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Soil methane generation mainly driven by soil prokaryotic microbes can be coupled with the degradation of petroleum hydrocarbons (PHCs); however, the relationship between prokaryotic community structure and methane production activity in soil with the potential risk of PHC contamination is seldom reported. In this study, 3 soil samples (CS-1 to CS-3) in the area nearby an exploratory gas well and 5 soil samples (DC-1 to DC-5) in a drill cutting dump area were obtained from the Fuling shale gas field (Chongqing City, China). Then, the prokaryotic community structure was examined by Illumina Miseq sequencing, and the linkage between soil methane production rate (MPR) and prokaryotic community composition was analyzed. The results indicated that 2 samples (DC-4 and DC-5) collected from the drill cutting dump area had significantly higher MPR than the other samples, and a significant and positive relationship (r = 0.44, P

Soil autotrophic bacteria are important for sequestrating atmospheric CO2 and thus contribute to the global carbon cycle. However, information is limited on how chemical fertilization can alter the composition of soil autotrophic bacteria. In this study, the cbbL gene (a key gene in the Calvin-Benson-Bassham cycle that encodes the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, RubisCO) was used to study the abundance and composition of autotrophic bacteria in an entisol with 26 years of continuous fertilization. In this process, five chemical fertilization regimes were examined: without fertilization control (CT), nitrogen and phosphorus fertilization (NP), N and potassium fertilization (NK), PK fertilization (PK), and NPK fertilization (NPK). The results showed that after the 26-year (1991–2017) period, the CT with a neutral pH (7.55) had a higher RubisCO activity than did the chemical fertilizer treatments, but the cbbL abundance was the lowest under CT. Soil RubisCO activity positively correlated with soil pH (R2 = 0.67, P

Purpose This study aimed to evaluate the efficacy and safety of intratumoral IL-12 gene therapy in an HCC-hu-PBL-NOD/SCID mouse model. Materials and methods The HCC murine model was generated in NOD/SCID mice, and mice with grafted tumors were injected intraperitoneally with 2 × 107 human peripheral blood lymphocytes 14 days after modeling. After 4 days, mice were randomly divided into the 9597/IL-12 group, the 9597/plasmid group and the PBS group. The changes of tumor volume were measured and mouse peripheral blood was sampled post-treatment for ELISA and CBA analyses, and the grafted tumors were collected 28 days post-treatment for immunohistochemistry, ELISA, CBA and detection of cell cycle and apoptosis. Results The tumor volume was smaller in the 9597/IL-12 group than in the 9597/plasmid and PBS groups on days 7, 14, 21, and 28 post-treatment (P < 0.05). Higher IL-12 levels were detected in the peripheral blood and the supernatants of grafted tumor homogenates in the 9597/IL-12 group than in the 9597/plasmid and PBS groups 7, 14, 21 and 28 days post-treatment (P < 0.05). IHC revealed higher counts of CD3+T cells, CD4+T helper cells, IFN-γ Th1 cells+ and S-100 protein positive dentric cells and lower MVD in the 9597/IL-12 group than in the 9597/plasmid and PBS groups (P < 0.05). Flow cytometry showed a significantly higher proportion of HCC cells at the G0/G1 phase and a significantly lower proportion of HCC cells at the S phase in the 9597/IL-12 group than in the PBS group (P < 0.05) and a greater apoptotic rate of HCC cells in the 9597/IL-12 group than in the 9597/plasmid and PBS groups (P < 0.05). Conclusion Intratumoral IL-12 gene therapy may inhibit tumorigenesis with mild adverse effects in a HCC-hu-PBL-NOD/SCID murine model through inhibiting angiogenesis, arresting cells in G0/G1 phase and inducing apoptosis.

