Analysis of fermentation control factors on volatile compounds of primary microorganisms in Jiang-flavor Daqu.

Chinese Jiang-flavor Baijiu is the most widely consumed liquor. Jiang-flavor Daqu, a fermentation starter, is important sources of key flavors of Jiang-flavor Baijiu. Some microbes play significant roles in flavor formation of Daqu. In order to clarify the microbial population that promotes the formation of Daqu flavor, we use high throughput sequencing technology combined with headspace solid-phase microextraction gas chromatography-mass spectrometry to investigate microbial population and volatile compounds in Jiang-flavor Daqu. In addition, the dynamic changes of physicochemical factors and enzyme activities in Jiang-flavor Daqu were investigated. Correlations between microbial population, volatile compounds, physicochemical factors, and enzyme activities of Jiang-flavor Daqu were disclosed by redundancy analysis and Spearman correlation analysis. A total of 66 volatile compounds were identified and 14 primary microorganisms were selected. Results showed that high temperature environment could promote the formation of acids, aldehydes and ketones, phenols, furans by affecting the growth of Monascus, Trichomonascus, Cutaneotrichosporon, Wallemia, Millerozyma, Nigrospora, Cladosporium, Bacillus, and Pediococcus in the early fermentation stage. While high nitrogen environment was more suitable for the growth of Virgibacillus and Kroppenstedtia, who could promote the formation of pyrazines in the late fermentation stage. PRACTICAL APPLICATIONS: This study has provided a scientific basis for the directed regulation of Daqu fermentation through physicochemical factors, developed scientific basis for artificially constructing Daqu microbial population and obtaining an easy-to-operate, reproducible fermentation system for Daqu production.

Development of a novel latent electrochemical molecular substrate for the real-time monitoring of the tumor marker aminopeptidase N in live cells, whole blood and urine.

Aminopeptidase N (APN/CD13) plays an important role in the growth and metastasis, of tumor, and is a potential biomarker for the post-treatment surveillance of cancer reoccurrence and progression of various malignancies. Thus, we have designed and prepared a convenient and ultrasensitive APN-targeting activity-based ratiometric electrochemical molecular substrate (Ala-AFC) for direct real-time monitoring of APN activity in biosamples. The APN in our experiment was used to hydrolyze the alanine moiety of the Ala-AFC probe and, as a result of this hydrolysis, realize concomitantly a cascade reaction to unmask the electrochemical reporter N-alkylated amino ferrocene (AAF). The Ala-AFC probe exhibited high sensitivity with a wide detection range of 0.05-110 ng mL-1 and a low limit of detection of 23.18 pg mL-1. The electrochemical signals were found to be distinctly specific for APN and free of interference from other electroactive biological species. Furthermore, the Ala-AFC probe was employed to monitor and quantify, in real-time, the activity of APN in tumor cells, whole blood, and urine. In addition, the results of our direct electrochemical quantifications of the amount of APN in whole blood and urine were found to be consistent with the results of the use of commercially available fluorometric assay kits to sense APN in serum and urine. Thus our approach shows promise as a point-of-care tool for cancer diagnostics and post-treatment surveillance of cancer reoccurrence.

Upregulating KTN1 promotes Hepatocellular Carcinoma progression.

