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What is known and the objective Our previous studies have shown that saponins of Sanguisorba parviflora (Maxim) Takeda (Sp. T) relieved cyclophosphamide-induced myelosuppression in mice with leukopenia. The hematopoietic cell-specific protein 1-associated protein X-1 (HAX-1) participated in the survival of neutrophils through the regulation of mitochondrial function. This study aimed to comprehensively identify the role of HAX-1 in Sp. T to alleviate leukopenia. Methods HAX-1 expression was examined in the peripheral blood neutrophils using real-time polymerase chain reaction (PCR), Western blot analysis and immunohistochemical staining. Neutrophil apoptosis was measured by flow cytometry. Mitochondrial function was evaluated via reactive oxygen species (ROS) generation and mitochondrial membrane potential (ΔΨm) integrity. Results and discussion Our study indicated that the expression of the HAX-1 gene was significantly decreased in the peripheral blood neutrophils of leukopenia patients compared with healthy donors. The saponins of Sp. T induced HAX-1 expression and promoted myeloid progenitor cell (mEB8-ER cell) viability, while overexpression of HAX-1 reduced the production of reactive oxygen species (ROS) and maintained the integrity of the mitochondrial membrane potential. Cyclophosphamide-induced mitochondrial dysfunction and apoptosis could be abrogated by treatment with Sp. T or the addition of metformin. What is new and our conclusion Our data support a mechanism where Sp. T protects against chemotherapy-induced leukopenia by regulating HAX-1 gene expression in a mitochondrial-dependent manner.

MiRNA-126 (miR-126) has been shown to be involved in various malignancies as well as other biological processes. However, currently, its role in esophageal squamous cell carcinoma (ESCC) is not well understood. The present study is focused on the mechanisms that underlie the effect of miR-126 on cell survival and death (apoptosis and autophagy) in ESCC cells. MiR-126 expression was found to be enhanced in ESCC cells and tissues. Downregulation of miR-126 suppressed cell survival, and TUNEL staining indicated that miR-126 inhibition promoted ESCC cell death. In addition, the production of LC3B and p62 proteins, two autophagy signals, was reduced following miR-126 inhibition. A dual luciferase reporter assay demonstrated that the STAT3 3’-UTR is a direct target of miR-126. Furthermore, STAT3 knock-down rescued the effects on autophagy and apoptosis caused by miR-126 inhibition in ESCC cells. The results of this study may provide some insight into the molecular and biological mechanisms underlying ESCC generation and contribute to the development of novel therapeutic approaches for ESCC.

Two novel, Gram-stain-positive, non-motile, facultatively anaerobic, rod-shaped bacteria (strains 2129T and 2119) were isolated from the faeces of Tibetan antelopes (Pantholops hodgsonii) on the Qinghai-Tibet Plateau, PR China. The 16S rRNA gene sequences of the strains showed highest similarity values to Actinomyces timonensis DSM 23838T (92.9 and 92.8 %, respectively), and phylogenetic analysis based on 16S rRNA gene and genomic sequences indicated that strains 2129T and 2119 represent a new lineage. Strains 2129T and 2119 could ferment d-adonitol and d-xylose, but were unable to utilize d-mannose and d-melibiose nor produce esterase (C4) and proline arylamidase. The G+C contents of the two strains were both 69.0 mol%. Their genomes exhibited less than 40.4 % relatedness in DNA-DNA hybridization tests (below 70 % as the recommended threshold for new species) with all available genomes of the genus Actinomyces in the NCBI database. The major fatty acids of the two strains were C18 : 1ω9c and C16 : 0, and the major polar lipids were diphosphatidylglycerol, glycolipid, phosphatidylinositol, phosphatidyl inositol mannoside and phosphoglycolipid. Based on the results of genotypic, phenotypic and biochemical analyses, it is proposed that the two unidentified bacteria be classified as representing a novel species, Actinomyces lilanjuaniae sp. nov. The type strain is 2129T (=CGMCC 4.7483T=DSM 106426T).