Jieyu Li,1–3,* Shenglan Huang,1,* Zhifeng Zhou,2,3 Wansong Lin,2,3 Shuping Chen,2,3 Mingshui Chen,2,3 Yunbin Ye1–3 1School of Basic Medical Sciences, Fujian Medical University, Fuzhou 350108, China; 2Laboratory of Immuno-Oncology, Fujian Cancer Hospital & Fujian Medical University Cancer Hospital, Fuzhou 350014, China; 3Fujian Key Laboratory of Translational Cancer Medicine, Fuzhou 350014, China *These authors contributed equally to this work Background: Dendritic cell (DC)-derived exosomes (Dexs) have been proved to induce and enhance antigen-specific T cell responses in vivo, and previous clinical trials have shown the feasibility and safety of Dexs in multiple human cancers. However, there is little knowledge on the efficacy of Dexs against hepatocellular carcinoma (HCC) until now.Methods: In this study, human peripheral blood-derived DCs were loaded with recombinant adeno-associated viral vector (rAAV)-carrying alpha-fetoprotein (AFP) gene (rAAV/AFP), and high-purity Dexs were generated. Then naive Tcells were stimulated with Dexs to investigate the specific T cell-mediated immune responses against HCC. Results: Our findings showed that Dexs were effective to stimulate naive T cell proliferation and induce T cell activation to become antigen-specific cytotoxic T lymphocytes (CTLs), thereby exhibiting antitumor immune responses against HCC. In addition, Dex-sensitized DC precursors seemed more effective to trigger major histocompatibility complex class I (MHC I)-restricted CTL response and allow DCs to make full use of the minor antigen peptides, thereby maximally activating specific immune responses against HCC.Conclusion: It is concluded that Dexs, which combine the advantages of DCs and cell-free vectors, are promising to completely, or at least in part, replace mature DCs (mDCs) to function as cancer vaccines or natural antitumor adjuvant. Keywords: hepatocellular carcinoma, dendritic cell, exosome, immune response, cytotoxic T lymphocyte

To improve the properties of silkworm ( Bombyx mori ) pupa protein (SPP), the effects of different modification techniques included enzymolysis, ultrasound, and micronization on the molecular structure and bioactivity of SPP has been evaluated in this study. The results showed that enzymolysis modification led to the most substantial change in the area and shape of the infrared (IR) absorption peaks, decreased in size with an increased number of cracks of modified protein by scanning electron microscopy images, followed by ultrasound and micronization modification. After modification by enzymolysis, ultrasound, and micronization, the sulfhydryl concentrations of SPP were increased by 48.56%, 34.82%, and 12.46%, respectively; disulfide bond concentrations were reduced by 40.47%, 10.79%, and 11.72%, respectively. Furthermore, the ratios of essential amino acids among total amino acids increased by 13.85%, 2.22%, and 6.93%, respectively. Antioxidant activity and ACE-inhibitory activity of SPP also increased notably after modifications.

Agricultural soils in oilfields have high risk for polycyclic aromatic hydrocarbon (PAH) pollution. In this study, from the Jianghan Oilfield (Hubei Province, China) with a history of >50 years, 7 soil samples (OS-1 to OS-7) were collected. Subsequently, the bacterial, archaeal, and fungal community structures were investigated by Illumina MiSeq sequencing, and the relationship between microbial community structure and soil PAH content was analyzed. The results indicated that bacterial and archaeal Chao 1 indices showed a significantly negative relationship with soil PAH content, and only the bacterial Shannon index had a significantly negative relationship with soil PAH content. Moreover, the community structure of bacteria (r 2 = 0.9001, p = 0.013) showed a stronger correlation with PAH content than that of fungi (r 2 = 0.7357, p = 0.045), and no significant relationship was found between archaeal community structure (r 2 = 0.4553, p = 0.262) and soil PAH content. In addition, the relative greater abundances of some bacterial genus belonging to Actinobacteria (Mycobacterium and Micromonospora) and Proteobacteria (Pseudomonas, Lysobacter, Idiomarina, Oxalobacteraceae, and Massilia), fungal genus belonging to Ascomycota (Sordariales and Pleosporales), and archaeal phylum (Euryarchaeota) were detected in the soil samples (OS-3 and OS-5) with greater PAH content. In summary, soil PAHs showed an obvious influence and selectivity on the soil microbiota. Furthermore, compared with fungi and archaea, bacteria was more sensitive to soil PAH pollution, and the diversity indices and community structure of bacteria both might be suitable indicators for assessment of soil PAH stress on the soil ecosystem.