Background: Hepatocellular carcinoma (HCC) presents a common malignant tumor worldwide. Although kinectin 1 (KTN1) is the most frequently identified antigen in HCC tissues, the detailed roles of KTN1 in HCC remain unknown. This study seeks to clarify the expression status and clinical value of KTN1 in HCC and to explore the complicated biological functions of KTN1 and its underlying mechanisms. Methods: In-house reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of KTN1 in HCC tissues. External gene microarrays and RNA-sequencing datasets were downloaded to confirm the expression patterns of KTN1. The prognostic ability of KTN1 in HCC was assessed by a Kaplan-Meier curve and a hazard ratio forest plot. The CRISPR/Cas9 gene-editing system was used to knock out KTN1 in Huh7 cells, which was verified by PCR-Sanger sequencing and western blotting. Assays of cell migration, invasion, viability, cell cycle, and apoptosis were conducted to explore the biological functions. RNA sequencing was performed to quantitatively analyze the functional deregulation in KTN1-knockout cells compared to Huh7-wild-type cells. Upregulated genes that co-expressed with KTN1 were identified from HCC tissues and were functionally annotated. Results: KTN1 expression was increased in HCC tissues (standardized mean difference [SMD] = 0.20 [0.04, 0.37]). High KTN1 expression was significantly correlated with poorer prognosis of HCC patients, and KTN1 may be an independent risk factor for HCC (pooled HRs = 1.31 [1.05, 1.64]). After KTN1-knockout, the viability, migration, and invasion ability of HCC cells were inhibited. The proportion of HCC cells in the G0-G1 phases increased after KTN1 knockout, which also elevated the apoptosis rates in HCC cells. Several cascades, including innate immune response, chemical carcinogenesis, and positive regulation of transcription by RNA polymerase II, were dramatically changed after KTN1 knockout. KTN1 primarily participated in the cell cycle, DNA replication, and microRNAs in cancer pathways in HCC tissues. Conclusion: Upregulation of KTN1 served as a promising prognosticator in HCC patients. KTN1 promotes the occurrence and deterioration of HCC by mediating cell survival, migration, invasion, cell cycle activation, and apoptotic inhibition. KTN1 may be a therapeutic target in HCC patients.

Laminarin from Seaweed (Laminaria japonica) Inhibits Hepatocellular Carcinoma Through Upregulating Senescence Marker Protein-30.

Objective: This study aimed at investigating the specific roles of laminarin from seaweed (Laminaria japonica) in hepatocellular carcinoma (HCC) and its potential mechanisms related to senescence marker protein-30 (SMP-30). Materials and Methods: Human HCC cell lines, including Bel-7404 and HepG2, were incubated with different concentrations of laminarin (0, 5, 15, 25, 35, and 45 mg/mL). The cell viability and apoptosis rates were detected by WST-8 cell proliferation assay and flow cytometry, respectively. Hepa 1-6 tumor-bearing mice were injected with different concentrations of laminarin (400, 800, and 1200 mg/kg·d), and tumor volume and weight were measured. The expression of SMP-30 was detected in laminarin-treated Bel-7404 and HepG2 HCC cells and LO2 normal liver cells by quantitative real-time PCR and Western blotting. Results: The treatment with laminarin (48 h) significantly decreased the viability and increased the apoptosis rates of Bel-7404 and HepG2 cells in a dose-dependent manner. The injection of laminarin also significantly decreased the tumor volumes (beginning on the 10th day) and tumor weights (30 d post-injection) of mice in a dose-dependent manner. In addition, the treatment with laminarin (35 mg/mL for 48 h) significantly upregulated SMP-30 in Bel-7404 and HepG2 cells but not in LO2 cells. Conclusion: Laminarin inhibited the proliferation of Bel-7404 and HepG2 cells and inhibited the growth of tumors in Hepa 1-6 tumor-bearing mice by upregulating SMP-30.

Electrochemical substrate for active profiling of cellular surface leucine aminopeptidase activity and drug resistance in cancer cells.

Leucine aminopeptidase (LAP) is an essential proteolytic enzyme and potential biomarker for liver malignancy. Overexpression of LAP is directly linked with some fatal physiological and pathological disorders. In this regard, we have designed an activity based electrochemical substrate leucine-benzyl ferrocene carbamate (Leu-FC) for selective profiling of LAP activity in live cells. In practice, LAP instantaneously hydrolyze the Leu residue of the substrate Leu-FC to eliminate the unmasked electrochemical reporter amino ferrocene via predefined self-immolative cascade. The electrochemical signal is distinctly specific for LAP and free of other electroactive biological interference. The substrate Leu-FC empowered sensor displayed broad dynamic range with admirable detection limits. On top of this, the probe Leu-FC was employed in real-time active profiling of cellular LAP activity in HepG2 cells and effect of LAP inhibitor. In extent, the substrate Leu-FC can effectively monitor cisplatin induced overexpression of LAP activity in HepG2 cells in presence and absence of bestatin. The sensor showcased an excellent reliability towards monitoring cellular LAP activity in HepG2 cells. Unlike the traditional antibody-based immunoassays, our approach is capable of monitoring in-situ activity of LAP in live cells.