This research aims to investigate the ameliorative potential of baicalin on lipopolysaccharide-induced acute lung injury by the suppression of oxidative stress and inflammation via the activation of the nuclear erythroid factor 2 (Nrf2)-mediated heme oxygenase-1 (HO-1) signaling pathway. Specific pathogen-free male mice, weighing between 25 and 30 g, were divided into the following four groups of 10 mice each: the control group, LPS group, LPS + baicalin group, and baicalin group. Bronchoalveolar lavage fluid (BALF), blood, and tissue were collected on the 16th day and used for hematological (total leukocyte, macrophage, neutrophil, and lymphocyte counts in both blood and BALF, biochemical (antioxidant enzymes, MDA, Nrf2, and HO-1), and histological analyses. The protective effect of baicalin on lipopolysaccharide-induced acute lung injury is based on its antioxidative stress capabilities that are mediated partly by the Nrf2/HO-1 signaling pathway. Baicalin pretreatment significantly decreased the rise in the lung injury score; total leukocyte, neutrophil, lymphocyte, and macrophage counts; pro-inflammatory mediators, tumor necrosis factor (TNF-α), interleukins (IL-6 and IL-1β); biosynthesis of oxidative products, e.g., malondialdehyde (MDA); and restoration of antioxidative enzyme (superoxide dismutase and catalase) activities by improving the expression of nuclear Nrf2 and cytosolic HO-1 in lipopolysaccharide-induced acute lung injury. The protective effects of baicalin are partly due to its antioxidant and anti-inflammatory effects. Our findings indicate that baicalin protects against lipopolysaccharide-induced severe lung injury by enhancing antioxidant systems and significantly reducing both inflammatory cells and mediators via the Nrf2-mediated HO-1 signaling pathway.

Wildlife is a reservoir of emerging infectious diseases of humans and domestic animals. Marmota himalayana mainly resides 2800–4000 m above sea level in the Qinghai-Tibetan Plateau, and is the primary animal reservoir of plague pathogen Yersinia pestis. Recently we isolated a new species, Escherichia marmotae from the faeces of M. himalayana. In this study we characterised E. marmotae by genomic analysis and in vitro virulence testing to determine its potential as a human pathogen. We sequenced the genomes of the seven E. marmotae strains and found that they contained a plasmid that carried a Shigella-like type III secretion system (T3SS) and their effectors, and shared the same O antigen gene cluster as Shigella dysenterae 8 and E. coli O38. We also showed that E. marmotae was invasive to HEp-2 cells although it was much less invasive than Shigella. Thus E. marmotae is likely to be an invasive pathogen. However, E. marmotae has a truncated IpaA invasin, and lacks the environmental response regulator VirF and the IcsA-actin based intracellular motility, rendering it far less invasive in comparison to Shigella. E. marmotae also carried a diverse set of virulence factors in addition to the T3SS, including an IS1414 encoded enterotoxin gene astA with 37 copies, E. coli virulence genes lifA/efa, cif, and epeA, and the sfp gene cluster, Yersinia T3SS effector yopJ, one Type II secretion system and two Type VI secretion systems. Therefore, E. marmotae is a potential invasive pathogen.

Background: Non-small cell lung cancer (NSCLC) has a significant impact on human health. The aim of this study was to explore the role of long non-coding RNA DLX6-AS1 in the proliferation, migration, and invasion of NSCLC cells. Methods: The expression of DLX6-AS1 in NSCLC tumor tissues and cell lines was examined by qRT-PCR. The effects of DLX6-AS1 knockdown on cell proliferation, migration, and invasion were assessed by Cell Counting Kit-8, wound healing, and transwell assays, respectively. Bioinformatics analyses, luciferase reporter assays, and RNA pull-down assays were employed to examine the mechanism by which DLX6-AS1 exerted its oncogenesis effects in NSCLC. The anti-tumor effect of silencing DLX6-AS1 in vivo was also evaluated. Results: DLX6-AS1 was over-expressed in NSCLC tumor tissues and cell lines and its level of expression was found to be associated with tumor size and advanced clinical stage in patients with NSCLC. Downregulation of DLX6-AS1 inhibited cell proliferation, cell clone formation, migration, and invasion of NSCLC cells. DLX6-AS1 was found to interact with miR-27b-3p/GSPT1. DLX6-AS1 expression was negatively correlated with miR-27b-3p expression, but positively correlated with GSPT1 expression in NSCLC samples. DLX6-AS1 knockdown also effectively suppressed tumor growth in an in vivo xenograft model. Conclusion: DLX6-AS1 regulated NSCLC progression by targeting the miR-27b-3p/GSPT1 axis, which may provide novel insights for NSCLC prognosis and therapy.

