Ascorbic acid-mediated reactive oxygen species homeostasis modulates the switch from tapetal cell division to cell differentiation in Arabidopsis.

The major antioxidant L-ascorbic acid (AsA) plays important roles in plant growth, development, and stress responses. However, the importance of AsA concentration and the regulation of AsA metabolism in plant reproduction remain unclear. In Arabidopsis (Arabidopsis thaliana) anthers, the tapetum monolayer undergoes cell differentiation to support pollen development. Here, we report that a transcription factor, DEFECTIVE IN TAPETAL DEVELOPMENT AND FUNCTION 1 (TDF1), inhibits tapetal cell division leading to cell differentiation. We identified SKEWED5-SIMILAR 18 (SKS18) as a downstream target of TDF1. Enzymatic assays showed that SKS18, annotated as a multicopper oxidase-like protein, has ascorbate oxidase activity, leading to AsA oxidation. We also show that VITAMIN C DEFECTIVE1 (VTC1), an AsA biosynthetic enzyme, is negatively controlled by TDF1 to maintain proper AsA contents. Consistently, either knockout of SKS18 or VTC1 overexpression raised AsA concentrations, resulting in extra tapetal cells, while SKS18 overexpression in tdf1 or the vtc1-3 tdf1 double mutant mitigated their defective tapetum. We observed that high AsA concentrations caused lower accumulation of reactive oxygen species (ROS) in tapetal cells. Overexpression of ROS scavenging genes in tapetum restored excess cell divisions. Thus, our findings demonstrate that TDF1-regulated AsA balances cell division and cell differentiation in the tapetum through governing ROS homeostasis.

Retraction Note to: Effect of miR-451 on the Biological Behavior of the Esophageal Carcinoma Cell Line EC9706.

The Editor-in-Chief has retracted this article [1] because Figure 8 overlaps with Figure 6b of [2] and Figure 6 overlaps with Figure 3 of [3] and Figure 3 of [4].

Exploring ecological effects of arsenic and cadmium combined exposure on cropland soil: from multilevel organisms to soil functioning by multi-omics coupled with high-throughput quantitative PCR.

Arsenic (As) and cadmium (Cd) pose potential ecological threats to cropland soils; however, few studies have investigated their combined effects on multilevel organisms and soil functioning. Here, we used collembolans and soil microbiota as test organisms to examine their responses to soil As and Cd co-contamination at the gene, individual, and community levels, respectively, and further uncovered ecological relationships between pollutants, multilevel organisms, and soil functioning. At the gene level, collembolan transcriptome revealed that elevated As concentrations stimulated As-detoxifying genes AS3MT and GST, whereas the concurrent Cd restrained GST gene expression. At the individual level, collembolan reproduction was sensitive to pollutants while collembolan survival wasn't. At the community level, significant but inconsistent correlations were observed between the biodiversity of different soil keystone microbial clusters and soil As levels. Moreover, soil functioning related to nutrient (e.g., carbon, nitrogen, phosphorus, and sulfur) cycles was inhibited under As and Cd co-exposure only through the mediation of plant pathogens. Overall, these findings suggested multilevel bioindicators (i.e., AS3MT gene expression in collembolans, collembolan reproduction, and biodiversity of soil keystone microbial clusters) in cropland soils co-contaminated with As and Cd, thus improving the understanding of the ecotoxicological impact of heavy metal co-contamination on soil ecosystems.

Effect of miR-451 on the biological behavior of the esophageal carcinoma cell line EC9706.

BACKGROUND: MicroRNAs play important roles in coordinating a variety of cellular processes. Abnormal expression of miRNAs has been linked to several cancers. However, the functional role of miR-451 in esophageal squamous cell carcinoma remains unclear. AIMS: The present study explored the effects of miR-451 on the biological behavior of the esophageal carcinoma cell line EC9706. METHODS: Synthetic miR-451 mimics were transfected into EC9706 cells using Lipofectamine™ 2000. The expression of miR-451 was analyzed by RT-PCR and the expressions of Bcl-2, AKT and phosphorylated AKT were analyzed by Western blotting. The MTT assay, soft agar colony formation assay, transwell assay and FACS were used to assess the effect of miR-451 on EC9706 cell proliferation, invasion, metastasis and apoptosis. Tumor growth was assessed by subcutaneous inoculation of cells into BALB/c nude mice. RESULTS: In comparison to the controls, a significant increase in the expression of miR-451 was associated with significantly decreased expressions of Bcl-2, AKT and p-AKT, and a significant increase in the apoptosis rate. The number of cell clones was significantly decreased by miR-451 expression, which also caused the inhibition of cell proliferation. The average number of cells penetrating the matrigel was significantly lower than the controls. Injection of miR-451 inhibited tumor growth in a xenograft model. CONCLUSIONS: Upregulated expression of miR-451 induced apoptosis and suppressed cell proliferation, invasion and metastasis in the esophageal carcinoma cell line EC9706. In addition, injection of miR-451 inhibited tumor growth in a xenograft model of esophageal cancer.