Delta 6-fatty acid desaturase is a membrane-bound enzyme, which is the rate-limiting factor in the biosynthesis of polyunsaturated fatty acids. In this study, a novel delta 6-desaturase gene was cloned from Bombyx mori (BmD6DES). Sequencing analysis revealed that BmD6DES has an open reading frame of 1357 bp that encodes 448 amino acids. Heterologous expression in yeast demonstrated that BmD6DES could synthesize γ-linolenic acid (GLA, 18:3, Δ6,9,12) by utilizing the endogenous substrate linoleic acid (LA, 18:2, Δ9,12). We found that BmD6DES transcripts were distributed in almost all B. mori tissues, with high expression levels observed at the 5th instar larval, pupal, and adult moth stages. A functional analysis of BmD6DES was performed by measuring mRNA levels after temperature stress, fungal infection, and RNA interference (RNAi). The results indicated that the highest expression of BmD6DES was observed at low temperatures (0 °C) and 6 h to 36 h after fungal infection. qPCR analysis demonstrated that BmD6DES mRNA levels in pupa after BmD6DES RNAi treatment were significantly reduced from 12 h to 72 h compared to those in the control group. Our findings suggest that BmD6DES not only induces the formation of the third carbon–carbon double bond in the LA carbon chain, but also leads to sensitivity to low-temperature stress and fungal infection. These results imply that BmD6DES is a key gene in the γ-linolenic acid pathway during B. mori development.

Non-small cell lung cancer (NSCLC) accounting for 85 % of all lung cancer was one of the main causes of death worldwide. In this study, we investigated the role of circRNA_102179 in NSCLC development. The levels of circRNA_102179 in NSCLC tissues and cell lines were determined by quantitative real-time PCR assay (qRT-PCR). CCK8 and colony formation assays were applied to explore the effect of circRNA_102179 on the growth of NSCLC cells in vitro. Transwell assay was utilized to analyze the impact of circRNA_102179 on the migration and invasion of NSCLC cells. Target prediction and luciferase reporter assay were used to identify the interacting miRNA of circRNA_102179. The interaction among circRNA_102179/ miR-330-5p/HMGB3 was further validated by colony formation and Transwell invasion assays. Finally, the mouse xenograft NSCLC model was used to explore the role of circRNA_102179 in the tumor growth of NSCLC cells in vivo. CircRNA_102179 was overexpressed in NSCLC tissues and cells compared with normal lung tissues and human bronchial epithelial cells (HBEs). The down-regulation of circRNA_102179 markedly reduced the proliferation, migration, and invasion of NSCLC cells. Moreover, down-expression of circRNA_102179 significantly increased the level of miR-330-5p/HMGB3 in NSCLC cells. Further functional experiments indicated that over-expression of miR-330-5p reversed the inhibitory effect of circRNA_102179 on NSCLC cells growth, migration, and invasion. Our results reveal that circRNA_102179 facilitates the proliferation, migration, and invasion of NSCLC cell via modulating miR-330-5p/ HMGB3 axis in NSCLC cells.

MUC1 is a tumor-associated antigen (TAA) overexpressed in many tumor types, which makes it an attractive target for cancer immunotherapy. However, this marker is a non-mutated antigen without high immunogenicity. In this study, we designed several new altered peptides by replacing amino acids in their sequences, which were derived from a low-affinity MUC1 peptide, thus bypassing immune tolerance. Compared to the wild-type (WT) peptide, the altered MUC1 peptides (MUC11081-1089L2, MUC11156-1164L2, MUC11068-1076Y1) showed higher affinity to the HLA-A0201 molecule and stronger immunogenicity. Furthermore, these altered peptides resulted in the generation of more cytotoxic T lymphocytes (CTLs) that could cross-recognize gastric cancer cells expressing WT MUC1 peptides, in an HLA-A0201-restricted manner. In addition, M1.1 (MUC1950-958), a promising antitumor peptide that has been tested in multiple tumors, was not able to induce stronger antitumor responses. Collectively, our results demonstrated that altered peptides from MUC1, as potential HLA-A0201-restricted CTL epitopes, could serve as peptide vaccines or constitute components of peptide-loaded dendritic cell vaccines for gastric cancer treatment.