What is known and objective We have previously shown that the saponins of Sanguisorba parviflora (Maxim.) Takeda (Sp. T) relieved cyclophosphamide-induced myelosuppression in leukopenic mice. Haematopoietic cell-specific protein 1-associated protein X-1 (HAX-1) participated in the survival of neutrophils through the regulation of mitochondrial function. The aim of the present study was to comprehensively identify the role of HAX-1 in the mechanism of leukopenia alleviation by Sp. T. Methods HAX-1 gene and protein expression levels in peripheral blood neutrophils were examined using real-time quantitative reverse transcription-polymerase chain reaction, western blot and immunohistochemical assays. Neutrophil apoptosis was measured using flow cytometry. Mitochondrial function was determined via assessments of the reactive oxygen species (ROS) generation and mitochondrial membrane potential (ΔΨm) integrity levels. Results and discussion The HAX-1 gene expression level in the peripheral blood neutrophils was significantly lower in patients with leukopenia than in healthy donors. The saponins of Sp. T induced HAX-1 expression and promoted myeloid progenitor cell (mEB8-ER cell) viability. HAX-1 overexpression reduced the production of ROS and maintained ΔΨm integrity. Cyclophosphamide-induced mitochondrial dysfunction and apoptosis could be abrogated by treatment with Sp. T or metformin. What is new and conclusion Our data suggest a mechanism through which Sp. T protects against chemotherapy-induced leukopenia by regulating HAX-1 gene expression in a mitochondrial-dependent manner.

A novel, Gram-stain-positive, catalase-positive, non-spore-forming, short rod-shaped strain (VUL4_3T) was isolated from rectal swabs of Old World vultures (Aegypius monachus) from the Tibet–Qinghai Plateau, China. Based on the results of biochemical tests and 16S rRNA gene sequence comparison, strain VUL4_3T was determined to be a member of the genus Actinomyces that is closely related to the type strains of Actinomyces liubingyangii (97.7 % 16S rRNA gene sequence similarity) and Actinomyces marimammalium (96.5 %). Optimal growth occurred at 37 °C, pH 6–7 and with 1 % (w/v) NaCl. The typical major cellular fatty acids of strain VUL4_3T were C18 : 1ω9c, C16 : 0 and C18 : 0. The VUL4_3T genome contained 2 207 832 bp with an average G+C content of 51.9 mol%. DNA–DNA hybridization values between strain VUL4_3T and the above two species of the genus Actinomyces showed less than 32 % DNA–DNA relatedness, supporting a novel species status of strain VUL4_3T. Based on the phenotypic data and phylogenetic inference, the novel species Actinomyces tangfeifanii sp. nov. is proposed. The type strain is VUL4_3T (=CGMCC 4.7369T=DSM 103436T).

Pancreatic cancer is one of the deadliest cancers with rapid disease progression. Further elucidation of its underlying molecular mechanisms and novel biomarkers for early detection is necessary. Exosomes are small extracellular vesicles that are released by multiple cell types acting as message carriers during intercellular communication and are promising biomarker candidates. However, the role of pancreatic cancer cell-derived exosomes in cancer progression and the application of these vesicles as novel diagnostic biomarkers have not been fully studied. In this study, we found that PC-1.0 (a highly malignant pancreatic cell line) cell-derived exosomes could be taken up by and enhance PC-1 (a moderately malignant pancreatic cell line) cell proliferation, migration and invasion abilities. We identified ZIP4 as the most upregulated exosomal protein in PC-1.0 cells from our proteomic analysis. In vitro and in vivo (a subcutaneous BALB/c nude mouse model) studies showed that exosomal ZIP4 can significantly promote pancreatic cancer growth. Using clinical blood samples, we compared the diagnostic values of serum exosomal ZIP4 levels between malignant pancreatic cancer patients (n = 24) and benign pancreatic disease patients (n = 32, AUC = .89), and between biliary disease patients (n = 32, AUC = .8112) and healthy controls (n = 46, AUC = .8931). In conclusion, exosomal ZIP4 promotes cancer growth and is a novel diagnostic biomarker for pancreatic cancer.

