of disability. This study was approved by the Ethics Committee of China-Japan Union Hospital of Jilin University, China (approval No. 201702202) on February 22, 2017.

[In vitro construction and confirmation of adenovirus containing a mifepristone (RU486)-inducible regulation system for NRF2 gene].

OBJECTIVE: To construct an adenovirus containing a mifepristone (RU486)-inducible regulation system for NRF2 gene, express the product in H460 cell and verify whether the mentioned system can control the gene expression and assess its efficiency. METHODS: A RU486-inducible regulation system for Nrf2 gene was introduced into an adenovirus. The confirmation was performed through the LUC and Dsred genes. And the expression pattern of Nrf2 at the viral level was examined by Western blot and RT-PCR (reverse transcription-polymerase chain reaction). RESULTS: The expressions of LUC and Dsred showed a rising trend with the incremental dose of RU486. After the transfection H460 cell with Ad-RUNrf2, the results of RT-PCR and Western blot demonstrated that the expression of Nrf2 was elevated with a rising dose of RU486. After the removal of RU486, the expression of Nrf2 was reduced. CONCLUSION: The construction of an adenovirus carrying Nrf2 gene regulated by a RU486-inducible system is successful, and RU486 can adjust the cellular expression of Nrf2 factor. The LUC and the Dsred expression assumes the dosage dependence along with RU486 to increase; after the Ad-RUNrf2 infects the H460 cell, through RTPCR and Western the Blot result demonstrated that the expression of Nrf2 increases along with the RU486 dosage increases, after removing RU486, the Nrf2 expression is weaken. Showing the construction of the adenovirus carrying Nrf2 gene regulated by the mifepristone (RU486)-inducible system is successful, and RU486 can adjust the Nrf2 factor in the cell the expression.

[Establishment and characterization of a human gallbladder carcinoma cell line EH-GB1 originated from a metastatic tumor].

OBJECTIVE: To establish a human gallbladder carcinoma cell line derived from a metastatic gallbladder carcinoma and identify its biological characteristics. METHODS: Tissue samples were separated from the surgical specimen obtained from a patient with metastatic carcinoma and single-cell suspension was prepared. Then the cells were cultured in DMEM medium supplemented with 15% fetal bovine serum. The morphology of tumor cells was observed under an electron microscope. The cell growth curve was plotted. The tumorigenicity of the cell line was studied by subcutaneous inoculation in SCID mice. The cells were infected by lentiviral vector carrying fluorescent report genes (lenti-GFP and lenti-Red2) separately for expressions of GFP and Red2, respectively. RESULTS: A novel metastatic gallbladder carcinoma cell line was successfully established and named "EH-GB1". It could be passaged for over 20 generations with typical malignant epithelial morphology and a stable growth cycle of 24 h. Tumors were formed in all of the 10 SCID mice inoculated with EH-GB1 cells subcutaneously, and the tumor cells were tumor marker CA19-9-positive. Continuous expressions of fluorescent report genes were observed in EH-GB1 cells infected by lenti-GFP and lenti-Red2. CONCLUSION: EH-GB1 cells might be the first stable cell line of human gallbladder carcinoma established from a metastatic focus of gallbladder carcinoma. This cell line with continuous expressions of GFP and Red2 might be a novel and perfect experimental model for clinical and basic research on gallbladder carcinoma.

A SIRPα-Fc fusion protein enhances the antitumor effect of oncolytic adenovirus against ovarian cancer.

Oncolytic viruses armed with therapeutic transgenes of interest show great potential in cancer immunotherapy. Here, a novel oncolytic adenovirus carrying a signal regulatory protein-α (SIRPα)-IgG1 Fc fusion gene (termed SG635-SF) was constructed, which could block the CD47 'don't eat me' signal of cancer cells. A strong promoter sequence (CCAU) was chosen to control the expression of the SF fusion protein, and a 5/35 chimeric fiber was utilized to enhance the efficiency of infection. As a result, SG635-SF was found to specifically proliferate in hTERT-positive cancer cells and largely increased the abundance of the SF gene. The SF fusion protein was effectively detected, and CD47 was successfully blocked in SK-OV3 and HO8910 ovarian cancer cells expressing high levels of CD47. Although the ability to induce cell cycle arrest and cell death was comparable to that of the control empty SG635 oncolytic adenovirus in vitro, the antitumor effect of SG635-SF was significantly superior to that of SG635 in vivo. Furthermore, CD47 was largely blocked and macrophage infiltration distinctly increased in xenograft tissues of SK-OV3 cells but not in those of CD47-negative HepG2 cells, indicating that the enhanced antitumor effect of SG635-SF was CD47-dependent. Collectively, these findings highlight a potent antitumor effect of SG635-SF in the treatment of CD47-positive cancers.