[Prevention from PICC-related venous thrombosis in the upper limbs of malignant tumor patients with moxibustion combined with plucking at Jiquan (HT 1): a randomized controlled trial].

OBJECTIVE: To observe the clinical effect of moxibustion combined with plucking technique at Jiquan (HT 1) for preventing peripherally inserted central catheter (PICC)-related venous thrombosis in the upper limbs of malignant tumor patients. METHODS: A total of 80 malignant tumor patients undergoing PICC were randomized into an observation group and a control group, 40 cases in each one. In the control group, the routine care for PICC was exerted. In the observation group, besides the routine care, moxibustion combined with plucking technique at Jiquan (HT 1) was added. Mild moxibustion was exerted along the venous distribution of PICC (avoiding the entry site) for 10 to 15 min, and then, the circling moxibustion was applied to Quchi (LI 11), Xuehai (SP 10) and Tianfu (LU 3), 3 to 5 min at each acupoint. Finally, plucking technique was given at Jiquan (HT 1) for 5 to 10 min. This combined therapy was intervened since the 2nd day of PICC placement, once daily, 5 times a week, for 3 weeks totally. The incidence of the PICC-related venous thrombosis in the upper limbs was compared between the two groups on day 42 of placement. On day 2, 7, 14, 21, 28, 35 and 42 of PICC placement, the peak systolic velocity (PSV) and the end-diastolic velocity (EDV) of the subclavicular vein on the placement side were observed separately in the two groups. RESULTS: The incidence of the PICC-related venous thrombosis in the upper limbs in the observation group was lower than that in the control group (2.5% [1/40] vs 17.5% [7/40], P<0.05). From day 7 to 35 of PICC placement, PSV of the subclavicular vein on the placement side was higher than that on the day 2 of PICC placement in the observation group (P<0.05). On day 28 and 42 of PICC placement, PSV of the subclavicular vein on the placement side was lower than that on the day 2 of PICC placement in the control group (P<0.05). In the observation group, EDV of the subclavicular vein on the placement side was higher than that on the day 2 of PICC placement from day 7 to 28 of PICC placement (P<0.05). In the control group, EDV of the subclavicular vein on the placement side from day 28 to 42 of PICC placement was lower than that on the day 2 of PICC placement (P<0.05). From day 7 to 42 of PICC placement, PSV and EDV of the subclavicular vein on the placement side in the observation group were all higher than those in the control group (P<0.01, P<0.05). CONCLUSION: The combined treatment of moxibustion with plucking technique at Jiquan (HT 1) can effectively prevent PICC-related venous thrombosis in the upper limbs and improve venous blood flow velocity in malignant tumor patients.

Room-temperature ppb-level SO2 gas sensors based on RGO/WO3 and MWCNTs/WO3 nanocomposites.

ppb-level SO2 gas sensors for use at room temperature were fabricated using an in situ one-pot polyol method combined with metal organic decomposition (MOD) of nanocomposite films of multi-walled carbon nanotubes/WO3 (MWCNTs/WO3) and reduced graphene oxide/WO3 (RGO/WO3) on an alumina substrate. Comparative gas sensing results showed that the SO2 gas sensor based on the RGO/WO3 nanocomposite film exhibited a higher response compared with the MWCNTs/WO3 nanocomposite film and pristine WO3 film, sensing SO2 gas at very low (ppb-level) concentrations at room temperature. The SO2 gas sensor based on the RGO/WO3 nanocomposite film also had fast response and recovery times, good reproducibility, and the lowest detection limit. The formation of new conducting pathways and the spread of the depletion layers at the interface of the doped MWCNTs or RGO and the WO3 matrix upon interaction with SO2 gas were responsible for the enhanced response.

In vivo and in vitro antitumor effects of a staphylococcal enterotoxin A mutant (SEA-H61D).