Two strains (pika_113T and pika_114) of a previously undescribed Actinomyces -like bacterium were recovered from the intestinal contents of plateau pika (Ochotona curzoniae) on the Tibet-Qinghai Plateau, China. Results from biochemical characterization indicated that the two strains were phenotypically homogeneous and distinct from other previously described species of the genus Actinomyces . Based on the comparison of 16S rRNA gene sequences and genome analysis, the bacteria were determined to be a hitherto unknown subline within the genus Actinomyces , being most closely related to type strains of Actinomyces denticolens and Actinomyces timonensis with a respective 97.2 and 97.1 % similarity in their 16S rRNA gene sequences. Phylogenetic analyses confirmed that pika_113T was well separated from any other recognized species of the genus Actinomyces and within the cluster with A. denticolens and A. timonensis. The genome of strain pika_113T displayed less than 42 % relatedness in DNA–DNA hybridization with all the available genomes of existing species of the genus Actinomyces in the NCBI database. Collectively, based on the phenotypic characteristics and phylogenetic analyses results, we propose the novel isolates as representatives of Actinomyces gaoshouyii sp. nov. The type strain of Actinomyces gaoshouyii is pika_113T (=CGMCC 4.7372T=DSM 104049T), with a genomic DNA G+C content of 71 mol%.

Background Trichinella spiralis is a kind of intestinal nematode that can strongly modulate the host immune system. However, the effects of T. spiralis infection on the intestinal flora are poorly understood. This study aimed to explore the effect of T. spiralis infection on the intestinal flora. Methods The intestinal contents of T. spiralis infected mice were examined through high-throughput sequencing (Illumina) of the V3–V4 hypervariable region in bacterial 16S rRNA gene. The sequences were analyzed using the QIIME software package and other bioinformatics methods. Results Altogether 2,899,062 sequences were generated from the samples collected from different intestinal regions at various infection time points; the 44,843 Operational Taxonomic Unit (OTUs) analysis showed that T. spiralis infection would decrease the diversity of intestinal flora in the infected mice relative to that in the uninfected ones, especially in the large intestine and feces. Further analysis indicated that, the genera Oscillospira from the phylum Firmicutes showed a higher abundance in the helminth-infected small and larger intestines; the genera Bacteroides from the phyla Bacteroides, the genera Lactobacillus from the phyla Firmicutes, the genera Escherichia from the phyla Proteobacteria, and the genera Akkermansia from the phyla Verrucomicrobia displayed increased abundances in the T. spiralis positive fecal samples compared with those in the negative samples. Conclusions T. spiralis infection decreases the diversity of the intestinal flora in the infected mouse. However, it remains unclear about the association between the changes in intestinal flora caused by T. spiralis infection and the parasite pathogenesis, which should be further examined.