A novel triple-regulated oncolytic adenovirus carrying PDCD5 gene exerts potent antitumor efficacy on common human leukemic cell lines.

PDCD5 (programmed cell death 5) accelerates apoptosis of certain tumor cells and the replication-defective Ad-PDCD5 may be a promising agent for enhancing chemosensitivity. In this study, a triple-regulated conditionally replicating adenoviruses (CRAd) carrying PDCD5 gene expression cassette, SG611-PDCD5, was engineered. In SG611-PDCD5, the E1a gene with a deletion of 24 nucleotides within CR2 region is controlled under the human telomerase reverse transcriptase (hTERT) promoter, the E1b gene expression is directed by the hypoxia response element (HRE), whereas the PDCD5 gene is controlled by the cytomegalovirus promoter. The tumor-selective replication of this virus and its antitumor efficacy were characterized in several leukemic cell lines in vitro and in xenograft models of human leukemic cell line in nude mice. It was found by RQ-RT-PCR assay that SG611-PDCD5 expressed PDCD5 efficiently in leukemic cells. In K562 tumor xenograft models, SG611-PDCD5 displayed a tumor killing capacity. At a dose of 1 x 10(9) plaque-forming units, SG611-PDCD5 alone could completely inhibit the tumor growth and more effective than replication-defective Ad-PDCD5. Histopathologic examination revealed that SG611-PDCD5 administration resulted in leukemic cell apoptosis. We concluded that the triple-regulated SG611-PDCD5, as a more potent and safer antitumor therapeutic, could provide a new strategy for leukemia biotherapy.

A gutless adenoviral vector expressing full-length anti-Her2 antibody.

1. Therapeutic monoclonal antibodies are increasingly being used in clinical cancer treatment, but their complex technology and high cost limit their use. Helper-dependent (HD) adenoviruses are among the most efficient and safe gene therapy vectors capable of mediating long-term expression. 2. Using Gateway (Invitrogen, San Diego, CA, USA) cloning technology, we constructed an HD­trastuzumab (TAb) plasmid carrying the full-length anti-HER2 antibody gene. Using an efficient recombinase, namely in vitro-evolved Flippase-expressing recombinase, to excise the helper virus packaging signal in producer cells, we developed a scalable HD vector production method. Antibody expression of HD-TAb in vitro was detected by ELISA and western blot. 3. The full-length antibody gene delivery system allowed for continuous production of a full-length antibody at a high concentration. Bioactive antibody macromolecules were generated via gene transfer in vitro. 4. In conclusion, HD adenoviral vectors can stably express a full-length antibody for prolonged periods without the difficulties associated with sophisticated antibody manufacture techniques and at a much lower cost. As a promising tool for gene therapy, this novel system can shorten the duration and reduce the expense of antibody development.

Antitumor activity of EGFR-specific CAR T cells against non-small-cell lung cancer cells in vitro and in mice.

Effective control of non-small-cell lung cancer (NSCLC) remains clinically challenging, especially during advanced stages of the disease. This study developed an adoptive T-cell treatment through expression of a chimeric antigen receptor (CAR) to target human epidermal growth factor receptor (EGFR) in NSCLC. We optimized the non-viral piggyBac transposon system to engineer human T cells for the expression of EGFR-CAR, consisting of EGFR scFv, transmembrane domain, and intracellular 4-1BB-CD3ζ signaling domains. The modified CAR T cells exhibited expansion capability and anticancer efficacy in a time- and antigen-dependent manner in vitro as well as regression of EGFR-positive human lung cancer xenografts in vivo. EGFR-CAR T therapy is a promising strategy to improve the efficacy and potency of the adoptive immunotherapy in NSCLC. Moreover, EGFR-CAR T therapy could become a clinical application for NSCLC patients in the future.

11R-P53 and GM-CSF Expressing Oncolytic Adenovirus Target Cancer Stem Cells with Enhanced Synergistic Activity.