In this study, we evaluated SEA-H61D, a staphylococcal enterotoxin A mutant without emetic activity, as an antitumor agent in vitro and in vivo. It showed that SEA-H61D could significantly inhibit the growth of many cancer cell lines in vitro at very low concentrations by activating human peripheral blood mononuclear cells (PBMCs). CD4+ and CD8+ T lymphocytes could be activated at a dose between 125 and 500 µg/kg. Systemic administration of SEA-H61D in vivo significantly inhibited tumor growth, with the treated group undergoing tumor necrosis and showing a strong infiltration of lymphocytes to the tumor area.

[The effects of ginsenosides Rg3 on the expressions of VEGF and KDR in human lung squamous cancer cells].

OBJECTIVE: To investigate the inhibitive effects of Ginsenosides Rg3 (GS-Rg3) in the process of tumor angiogenesis and the effects on the expressions of VEGF and its receptor KDR in human lung squamous cancer SK-MES-1 cell line. METHODS: Human lung cancer SK-MES-1 cells were cultured in vitro and immunocytochemistry and RT-PCR methods were used to detect the effects of different concentrations of Rg3 on the expressions of VEGF and KDR on SK-MES-1 cells. RESULTS: The immunocytochemistry results showed that the positive rates of VEGF protein in different group of SK-MES-1 cells were 81.33 +/- 9.04, 61.80 +/- 7.98, 43.80 +/- 5.25, 29.77 +/- 8.04, respectively. The positive rates of KDR protein in different group of SK-MES-1 cells were 65.51 +/- 7.45, 51.73 +/- 9.21, 34.87 +/- 6.15, 22.04 +/- 5.11, respectively. There were significant differences between each group. RT-PCR results suggested that with the increase of the concentration of Rg3, VEGF and KDR amplified bands gradually weakened. There were significant differences between each group. CONCLUSION: GS-Rg3 can down-regulate the expressions of KDR and VEGF protein and their mRNA in human lung squamous cancer SK-MES-1 cells. It may be one of the mechanisms in the process of inhibiting tumor angiogenesis.

Effects of Bifidobacterium infantis on cytokine-induced neutrophil chemoattractant and insulin-like growth factor-1 in the ileum of rats with endotoxin injury.

BACKGROUND: The digestive tract is the maximal immunizing tissue in the body, and mucosal integrity and functional status of the gut is very important to maintain a healthy organism. Severe infection is one of the most common causes of gastrointestinal dysfunction, and the pathogenesis is closely related to endotoxemia and intestinal barrier injury. Bifidobacterium is one of the main probiotics in the human body that is involved in digestion, absorption, metabolism, nutrition, and immunity. Bifidobacterium plays an important role in maintaining the intestinal mucosal barrier integrity. This study investigated the protective mechanism of Bifidobacterium during ileal injury in rats. AIM: To investigate the effects of Bifidobacterium on cytokine-induced neutrophil chemoattractant (CINC) and insulin-like growth factor 1 (IGF-1) in the ileum of rats with endotoxin injury. METHODS: Preweaning rats were randomly divided into three groups: Control (group C), model (group E) and treatment (group T). Group E was intraperitoneally injected with lipopolysaccharide (LPS) to create an animal model of intestinal injury. Group T was intragastrically administered Bifidobacterium suspension 7 d before LPS. Group C was intraperitoneally injected with normal saline. The rats were killed at 2, 6 or 12 h after LPS or physiological saline injection to collect ileal tissue samples. The expression of ileal CINC mRNA was evaluated by reverse transcription-polymerase chain reaction (RT-PCR), and expression of ileal IGF-1 protein and mRNA was detected by immunohistochemistry and RT-PCR, respectively. RESULTS: The ileum of rats in Group C did not express CINC mRNA, ileums from Group E expressed high levels, which was then significantly decreased in Group T (F = 23.947, P < 0.05). There was no significant difference in CINC mRNA expression at different times (F = 0.665, P > 0.05). There was a high level of IGF-1 brown granules in ileal crypts and epithelial cells in Group C, sparse staining in Group E, and dark, dense brown staining in Group T. There was a significant difference between Groups C and E and Groups E and T (P < 0.05). There was no significant difference in IGF-1 protein expression at different times (F = 1.269, P > 0.05). IGF-1 mRNA expression was significantly different among the three groups (P < 0.05), though not at different times (F = 0.086, P > 0.05). CONCLUSION: Expression of CINC mRNA increased in the ileum of preweaning rats with endotoxin injury, and exogenous administration of Bifidobacterium reduced CINC mRNA expression. IGF-1 protein and mRNA expression decreased in the ileum of preweaning rats with endotoxin injury, and exogenous administration of Bifidobacterium prevented the decrease in IGF-1 expression. Bifidobacterium may increase IGF-1 expression and enhance intestinal immune barrier function in rats with endotoxin injury.