Aims To explore the pro-metastatic role of exosomes derived from highly invasive pancreatic cancer cell and the associated aberrant expression of exosomal microRNAs (miRNAs). Main methods Weakly invasive PC-1 cells were treated with exosomes of highly invasive PC-1.0 cells to determine the pro-metastatic effect of PC-1.0 derived exosomes. The exosomal miRNA profile was further investigated using high-throughput sequencing. The level of miR-125b-5p in highly and weakly invasive pancreatic cancer cells was further determined. Pancreatic cancer cells transfected with miR-125b-5p mimic and inhibitor were used to explore the effect of miR-125b-5p on migration, invasion and epithelial-to-mesenchymal transition (EMT). Treatment with PC-1.0 derived exosome and Western blot assay were performed to validate STARD13 as a target of exosomal miR-125b-5p in pancreatic cancer. Key findings PC-1.0 derived exosomes promoted the migration and invasion of weakly invasive PC-1 cells. miRNA sequencing revealed 62 miRNAs upregulated in PC-1.0 derived exosomes. miR-125b-5p most significantly promoted migration and invasion and was associated with metastasis in pancreatic cancer. Further, miR-125b-5p was upregulated in highly invasive pancreatic cancer cells and increased migration, invasion, and EMT. Moreover, its upregulation was associated with activation of MEK2/ERK2 signaling. The tumor suppressor STARD13 was directly targeted by miR-125b-5p in pancreatic cancer, which was associated with good prognosis and was suppressed by exosomes derived from highly invasive cancer cells. Significance This study explored the pro-metastatic role of exosomes derived from highly invasive pancreatic cancer cells and the associated aberrant expression of exosomal miRNAs, which may help to elucidate the metastatic mechanism of pancreatic cancer.

Pancreatic cancer is an aggressive type of cancer with a poor prognosis, short survival rate and high mortality. Therefore, understanding the molecular mechanism underlying the aggressive growth of pancreatic cancer is of importance. An increasing number of studies suggest that numerous microRNAs (miRNAs/miRs) are associated with the tumorigenesis, progression and prognosis of tumors. In a recent study by the present authors, it was revealed that the expression of miR-221 was significantly downregulated in highly aggressive pancreatic cancer cells compared with weakly aggressive pancreatic cancer cells. In addition, miR-221 has been suggested as a novel tumor-associated miRNA, as it is involved in apoptosis, invasion, metastasis and autophagy of tumor cells. However, the function of miR-221 in pancreatic cancer remains yet to be investigated. In the present study, it was revealed that transfection with miR-221 mimic was able to significantly induce apoptosis and autophagy in pancreatic cancer cells compared with the negative control. The protein deacetylase histone deacetylase-6 (HDAC6) has emerged to be an important component in the cellular management of protein aggregates. Studies suggest that HDAC6 serves a function in the clearance of aggresomes, thereby implying a functional association between HDAC6 and autophagy. In the present study, it was revealed that transfection with miR-221 mimic was able to suppress the protein expression of HDAC6 in pancreatic cancer cells compared with the negative control. Immunofluorescence data suggested that the inhibition of HDAC6 was able to induce autophagy in pancreatic cancer cells. Additionally, the results of the present study suggest that the downregulation of miR-221 expression may serve an oncogenic function in the apoptosis and autophagy of pancreatic cancer cells by inducing the expression of HDAC6.

Pancreatic cancer is one of the most malignant tumors. Invasion and metastasis can occur in the early stage of pancreatic cancer, contributing to the poor prognosis. Accordingly, in this study, we evaluated the molecular mechanisms underlying invasion and metastasis. Using mass spectrometry, we found that Integrin alpha 6 (ITGA6) was more highly expressed in a highly invasive pancreatic cancer cell line (PC-1.0) than in a less invasive cell line (PC-1). Through in vitro and in vivo experiments, we observed significant decreases in invasion and metastasis in pancreatic cancer cells after inhibiting ITGA6. Based on data in TCGA, high ITGA6 expression significantly predicted poor prognosis. By using Co-IP combined mass spectrometry, we found that ribosomal protein SA (RPSA), which was also highly expressed in PC-1.0, interacted with ITGA6. Similar to ITGA6, high RPSA expression promoted invasion and metastasis and indicated poor prognosis. Interestingly, although ITGA6 and RPSA interacted, they did not mutually regulate each other. ITGA6 and RPSA affected invasion and metastasis via the PI3K and MAPK signaling pathways, respectively. Inhibiting ITGA6 significantly reduced the expression of p-AKT, while inhibiting RPSA led to the downregulation of p-ERK1/2. Compared with the inhibition of ITGA6 or RPSA alone, the downregulation of both ITGA6 and RPSA weakened invasion and metastasis to a greater extent and led to the simultaneous downregulation of p-AKT and p-ERK1/2. Our research indicates that the development of drugs targeting both ITGA6 and RPSA may be an effective strategy for the treatment of pancreatic cancer.