Targeting cancer stem cells with oncolytic virus (OV) holds great potential for thorough elimination of cancer cells. Based on our previous studies, we here established 11R-P53 and mGM-CSF carrying oncolytic adenovirus (OAV) SG655-mGMP and investigated its therapeutic effect on hepatocellular carcinoma stem cells Hep3B-C and teratoma stem cells ECCG5. Firstly, the augmenting effect of 11R in our construct was tested and confirmed by examining the expression of EGFP with Fluorescence and FCM assays after transfecting Hep3B-C and ECCG5 cells with OVA SG7605-EGFP and SG7605-11R-EGFP. Secondly, the expressions of 11R-P53 and GM-CSF in Hep3B-C and ECCG5 cells after transfection with OAV SG655-mGMP were detected by Western blot and Elisa assays, respectively. Thirdly, the enhanced growth inhibitory and augmented apoptosis inducing effects of OAV SG655-mGMP on Hep3B-C and ECCG5 cells were tested with FCM assays by comparing with the control, wild type 5 adenovirus, 11R-P53 carrying OVA in vitro. Lastly, the in vivo therapeutic effect of OAV SG655-mGMP toward ECCG5 cell-formed xenografts was studied by measuring tumor volumes post different treatments with PBS, OAV SG655-11R-P53, OAV SG655-mGM-CSF and OAV SG655-mGMP. Treatment with OAV SG655-mGMP induced significant xenograft growth inhibition, inflammation factor AIF1 expression and immune cells infiltration. Therefore, our OAV SG655-mGMP provides a novel platform to arm OVs to target cancer stem cells.

Antitumor activity of an hTERT promoter-regulated tumor-selective oncolytic adenovirus in human hepatocellular carcinoma.

AIM: To construct a tumor-selective replication-competent adenovirus (RCAd), SG300, using a modified promoter of human telomerase reverse transcriptase (hTERT). METHODS: The antitumor efficacy of SG300 in hepatocellular carcinoma was assessed in vitro and in vivo. In vitro cell viability by MTT assay was used to assess the tumor-selective oncolysis and safety features of SG300, and in vivo antitumor activity of SG300 was assessed in established hepatocellular carcinoma models in nude mice. RESULTS: SG300 could lyse hepatocellular carcinoma cells at a low multiplicity of infection (MOI), but could not affect growth of normal cells even at a high MOI. Both in Hep3B and SMMC-7721 xenograft models of hepatocellular carcinoma, SG300 had an obvious antitumor effect, resulting in a decrease in tumor volume. Its selective oncolysis to tumor cells and safety to normal cells was also superior to that of ONYX-015. Pathological examination of tumor specimens showed that SG300 replicated selectively in cancer cells and resulted in apoptosis and necrosis of cancer cells. CONCLUSION: hTERT promoter-regulated replicative adenovirus SG300 has a better cancer-selective replication-competent ability, and can specifically kill a wide range of cancer cells with positive telomerase activity, and thus has better potential for targeting therapy of hepatocellular carcinoma.

Evaluation of antibody level against Fusobacterium nucleatum in the serological diagnosis of colorectal cancer.

Fusobacterium nucleatum (F. nucleatum, Fn) is associated with the colorectal cancer (CRC). Fn-infection could induce significant levels of serum Fn-specific antibodies in human and mice. The objective of this study was to identify Fn-infection that elicit a humoral response in patients with CRC and evaluate the diagnostic performance of serum anti-Fn antibodies. In this work, we showed the mean absorbance value of anti-Fn-IgA and -IgG in the CRC group were significantly higher than those in the benign colon disease group and healthy control group (P < 0.001). The sensitivity and specificity of ELISA for the detection of anti-Fn-IgA were 36.43% and 92.71% based on the optimal cut-off. The combination of anti-Fn-IgA and carcino-embryonic antigen (CEA) was better for diagnosing CRC (Sen: 53.10%, Spe: 96.41%; AUC = 0.848). Furthermore, combining anti-Fn-IgA with CEA and carbohydrate antigen 19-9 (CA19-9) (Sen: 40.00%, Spe: 94.22%; AUC = 0.743) had the better ability to classify CRC patients with stages I-II. These results suggested that Fn-infection elicited high level of serum anti-Fn antibodies in CRC patients, and serum anti-Fn-IgA level may be a potential diagnosing biomarker for CRC. Serum anti-Fn-IgA in combination with CEA and CA19-9 increases the sensitivity of detecting early CRC.

Yellow Pigment Aurovertins Mediate Interactions between the Pathogenic Fungus Pochonia chlamydosporia and Its Nematode Host.