Overexpression of MAGE-D4 in colorectal cancer is a potentially prognostic biomarker and immunotherapy target.

Melanoma-associated antigen D4 (MAGE-D4) is a novel member of MAGE family. This study aimed to examine the expression and immunogenicity of MAGE-D4 in colorectal cancer (CRC) to determine its potential as a prognosis and immunotherapeutic target. The expression of MAGE-D4 mRNA and protein was determined by RT-PCR and immunohistochemistry (IHC) in CRCs with paired adjacent non-tumor tissues, colorectal adenomas and normal colorectal tissues, respectively. Sera from 64 CRC patients were tested for MAGE-D4 antibody by ELISA. MAGE-D4 mRNA was more frequently expressed in CRCs (76.7%, 46/60) than in adjacent non-tumor tissues (15.0%, 9/60). MAGE-D4 protein was detected in all the CRC tissues tested, 70.0% of which showed high expression. There was no MAGE-D4 protein detected in any paired adjacent non-tumor tissue. No MAGE-D4 expression was found in colorectal adenomas and normal colorectal tissues by either RT-PCR or immunohistochemistry. Patients with high MAGE-D4 protein expression had significantly shorter overall survival than those with low MAGE-D4 protein expression (median, 68.6 vs 122.2 months; P=0.030). Furthermore, multivariate analysis exhibited high MAGE-D4 protein expression had a trend toward an independent prognostic factor (hazard ratio: 6.124; P=0.050). Humoral immunity to MAGE-D4 was detected in 12 of 64 (18.8%) CRC patients' sera but not in 77 healthy donors. There was no correlation between MAGE-D4 expression, serum antibody and clinicopathological parameters. These findings suggest MAGE-D4 may serve as a potentially prognostic biomarker and an attractive target of immunotherapy in CRC.

MAGED4 expression in glioma and upregulation in glioma cell lines with 5-aza-2'-deoxycytidine treatment.

Melanoma-associated antigen (MAGE) family genes have been considered as potentially promising targets for anticancer immunotherapy. MAGED4 was originally identified as a glioma-specific antigen. Current knowledge about MAGED4 expression in glioma is only based on mRNA analysis and MAGED4 protein expression has not been elucidated. In the present study, we investigated this point and found that MAGED4 mRNA and protein were absent or very lowly expressed in various normal tissues and glioma cell line SHG44, but overexpressed in glioma cell lines A172,U251,U87-MG as well as glioma tissues, with significant heterogeneity. Furthermore, MAGED4 protein expression was positively correlated with the glioma type and grade. We also found that the expression of MAGED4 inversely correlated with the overall methylation status of the MAGED4 promoter CpG island. Furthermore, when SHG44 and A172 with higher methylation were treated with the DNA demethylating agent 5-aza-2'-deoxycytidine (5-AZA-CdR) reactivation of MAGED4 mRNA was mediated by significant demethylation in SHG44 instead of A172. However, 5-AZA-CdR treatment had no effect on MAGED4 protein in both SHG44 and A172 cells. In conclusion, MAGED4 is frequently and highly expressed in glioma and is partly regulated by DNA methylation. The results suggest that MAGED4 might be a promising target for glioma immunotherapy combined with 5-AZA-CdR to enhance its expression and eliminate intratumor heterogeneity.

Knockdown of GCF2/LRRFIP1 by RNAi causes cell growth inhibition and increased apoptosis in human hepatoma HepG2 cells.