Epidemiological analysis of group A streptococci recovered from patients in China.

Since the mid-1980s, there has been a resurgence of severe forms of invasive group A streptococcal (GAS) disease in many countries and regions. However, there has not been any systemic epidemiologic analysis of GAS disease reported in mainland China. To analyse the molecular epidemiology of GAS disease, 86 strains from patients in different regions of mainland China were collected. The collection sites included blood, pus, wounds, the epipharynx and other sites. A total of 21 different emm types were identified in the isolates. In both invasive and non-invasive isolates, M1 (29.1%) and M12 (23.3%) were the most prevalent types, a different distribution to M type distributions reported in other countries. Furthermore, minor emm gene sequence alterations were noted for six types. Several important GAS virulence factors were detected by PCR using specific primers. The speB and slo genes were detected in all isolates and were species specific. Four superantigen genes, speA, speC, smeZ and ssa, were found in 52% (45/86), 51% (44/86), 82% (71/86) and 23% (27/86) of isolates, respectively. M1 isolates harboured more speA (84%) and fewer speC genes (44%), while M12 isolates had fewer speA (35%) and more speC genes (100%). There was also an association between some virulence genes and isolation sites, perhaps due to the correlation between the emm type distribution and virulence gene occurrence. For two important virulence genes related to necrotizing fasciitis, the sil gene was only carried by 11 of 86 isolates, and no sil gene contained the start codon ATA. The sla gene rarely occurred in GAS isolates, only four of 86 GAS strains being positive, including two isolates obtained from blood. In antimicrobial susceptibility tests, the overall rate of drug resistance in GAS isolates was higher than reported rates in other countries, and the resistance rates to erythromycin, tetracycline and clindamycin were 91.8, 93.4 and 80%, respectively. This epidemiological study may help to understand the pathogenesis of GAS disease and aid in vaccine development.

Improved stability and yield of Fv targeted superantigen by introducing both linker and disulfide bond into the targeting moiety.

Bacterial superantigens (SAg) are the most potent activators of human T lymphocytes and recombinant immunotoxin using bacterial SAg shows promising clinical values. To engineer superantigen for immunotherapy of hepatocellular carcinoma, we genetically fused the superantigen staphylococcus enterotoxin A (SEA(D(227)A)) to the single-chain disulfide-stabilized Fv (scdsFv) of anti-hepatoma monoclonal antibody HAb25 through a short peptide GGGSGGS. We expressed this recombinant protein in Escherichia coli and extract it from inclusion bodies. We found purified scdsFv-targeted SAg contains equivalent binding affinity with disulfide-stabilized Fv (dsFv) targeted SAg and single-chain Fvs (scFv) targeted SAg, but more stable and more suitable for large scale production. The MTS(3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliu m, inner salt) assay shows that the scdsFv-targeted SAg also shares the ability to activate a large number of T lymphocytes and has cytotoxic activity on human hepatoma cell line SMMC-7721. Therefore, this novel generation of recombinant immunotoxins using scdsFv has a high potential in hepato cancer treatment and the same strategy may also be applied to other cancer treatments.

Disulfide-stabilized single-chain antibody-targeted superantigen: construction of a prokaryotic expression system and its functional analysis.

AIM: To construct the expression vector of B3 (scdsFv)-SEA (D227A) and to identify its binding and cytotoxic ability to B3 antigen positive carcinoma cell lines. METHODS: This fusion protein was produced by a bacterial expression system in this study. It was expressed mainly in the inclusion body. The gene product was solubilized by guanidine hydrochloride, refolded by conventional dilution method, and purified using SP-sepharose cation chromatography. RESULTS: The expression vector B3 (scdsFv)-SEA-PET was constructed, the expression product existed mainly in the inclusion body, the refolding product retained the binding ability of the single-chain antibody and had cytotoxic effect on HT-29 colon carcinoma cells. The stability assay showed that the resulting protein was stable at 37 degrees. CONCLUSION: This genetically engineered B3 (scdsFv)-SEA fusion protein has bifunction of tumor targeting and tumor cell killing and shows its promises as an effective reagent for tumor-targeted immunotherapy.