Nematophagous fungi are globally distributed soil fungi and well-known natural predators of soil-dwelling nematodes. Pochonia chlamydosporia can be found in diverse nematode-suppressive soils as a parasite of nematode eggs and is one of the most studied potential biological control agents of nematodes. However, little is known about the functions of small molecules in the process of infection of nematodes by this parasitic fungus or about small-molecule-mediated interactions between the pathogenic fungus and its host. Our recent study demonstrated that a P. chlamydosporia strain isolated from root knots of tobacco infected by the root-knot nematode Meloidogyne incognita produced a class of yellow pigment metabolite aurovertins, which induced the death of the free-living nematode Panagrellus redivevus. Here we report that nematicidal P. chlamydosporia strains obtained from the nematode worms tended to yield a total yellow pigment aurovertin production exceeding the inhibitory concentration shown in nematicidal bioassays. Aurovertin D was abundant in the pigment metabolites of P. chlamydosporia strains. Aurovertin D showed strong toxicity toward the root-knot nematode M. incognita and exerted profound and detrimental effects on the viability of Caenorhabditis elegans even at a subinhibitory concentration. Evaluation of the nematode mutation in the ß subunit of F1-ATPase, together with the application of RNA interference in screening each subunit of F1FO-ATPase in the nematode worms, demonstrated that the ß subunit of F1-ATPase might not be the specific target for aurovertins in nematodes. The resistance of C. elegans daf-2(e1370) and the hypersensitivity of C. elegans daf-16(mu86) to aurovertin D indicated that DAF-16/FOXO transcription factor in nematodes was triggered in response to the aurovertin attack. These findings advance our understanding of the roles of aurovertin production in the interactions between nematodes and the pathogen fungus P. chlamydosporia.

[Gene therapy for ovarian cancers by adenovirus-mediated complete antibody gene].

OBJECTIVE: To study the feasibility of adenoviral transduction of Herceptin complete antibody gene and its effect on Her2 over-expressing cancer. METHODS: The genes of VH and VL from the monoclonal antibody Herceptin were cloned into the genome of replication-defective adenovirus by viral recombination technology to produce the recombinant adenovirus Ad-SG-Her. Normal human liver cells of the line L-02 were transfected with Ad-SG-Her and ELISA was used to detect the expression of Herceptin antibody 3 and 7 days after. Forty BALB/c nude mice were inoculated with Her2 high-expressing oophoroma cells of SK-OV-3 line and were randomized into 4 equal groups: group A injected with Ad-SG-Her at the dosage of: 2 x 10(9) plaque forming unit (pfu) through the caudal vein, group B injected with Ad-SG-Her at the dosage of 1 x 10(9) pfu, group C injected with Ad-SG-Her at the dosage of 5 x 10(8) pfu, and control group. On the days 3, 7, 10, 14, 21, 28, and 35 after the injection of virus, the antibody expression in the serum was measured by ELISA and the size of tumor was measured vernier caliper. Western blot and IFA was used to detect the specificity for Her2-overexpressing ovarian cancer cell lines SK-OV-3 and the integrity of complete antibody. Anti-tumor effects were also observed in nude mice bearing SK-OV-3 tumors. RESULTS: The constructed recombinant adenovirus Ad-SG-Her could express Herceptin efficiently both in vitro and in vivo. The biological activity of the expressed antibody was similar to that of the commercial Herceptin as shown by Western blotting, IFA, and ELISA. Herceptin expression of Ad-SG-Her was detected since day 3 after treatment in the groups A, B, and C and reached the peak on days 7 - 10. The expression lasted for four weeks or so. The expression level was significantly different between group A and the groups B and C (all P < 0.05), however, without a significant difference between the group B and group C. The antibody expression of group A might increase to 103.5 micro g/ml, high enough to inhibit tumor growth and induce tumor cell apoptosis. The antibody expression of the group B was below 40 micro g/ml, and that of the group C was below 30 micro g/ml. Furthermore the expressed antibody doses were statistically significantly different at different time points. Almost no tumor growth was seen in the group A during the observation period in comparison with the groups B and C and the control group (all P < 0.05). The tumor growth was almost not influenced in the group B and C and the control group. CONCLUSION: Ad-SG-Her efficiently expresses humanized complete Herceptin with effective bioactivity and induces long-term stable expression both in vitro and in vivo. The system may serve as a new antitumoral gene therapy strategy in antibody field.