Soil pH has been suggested as one of the most important factors affecting the ecological characteristics of soil ammonia-oxidizers (AO), which mediate the conversion of ammonia to nitrate via nitrite and contribute significantly to the leaching of nitrate to groundwater and the production of atmospheric nitrous oxide (N2O). However, the dynamics of the AO community in acidic purple soils, which are widely distributed in Southwest China, remain largely unknown. In this study, two typical purple soils with different pH values (acidic: ACI; alkaline: ALK) were collected and studied. The abundance of amoA (gene encoding ammonia monooxygenase) of ammonia-oxidizing bacteria (AOB) and archaea (AOA) and that of the cbbL gene (encoding ribulose-1,5-biphosphate carboxylase/oxygenase) were determined by real-time PCR, and the community structures of AOB and AOA were investigated by cloning and sequencing. The results revealed that abundances of AOB and AOA were significantly lower in the ACI purple soil sample than in the ALK sample, but a higher ratio of AOA to AOB was found in the ACI purple soil sample. No significant difference in the abundance of cbbL was found between the two soils, but the ratio of AOB and AOA amoA to cbbL genes in the ACI soil samples was higher than that in the ALK sample. Moreover, the ALK and ACI soils harbored contrasting community compositions of AO. AOB in the ALK were dominated by cluster 3a (87 %), while the percentage of cluster 3a decreased and clusters 9 and 10 accounted for almost 77 % of the AOB community in the ACI soil. Nitrososphaera and Nitrosotalea were the major AOA phylotypes in the ALK and ACI soils, respectively. In conclusion, our results revealed the potential relations among pH, AO, and total chemoautotrophic bacteria in soil and that pH might have an essential impact on the adaptation and selection of AO in purple soils.

Ammonia-oxidizing microorganisms, which drive the first and rate-limiting step in the process of nitrification, play an important role in soil N-cycling processes. However, little is known about the ammonia-oxidizing bacteria (AOB) and archaea (AOA) in purple soils, which are characterized as lithologic soils without distinct pedogenic horizons and are mainly distributed in the Sichuan basin of Southwestern China. Here, the abundance and community structure of AOB and AOA in a 21-year fertilization experiment were investigated by real-time PCR and terminal restriction fragment-length polymorphism (T-RFLP), respectively. In the present study, nine different fertilization treatments were examined: NPK fertilization plus pig manure (NPK + M), NPK fertilization (NPK), without fertilization (CK), pig manure fertilization (M), P with NH4Cl and KCl plus pig manure (Cl + M), PK fertilization (PK), NK fertilization (NK), NP fertilization (NP), and N fertilization (N). Our results indicate that N-fertilized treatments have higher AOB abundances than the control (CK) and treatments without N amendment. The lowest AOA abundance and AOA/AOB ratios were observed in the treatment (Cl + M) with long-term application of NH4Cl plus KCl. The AOB community structure under combined addition of N and P differed from that under other treatments. The Cl + M treatment had a distinct AOA community structure and higher diversity compared with other treatments. Moreover, higher potential nitrification rates (PNR) were found in the combined N and P addition treatment groups than that in the other treatment groups, and the lowest PNR and pH (

Squamous cell carcinoma (SCC) is histologically the most prominent type of cervical cancer. There is accumulating evidence suggesting that microRNAs (miRNAs) play important regulatory roles in the biological processes of cervical squamous cell carcinoma (CSCC). Deciphering the miRNA regulatory network in CSCC could deepen our understanding at the molecular level of CSCC initiation and progression. In the present study, we performed next‑generation sequencing (NGS) to profile miRNA expression in 3 pairs of early-stage CSCC samples. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify primary findings in another 20 pairs of CSCC samples. We identified 37 known miRNAs that exhibited significant alterations in expression (2-fold change or greater), among which 8 miRNAs were upregulated and 29 miRNAs were downregulated. Nine of these miRNAs were selected for further qRT-PCR validation. A novel miRNA candidate was also reported for the first time in the present study to be upregulated. The Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that its target genes were involved in MAPK, calcium and adherent junction signaling pathways. The present study systematically characterized the miRNA expression variation in early-stage CSCC and provides novel biomarkers for diagnosis and treatment as well as an opportunity for further investigation of the molecular mechanisms underlying the pathogenesis and development of CSCC.

The aim of this study was to investigate the protective effects of andrographolide (AP), a bioactive component isolated from Andrographis paniculata, on carbon tetrachloride (CCl(4))-induced liver injury as well as the possible mechanisms involved in this protection in mice. Acute liver injury was induced by CCl(4) intoxication in mice. Serum biological analysis, lipid peroxides and antioxidant estimation, histopathological studies, reverse transcription polymerase chain reaction (RT-PCR) and Western blot assay were carried out. CCl(4) treatment resulted in severe hepatic injury, as evidenced by significant elevation of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and typical histopathological changes, such as hepatocyte necrosis. Additionally, CCl(4) administration led to oxidative stress in mice, as indicated by a remarkable increase in the hepatic malondialdehyde (MDA) level, together with a significant decrease in liver reduced glutathione (GSH) content. However, CCl(4)-induced hepatotoxicity was significantly attenuated by pretreatment with AP, as demonstrated by significant reduction of serum ALT, AST levels and hepatic MDA activity, along with a remarkable increase in hepatic GSH content. Histopathological changes induced by CCl(4) were also ameliorated by AP pretreatment. The marked increase of tumor necrosis factor-α (TNF-α) induced by CCl(4) was attenuated by AP, and the dramatic elevation of heme oxygenase-1 (HO-1) at transcriptional and protein levels was augmented following AP pretreatment. AP can effectively prevent liver injury induced by CCl(4), which may be due to inhibition of oxidative stress and inflammatory responses.