RNAi-induced K-Ras gene silencing suppresses growth of EC9706 cells and enhances chemotherapy sensitivity of esophageal cancer.

To analyze the growth, proliferation, apoptosis, invasiveness and chemotherapy sensitivity of EC9706 cells after K-Ras gene silencing, an expression carrier pSilencer-siK-Ras was constructed, and the EC9706 cell line was transfected using a liposome technique. Six groups were established: Control, siRNA NC (transfected with empty vector pSilencer2.1); Ras siRNA (transfected with pSilencer-siK-Ras2); Paclitaxel; Paclitaxel + siRNA NC; and Ras siRNA +Paclitaxel. After the treatment, RT-PCR, Western blotting, MTT assay, flow cytometry and the Transwell technique were used to assess expression of K-Ras mRNA and protein in EC9706 cells, as well as cell growth, proliferation, apoptosis and invasiveness. The effect of Paclitaxel chemotherapy was also tested. pSilencer-siK-Ras2 effectively down-regulated expression of K-Ras mRNA and protein in EC9706 cells, growth being significantly inhibited. Flow cytometry indicated obvious apoptosis of cells in the experimental group, with arrest in the G1 phase; cell migration ability was also reduced. After pSilencer-siK-Ras2 transfection or the addition of Paclitaxel, EC9706 cells were suppressed to different extents; the suppressive effect was strengthened by combined treatment. The results suggested that RNAi-induced K-Ras gene silencing could enhance chemotherapy sensitivity of esophageal cancer.

[Expression, purification and protective antigen analysis of cell wall protein MRP of Streptococcus suis type 2].

AIM: To amplify the mrp gene of Streptococcus suis type 2 05ZYH33, express it in E.coli BL21 in order to acquire high purity recombinant protein MRP, then evaluate the protective antigen of recombinant protein MRP. METHODS: Using PCR technology to obtain the product of mrp gene of 05ZYH33, and then cloned it into the expression vector pET28a(+). The recombinant protein was purified by affinity chromatography, later immunized New Zealand rabbit to gain anti-serum, then test the anti-serum titer by ELISA. The opsonophagocytic killing test demonstrated the abilities of protective antigen of MRP. RESULTS: The truncated of MRP recombinant protein in E.coli BL21 expressed by inclusion bodies, and purified it in high purity. After immunoprotection, the survival condition of CD-1 was significantly elevated. The survival rate of wild-type strain 05ZYH33 in blood was apparently decreased after anti-serum opsonophagocyticed, but the mutant delta; MRP showed no differences. CONCLUSION: MRP represent an important protective antigen activity.

[Construction and activities of suilysin mutants].

AIM: To construct the suilysin mutant without hemolytic activity and evaluate its functions. METHODS: The proline in 353 site of suilysin was site-directed mutated to alanine, leucine and valine, respectively. The recombinant mutants were renaturated and purified by immobilized metal ion affinity chromatography, and the purified proteins were evaluated in the hemolytic activity and immunogenicity. RESULTS: We obtained three mutants, SLY(P353A), SLY(P353L) and SLY(P353V). The SLY(P353V) mutant had non-hemolytic activity. Western blotting and animal experiments showed that SLY(P353V) mutant still had immunogenicity. CONCLUSION: Suilysin mutant SLY(P353V) has no hemolytic activity but remains immunogenicity.

[Purification and biological activities analysis of streptococcus suis Serotype 2 suilysin].

AIM: To explore the purification methods of wild-type and recombinant suilysin and to evaluate their biological activities. METHODS: Wild-type suilysin was purified by ammonium sulfate precipitation, anion-exchange chromatography and hydrophobic chromatography in turn, while recombinant suilysin was first refolded and purified by immobilized metal ion affinity chromatography, and further purified by Thiopropyl Sepharose 6B. The biological activities were evaluated by hemolysis test, cytotoxicity assay. RESULTS: Both prepared wild-type and recombinant suilysin, with purify over 90%, have hemolysis activity and could injure target cells at high concentration while cholesterol could completely inhibit their activities. CONCLUSION: Recombinant suilysin has similar biological activities with wild-type suilysin, and this work contributed to further study the functions of suilysin on pathogenesis of steptococcus suis.