BACKGROUND: GC-binding factor 2 (GCF2) is a transcriptional regulator that represses transcriptional activity of the epidermal growth factor receptor (EGFR) by binding to a specific GC-rich sequence in the EGFR gene promoter. In addition to this function, GCF2 has also been identified as a tumor-associated antigen and regarded as a potentially valuable serum biomarker for early human hepatocellular carcinoma (HCC) diagnosis. GCF2 is high expressed in most HCC tissues and cell lines including HepG2. This study focused on the influence of GCF2 on cell proliferation and apoptosis in HepG2 cells. MATERIALS AND METHODS: GCF2 expression at both mRNA and protein levels in HepG2 cells was detected with reverse transcription (RT) PCR and Western blotting, respectively. RNA interference (RNAi) technology was used to knock down GCF2 mRNA and protein expression. Afterwards, cell viability was analyzed with a Cell Counting Kit-8 (CCK-8), and cell apoptosis and caspase 3 activity by flow cytometry and with a Caspase 3 Activity Kit, respectively. RESULTS: Specific down-regulation of GCF2 expression caused cell growth inhibition, and increased apoptosis and caspase 3 activity in HepG2 cells. CONCLUSIONS: These primary results suggest that GCF2 may influence cell proliferation and apoptosis in HepG2 cells, and also provides a molecular basis for further investigation into the possible mechanism at proliferation and apoptosis in HCC.

A constructed HLA-A2-restricted pMAGE-A1(278-286) tetramer detects specific cytotoxic T lymphocytes in tumour tissues in situ.

OBJECTIVE: To construct a human leucocyte antigen (HLA)-A2-restricted peptide 278-286 of melanoma-associated antigen family A, 1 (pMAGE-A1(278-286)) tetramer to analyse the distribution of cytotoxic T lymphocytes (CTLs) in tumour tissue and tumour-adjacent normal tissue. METHODS: A HLA-A2-pMAGE-A1(278-286) tetramer was constructed. The distribution of pMAGE-A1(278-286)-specific CTLs was investigated in tumour tissues and tumour-adjacent normal tissues from patients with hepatocellular carcinoma using in situ HLA-A2-pMAGE-A1(278-286) tetramer staining. RESULTS: Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis indicated that HLA-A2 heavy and light chain proteins were successfully obtained. The successful construction of the HLA-A2-pMAGE-A1(278-286) monomer was confirmed with Western blot analysis using W6/32 antibody. Flow cytometry confirmed the specific binding of HLA-A2-pMAGE-A1(278-286) tetramer to pMAGE-A1(278-286)-specific CTLs. In situ HLA-A2-pMAGE-A1(278-286) tetramer staining demonstrated that the number of pMAGE-A1(278-286)-specific CTLs in tumour tissues was significantly higher than in tumour-adjacent normal tissues. CONCLUSIONS: The HLA-A2-pMAGE-A1(278-286) tetramer was useful for the detection of pMAGE-A1(278-286)-specific CTLs in both tumour tissues and tumour-adjacent normal tissues. In situ tetramer staining is a powerful tool for investigating the distribution of pMAGE-A1278-286-specific CTLs in the tumour microenvironment.

Artificial antigen-presenting cells plus IL-15 and IL-21 efficiently induce melanoma-specific cytotoxic CD8+ CD28+ T lymphocyte responses.

OBJECTIVE: To develop a novel artificial antigen-presenting system for efficiently inducing melanoma-specific CD8(+) CD28(+) cytotoxic T lymphocyte (CTL) responses. METHODS: Cell-sized Dynabeads® M-450 Epoxy beads coated with H-2K(b): Ig-TRP2180-188 and anti-CD28 antibody were used as artificial antigen-presenting cells (aAPCs) to induce melanoma-specific CD8(+)CD28(+) CTL responses with the help of IL-21 and IL-15. Dimer staining, proliferation, ELISPOT, and cytotoxicity experiments were conducted to evaluate the frequency and activity of induced CTLs. RESULTS: Dimer staining demonstrated that the new artificial antigen-presenting system efficiently induced melanoma TRP2-specific CD8(+)CD28(+)CTLs. Proliferation and ELISPOT assays indicated that the induced CTLs rapidly proliferate and produce increased IFN- γ under the stimulation of H-2K(b): Ig-TRP2-aAPCs, IL-15, and IL-21. In addition, cytotoxicity experiments showed that induced CTLs have specific killing activity of target cells. CONCLUSIONS: The new artificial antigen-presenting system including aAPCs plus IL-21 and IL-15 can induce a large number of antigen-specific CD8(+) CD28(+) CTLs against the melanoma. Our study provides evidence for a novel adoptive immunotherapy against tumors.