Two strains (VUL4_1T and VUL4_2) of Gram-staining-positive, catalase-negative, non-spore-forming short rods were isolated from rectal swabs of Old World vultures (Gypaetus barbatus) in the Tibet-Qinghai Plateau, China. Analysis of morphological characteristics and biochemical tests indicated that the two strains closely resembled each other but were distinct from other species of the genus Actinomyces previously described. Based on the results of 16S rRNA gene sequence comparison and genome analysis, strains were determined to be members of the genus Actinomyces , closely related to the type strains of Actinomyces marimammalium (96.4 % 16S rRNA gene sequence similarity), Actinomyces hongkongensis (92.4 %), Actinomyces hordeovulneris (92.3 %) and Actinomyces nasicola (92.2 %), respectively. Optimal growth conditions were 37 °C, pH 6–7, with 1 % (w/v) NaCl. Strain VUL4_1T contained C18 : 1ω9c and C16 : 0 as the major cellular fatty acids and diphosphatidylglycerol as the major component of the polar lipids. The genomic DNA G+C content of VUL4_1T was 54.9 mol%. Strain VUL4_1T showed less than 70 % DNA–DNA relatedness with other species of the genus Actinomyces , further supporting strain VUL4_1T as a representative of a novel species. Based on the phenotypic data and phylogenetic inference, a novel species, Actinomyces liubingyangii sp. nov., is proposed with VUL4_1T (=CGMCC 4.7370T=DSM 104050T) as the type strain.

Two strains of Gram-stain-positive, facultatively anaerobic, non-spore-forming short rods (VUL7T and VUL8) were isolated from rectal swabs of Old World vultures, namely Gyps himalayensis, in Tibet-Qinghai Plateau, China. Optimal growth occurred at 37 °C, pH 6–7, with 1 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences classified the two strains to the genus Actinomyces , with highest 16S rRNA gene sequence similarity (95 %) to type strains of Actinomyces haliotis , Actinomyces radicidentis and Actinomyces urogenitalis . The major cellular fatty acids were C18 : 1ω9c and C16 : 0. MK-10(H4) was the major respiratory quinone. The genomic DNA G+C content of the isolate was 54.4 mol%. DNA–DNA hybridization values with the most closely related species of the genus Actinomyces was 24.6 %. The two strains can be differentiated from the most closely related species such as A. haliotis , A. radicidentis, A. graevenitzii and A. urogenitalis by a list of carbohydrate fermentations and enzyme activities. On the basis of physiological, biochemical and phylogenetic analysis, strains VUL7T and VUL8 represent novel species of the genus Actinomyces , for which the name Actinomyces vulturis sp. nov. is proposed. The type strain is VUL7T (=CGMCC 4.7366T=DSM 103437T).

Pancreatic cancer is a highly metastatic and chemo-resistant disease. Secreted proteins involved in cell-cell interactions play an important role in changing the tumor microenvironment. Previous studies generally focus on the secretome of cancer cell line from serum-free media, due to the serious interference of fetal bovine serum (FBS). However, serum-starvation may alter expression patterns of secreted proteins. Hence, efforts to decrease the interference of serum in proteomic analysis of serum-containing media have been hampered to quantitatively measure the tumor secretion levels. Recently, the metabolic labeling, protein equalization, protein fractionation and filter-aided sample preparation (FASP) strategy (MLEFF) has been successfully used to avoid the disturbance of serum on secretome analysis. Here, this efficient method was applied for comparative secretome analysis of two hamster pancreatic cancer cells with differentially metastatic potentials, enabling the observation of 161 differentially expressed proteins, including 106 proteins that had been previously reported and detected in plasma. By integrated analysis of our data and publicly available bioinformatics resources, we found that a combination panel consisting of CDH3, PLAU, and LFNG might improve the prognosis of overall pancreatic cancer survival. These secreted proteins may serve as a potential therapeutic targets for pancreatic cancer metastasis.