[Expression, purification and activity assay of Streptococcus suis serotype 2 cell wall protein SSU1664].

AIM: To amplify the SSU1664 gene from Streptococcus suis serotype 2 strain 05ZY, to express the gene in E.coli, and to evaluate the activities of the recombinant protein. METHODS: SSU1664 gene was amplified by PCR using primers according to 05ZY genome sequences and cloned into the expression vector. The recombinant protein was purified by affinity chromatography and its immunogen activities were tested by Western blot and ELISA. RESULTS: SSU1664 gene could solublely express in E.coli BL21(DE3). Western blot analysis showed that the recombinant protein could react with rat serum immunized with Streptococcus suis, but not with non-immunized rat serum. ELISA assay showed that anti-SSU1664 IgM content in Streptococcus suis-infected patient was significantly higher than that in healthy donors. CONCLUSION: The recombinant SSU1664 protein has immunogen activity and might be one promising Streptococcus suis vaccine candidate and diagnosis marker of Streptococcus suis early infection.

Development of colloidal gold-based immunochromatographic assay for rapid detection of Streptococcus suis serotype 2.

To develop colloidal gold immunochromatographic strips for the direct detection of the Streptococcus suis serotype 2 antigen, colloidal gold was prepared by reduction of a gold salt with sodium citrate and coupled with polyclonal antibody against S. suis serotype 2. The optimal concentrations of the capture antibody and the coating antibody were determined to be 22mug/mL and 2.0mg/mL, respectively, and that of the blocking buffer was determined to be 1.5% bovine serum albumin. Different serotypes of S. suis and other related bacteria were used to evaluate the sensitivity, specificity, and stability of the immunochromatographic strips. The detection sensitivity was found to be as high as 10(6)CFU/mL. There was no cross-reaction of the antibodies with other serotypes of S. suis (except with SS1/2, which shares some common sugar residues or antigenic determinants with serotype 2) and other related bacteria. In conclusion, we developed colloidal gold immunochromatographic strips that had high sensitivity and specificity. This method proved to be feasible, convenient, rapid, and effective for detecting S. suis serotype 2.

[Ginsenoside Rg3 induces apoptosis of human lung squamous cell carcinoma SK-MES-1 cell line].

OBJECTIVE: To investigate the effect of ginsenoside Rg3 on the apoptosis and survivin expression in human lung squamous cell carcinoma cell line SK-MES-1. METHODS: SK-MES-1 cells were divided into Rg3 treatment group, blank control group and positive control (arsenic trioxide) group. The apoptotic rate of the cells in each group was determined using flow cytometry, and the expression of survivin protein and mRNA was detected by immunocytochemistry and RT-PCR, respectively. RESULTS: A 48-h treatment with Ginsenoside Rg3 induced increased apoptotic rate of SK-MES-1 cells in a dose-dependent manner. Ginsenoside Rg3 significantly downregulated the expressions of survivin protein and mRNA as compared with the expression levels in the blank control group (P<0.05). CONCLUSION: Ginsenoside Rg3 can induce the apoptosis of SK-MES-1 cells, the mechanism of which may involve inhibited survivin expression.

Proteome analysis of Streptococcus suis serotype 2.

Outbreaks in humans, caused by Streptococcus suis serotype 2 (SS2), were reported in 1998 and 2005 in China. However, the mechanism of SS2-associated infection remains unclear. For the first time, a 2-D gel approach combined with MS was used to establish a comprehensive 2-D reference map for aiding our understanding of the pathogenicity of SS2. The identification of 694 out of 834 processed spots revealed 373 proteins. Most of the identified proteins were located in the cytoplasm and were involved in energy metabolism, protein synthesis, and cellular processes. Proteins that were abundant in the 2-DE gels could be linked mainly to housekeeping functions in carbohydrate metabolism, protein quality control and translation. 2-DE of secretory proteins was performed using IPG strips of pH 4-7. Among the 102 protein spots processed, 87 spots representing 77 proteins were successfully identified. Some virulence-associated proteins of SS2 were found, including arginine deiminase, ornithine carbamoyl-transferase, carbamate kinase, muramidase-released protein precursor, extracellular factor, and suilysin. Enolase and endopeptidase have been proposed as putative virulence-associated factors in this study. The 2-D reference map might provide a powerful tool for analyzing the virulence factor and the regulatory network involved in the pathogenicity of this microorganism.