Soil samples were collected from three plots under different land utilization patterns including degradation, farming, and restoration. The abundances of methanotrophs were quantified using real-time polymerase chain reaction (PCR) based on the pmoA and 16S rRNA genes, and the community fingerprint was analyzed using denaturing gradient gel electrophoresis (DGGE) aiming at pmoA gene. Significantly lower 16S rRNA and pmoA genes copies were found in the degradation treatment than in farming and restoration. Higher abundances of Type I than those of Type II methanotrophs were detected in all treatments. The treatment of farming was clearly separated from degradation and restoration according to the DGGE profile by cluster analysis. The lowest diversity indices were observed in the F (farming plot), suggesting that the community structure was strongly affected by farming activities. There were significantly positive correlations between the copy numbers of pmoA also Type II-related 16S rRNA genes and soil available K content. Strong negative and positive correlations were found between Type I and soil pH, and available P content, respectively. We concluded that the vegetation cover or not, soil characteristics including pH and nutrients of P and K as a result of anthropogenic disturbance may be key factors affecting methanotrophic communities in upland soil.

Currently, molecular biologic techniques achieve a great development in studies of soil samples. The objective of this research is to improve methods for microbial prospecting of oil and gas by applying culture-independent techniques to soil sampled from above a known oil and gas field. Firstly, the community structure of soil bacteria above the Ban 876 Gas and Oil Field was analyzed based on 16S rRNA gene clone libraries. The soil bacteria communities were consistently different along the depth; however, Chloroflexi and Gemmatimonadetes were predominant and methanotrophs were minor in both bacteria libraries (DGS1 and DGS2). Secondly, the numbers of methane-oxidizing bacteria, quantified using a culture-dependent procedure and culture-independent group-specific real-time PCR (RT-PCR), respectively, were inconsistent with a quantify variance of one or two orders of magnitude. Special emphasis was given to the counting advantages of RT-PCR based on the methanotrophic pmoA gene. Finally, the diversity and distribution of methanotrophic communities in the soil samples were analyzed by constructing clone libraries of functional gene. All 508-bp inserts in clones phylogenetically belonged to the methanotrophic pmoA gene with similarities from 83% to 100%. However, most of the similarities were below 96%. Five clone libraries of methanotrophs clearly showed that the anomalous methanotrophs (Methylosinus and Methylocystis) occupy the studied area.

A simple and rapid isocratic LC/MS coupled with electrospray ionization (ESI) method for simultaneous separation and determination of adenine, hypoxanthine, adenosine and cordycepin in Cordyceps sinensis (Cs) and its substitutes was developed. 2-Chloroadenosine was used as internal standard for this assay. The optimum separation for these analytes was achieved using the mixture of water, methanol and formic acid (85:14:1, v/v/v) as a mobile phase and a 2.0 x 150 mm Shimadzu VP-ODS column. Selective ion monitoring (SIM) mode ([M+H]+ at m/z 136, 137, 268, 252 and 302) was used for quantitative analysis of above four active components. The regression equations were liner in the range of 1.4-140.0 microg ml(-1) for adenine, 0.6-117.5 microg ml(-1) for hypoxanthine, 0.5-128.5 microg ml(-1) for adenosine and 0.5-131.5 microg ml(-1) for cordycepin. The limits of quantitation (LOQ) and detection (LOD) were, respectively 1.4 and 0.5 microg ml(-1) for adenine, 0.6 and 0.2 microg ml(-1) for hypoxanthine, 0.5 and 0.1 microg ml(-1) for adenosine and cordycepin. The recoveries of four constituents were from 93.5 to 107.0%. The nucleoside contents of various types of natural Cs and its substitutes were determined and compared with this developed method.

Meeting abstracts In tumor microenvironment, CD4+CD25+CD127dim/- Regulatory T cells (Treg cells) belong to a group of negative regulatory cells, which play an important role in the mechanism of immune inhibition and immune escape of liver cancer [[1][1]]. In this study, we will explore the