Two bacterial strains were isolated from faecal samples of Tibetan antelopes. The isolates were Gram-stain-positive, catalase-negative, coccus-shaped organisms that were tentatively identified as representing a novel streptococcal species based on their morphological features, biochemical test results and phylogenomic findings. Comparative 16S rRNA gene sequencing studies confirmed that the organisms were members of the genus Streptococcus , but they did not correspond to any recognized species of the genus. The nearest phylogenetic relative of the unknown coccus was Streptococcus ursoris NUM 1615T (93.4 % 16S rRNA gene sequence similarity). Analysis of groEL and rpoB gene sequences of the novel isolates showed interspecies divergence of 27.0 and 22.2 %, respectively, from the type strain of its closest 16S rRNA gene phylogenetic relative, S. ursoris. The complete genome of strain TA 26T has been sequenced. Digital DNA–DNA hybridization studies between strain TA 26T and other species of the genus Streptococcus deposited in the GenBank database showed less than 70 % DNA–DNA relatedness, supporting a novel species status of the strain. On the basis of their genotypic and phenotypic differences from recognized Streptococcus species, the two isolates represent a novel species of the genus Streptococcus , for which the nameStreptococcus pantholopis sp. nov. (type strain TA 26T=CGMCC 1.15667T=DSM 102135T) is proposed.

NSFC [30700092]; Chinese Academy of Sciences ; China Postdoctoral Science Foundation [20060400529]; Areas of Excellence [AoE/P-04/2004]; Xiamen University

Mechanisms of abnormal protein phosphorylation that regulate cell invasion and metastasis in pancreatic cancer remain obscure. In this study, we used high-throughput phosphorylation array to test two pancreatic cancer cell lines (PC-1 cells with a low, and PC-1.0 cells with a high potential for invasion and metastasis). We noted that a total of 57 proteins revealed a differential expression (fold change ≥ 2.0). Six candidate proteins were further validated by western blot with results found to be accordance with the array. Of 57 proteins, 32 up-regulated proteins (e.g. CaMK1-α and P90RSK) were mainly involved in ErbB and neurotrophin signaling pathways as determined using DAVID software, while 25 down-regulated proteins (e.g. BID and BRCA1) were closely involved in apoptosis and p53 signaling pathways. Moreover, four proteins (AKT1, Chk2, p53 and P70S6K) with different phosphorylation sites were found, not only among up-regulated, but also among down-regulated proteins. Importantly, specific phosphorylation sites can affect cell biological functions. CentiScaPe software calculated topological characteristics of each node in the protein-protein interaction (PPI) network: we found that AKT1 owns the maximum node degrees and betweenness in the up-regulation protein PPI network (26 nodes, average path length: 1.89, node degrees: 6.62±4.18, betweenness: 22.23±35.72), and p53 in the down-regulation protein PPI network (17 nodes, average path length: 2.04, node degrees: 3.65±2.47, betweenness: 16.59±29.58). In conclusion, the identification of abnormal protein phosphorylation related to invasion and metastasis may allow us to identify new biomarkers in an effort to develop novel therapeutic drug targets for pancreatic cancer treatment.

The Old World vulture may carry and spread pathogens for emerging infections since they feed on the carcasses of dead animals and participate in the sky burials of humans, some of whom have died from communicable diseases. Therefore, we studied the precise fecal microbiome of the Old World vulture with metataxonomics, integrating the high-throughput sequencing of almost full-length small subunit ribosomal RNA (16S rRNA) gene amplicons in tandem with the operational phylogenetic unit (OPU) analysis strategy. Nine vultures of three species were sampled using rectal swabs on the Qinghai-Tibet Plateau, China. Using the Pacific Biosciences sequencing platform, we obtained 54 135 high-quality reads of 16S rRNA amplicons with an average of 1442±6.9 bp in length and 6015±1058 reads per vulture. Those sequences were classified into 314 OPUs, including 102 known species, 50 yet to be described species and 161 unknown new lineages of uncultured representatives. Forty-five species have been reported to be responsible for human outbreaks or infections, and 23 yet to be described species belong to genera that include pathogenic species. Only six species were common to all vultures. Clostridium perfringens was the most abundant and present in all vultures, accounting for 30.8% of the total reads. Therefore, using the new technology, we found that vultures are an important reservoir for C. perfringens as evidenced by the isolation of 107 strains encoding for virulence genes, representing 45 sequence types. Our study suggests that the soil-related C. perfringens and other pathogens could have a reservoir in vultures and other animals.