Knockdown of OY-TES-1 by RNAi causes cell cycle arrest and migration decrease in bone marrow-derived mesenchymal stem cells.

OY-TES-1 is a member of the CTA (cancer-testis antigen) group expressed in a variety of cancer and restrictedly expressed in adult normal tissues, except for testis. To determine whether MSCs (mesenchymal stem cells) express OY-TES-1 and its possible roles on MSCs, OY-TES-1 expression in MSCs isolated from human bone marrow was tested with RT (reverse transcription)-PCR, immunocytochemistry and Western blot. Using RNAi (RNA interference) technology, OY-TES-1 expression was knocked down followed by analysing cell viability, cell cycle, apoptosis and migration ability. MSCs expressed OY-TES-1 at both mRNA and protein levels. The down-regulation of OY-TES-1 expression in these MSCs caused cell growth inhibition, cell cycle arrest, apoptosis induction and migration ability attenuation. Through these primary results it was suggested that OY-TES-1 may influence the biological behaviour of MSCs.

Serum immunoreactivity of SMP30 and its tissues expression in hepatocellular carcinoma.

OBJECTIVES: To detect serum antibody against SMP30 in HCC patients and to evaluate its potential associations with HCC patient's clinical parameter and expression levels in HCC tissues. DESIGN AND METHODS: Serum antibody to SMP30 was tested by ELISA method; SMP30 mRNA and protein expression in HCC patients were analyzed using the methods of in situ nucleic acid hybridization and immunohistochemistry, respectively. RESULTS: The highest relevance of SMP30 antibody was associated with HCC (32.4%). The positive rate of SMP30 antibody was not related to the age of patients, tumor size, metastasis and infections of HBV, but the positive rate for SMP30 antibody in the HCC sera with alpha-fetoprotein (AFP) negative was higher (43.6%) compared with that AFP positive (26.2%). Both SMP30 mRNA and protein expression levels were downregulated in HCC and upregulated in adjacent tissues. CONCLUSIONS: SMP30 may be useful for HCC serologic screening, especially for the patients with AFP negative.

Identification of HCC-22-5 tumor-associated antigen and antibody response in patients.

BACKGROUND: Serological identification of antigens by recombinant expression cloning (SEREX) is a promising method used to analyze tumor-associated antigen (TAA). Nineteen primary HCC-associated antigens have been found from a HCC cDNA library using autogenous serum by the SEREX approach. We searched for HCC-associated antigens and applied them to HCC diagnosis. METHODS: Nine of 19 primaries HCC-associated antigens identified by SEREX method were tested their immune response again with distinct allogeneic sera. One of the screened HCC-associated antigens, HCC-22-5 was recombined and expressed and made the frequency analysis of its seropositivity in various patients using the methods of Western-blot and ELISA. RESULTS: SEREX analysis showed that 9 primary HCC-associated antigens had high-titered IgG antibody in the majority of HCC patients. Western-Blot method confirmed that 3/7 HCC patients had antibodies against HCC-22-5, which demonstrated that expressed HCC-22-5 antigen had the character of antigen. Sera samples from 341 patients and 80 normal individuals have been tested for autoantibodies against HCC-22-5 by ELISA method. The results found that 51/128 of HCC, 11/76 of chronic hepatitis, 11/22 of liver cirrhosis and 8/54 of nasopharynx cancer patients had antibodies against HCC-22-5. No antibody response to HCC-22-5 had been found in the sera of 7 lung cancers, 54 gastric-intestine patients and 80 normal individuals. The groups of HCC and liver cirrhosis had higher antibody positive rate than that of other groups (p<0.05). In the HCC sera with alpha-fetoprotein (AFP) negative, the positive rate of HCC-22-5 was as high as 78.9%. CONCLUSIONS: HCC-22-5 can be used for HCC serologic screening, especially for the patients with AFP negative.