The effect of monovalent ions on both the reactivity and global folding of the 8-17 DNAzyme is investigated, and the results are compared with those of the hammerhead ribozyme, which has similar size and secondary structure. In contrast to the hammerhead ribozyme, the 8-17 DNAzyme activity is not detectable in the presence of 4 M K(+), Rb(+), or Cs(+) or in the presence of 80 mM, [Co(NH(3))(6)](3+). Only 4 M Li(+), NH(4)(+) and, to a lesser extent, Na(+) conferred detectable activity. The observed rate constants (k(obs) approximately 10(-3) min(-1) for Li(+) and NH(4)(+)) are approximately 1000-fold lower than that in the presence of 10 mM Mg(2+), and approximately 200,000-fold slower than that in the presence of 100 microM Pb(2+). Since the hammerhead ribozyme displays monovalent ion-dependent activity that is often within approximately 10-fold of divalent metal ion-dependent activity, these results suggest that the 8-17 DNAzyme, obtained by in vitro selections, has evolved to have a more stringent divalent metal ion requirement for high activity as compared to the naturally occurring ribozymes, making the 8-17 DNAzyme an excellent choice as a Pb(2+) sensor with high selectivity. In contrast to the activity data, folding was observed in the presence of all the monovalent ions investigated, although those monovalent ions that do not support DNAzyme activity have weaker binding affinity (K(d) approximately 0.35 M for Rb(+) and Cs(+)), while those that confer DNAzyme activity possess stronger affinity (K(d) approximately 0.22 M for Li(+), Na(+) and NH(4)(+)). In addition, a correlation between metal ion charge density, binding affinity and enzyme activity was found among mono- and divalent metal ions except Pb(2+); higher charge density resulted in stronger affinity and higher activity, suggesting that the observed folding and activity is at least partially due to electrostatic interactions between ions and the DNAzyme. Finally, circular dichroism (CD) study has revealed Z-DNA formation with the monovalent metal ions, Zn(2+) and Mg(2+); the K(d) values obtained using CD were in the same range as those obtained from folding studies using FRET. However, Z-DNA formation was not observed with Pb(2+). These results indicate that Pb(2+)-dependent function follows a different mechanism from the monovalent metal ions and other divalent metal ions; in the presence of latter metal ions, metal-ion dependent folding and structural changes, including formation of Z-DNA, play an important role in the catalytic function of the 8-17 DNAzyme.

Although the atomic structures of actin, tropomyosin, and troponin have been solved separately, much remains unknown about their assembled structures in the thin filament. A variety of evidence indicates that the TnI C-terminus interacts with tropomyosin on the actin filament, and this interaction is a critical aspect of the regulation of muscle contraction. The present report concerns this TnI-tropomyosin interaction, and suggests that it involves the middle region of tropomyosin. Tropomyosin is a 40nm coiled coil protein, comprised of 7 successive quasi-repeating regions. To determine the involvement of these quasi repeats in TnI binding a panel of tropomysoins was studied in which different quasi repeats were deleted. C-terminal TnI peptide 131-210 (TnI C-term) had different effects on tropomyosin, depending upon the presence of different quasi repeats. When all tropomyosin quasi repeats are present, TnI C-term strengthens tropomyosin-actin binding affinity five-fold. Removal of tropomyosin quasi-repeats 2 and 3 diminishes this effect of TnI C-term slightly to 2.7 fold. In contrast, removal of tropomyosin repeats 3 and 4 completely abolished the TnI C-term effect on tropomyosin-actin binding. The behavior of tropomyosin missing repeats 2, 3 and 4 is very similar to the behavior of tropomyosin missing repeats 3 and 4. These findings suggest that in the normal thin filaments, which contain only one troponin for every tropomyosin, it is the middle, fourth quasi-repeat of tropomyosin which interacts with TnI C-terminus.