Development and evaluation of an up-converting phosphor technology-based lateral flow assay for rapid detection of Francisella tularensis.

Francisella tularensis is a potential biowarfare/bioterrorism agent and zoonotic pathogen that causes tularemia; thus, surveillance of F. tularensis and first-level emergency response using point-of-care testing (POCT) are essential. The UPT-LF POCT assay was established to quantitatively detect F. tularensis within 15 min, and the sensitivity of the assay was 10(4) CFU · mL(-1) (100 CFU/test). The linear quantitative range covered five orders of magnitude, and the coefficients of variation were less than 10%. Except Shigella dysenteriae, UPT-LF showed excellent specificity to four strains that are also potential biowarfare/bioterrorism agents and 13 food-borne pathogenic strains. Samples with pH 2-13, high ion strengths (≥ 2 mol · L(-1) solution of KCl and NaCl), high viscosities (≤ 50 mg · mL(-1) PEG20000 or ≥ 20% glycerol), and high concentrations of biomacromolecules (≥ 400 mg · mL(-1) bovine serum albumin or ≥ 80 mg · mL(-1) casein) showed little influence on the assay. For practical utilization, the tolerance limits for seven powders and eight viscera were determined, and operation errors of liquid measurement demonstrated a minor influence on the strip. Ftu-UPT-LF is a candidate POCT method because of its excellent sensitivity, specificity, and stability in complex samples, as well as low operation error.

[Development and systematical evaluation of an up-converting phosphor technology based lateral flow assay for quantitative detection of Burkholderia pseudomallei].

OBJECTIVE: To develop an up-converting phosphor technology-based lateral-flow (UPT-LF) assay for rapid quantitative detection of Burkholderia pseudomallei on site. METHODS: The strip Bps-UPT-LF strip was prepared with up-converting phosphor (UCP) particles as the bio-label using double-antibody sandwich method. Detection performance, including sensitivity, quantitative accuracy, precision, and specificity, were first evaluated using bacterial suspensions of Burkholderia pseudomallei, the related species and the strains which had similar routes of transmission with serial standard concentrations diluted by phosphate buffer, then biological and chemical reagents and simulated samples with series concentrations were employed for sample tolerance evaluation, while the operation error during on site detection was also evaluated through adjusting liquid measure. RESULTS: The whole detection was accomplished within 20 minutes, and the sensitivity was 10(4) CFU/ml with linear quantitative range from 10(4) CFU/ml to 10(7) CFU/ml, which covered four orders of magnitude. Bps-UPT-LF strip demonstrated high specificity with the absence of any false-positive result even at 10(7) and 10(8) CFU/ml of non-specific bacterial contamination. Not only Bps-UPT-LF strip could tolerate to high concentration of the extreme acid and basic matter (pH 1-12), saline matter (≤ 2 mol/L mixture of NaCl and KCl), viscous materials (≤ 50 g/L of PEG 20000 and ≤ 20% of glycerol) and bio-macromolecule (≥ 400 g/L of bovine serum albumin or ≥ 80 g/L of casein), but also it can directly detect animal, environmental and powder specimen, such as ≥ 400 g/L of milk powder, flour powder, fruit juice, fresh and decomposed viscera, and ≤ 200 g/L of putty powder, sucrose, gourmet powder, and soil. Operation errors of liquid measure had few effects on sensitivity and specificity, including -50%-200% of sample, -22%-44% of sample-treating buffer and -30%-30% of loading mixture. CONCLUSION: The good detection performance and tolerance performance bring the bright future for Bps-UPT-LF strip to detect Burkholderia pseudomallei on site rapidly and quantitatively for nature foci surveillance and anti-bioterrorism.

Decreased expression of SCUBE2 is associated with progression and prognosis in colorectal cancer.

Signal peptide-CUB-epidermal growth factor-like domain-containing protein 2 (SCUBE2), a member of the SCUBE protein family, is a secreted and membrane-associated multi-domain glycoprotein. SCUBE2 is known as a novel tumor suppressor and a useful prognostic marker in breast cancer. In the present study, we investigated the expression (including mRNA and protein levels) of SCUBE2 in colorectal cancer (CRC) and adjacent normal tissues, using quantitative real-time PCR, western blot analysis and immunohistochemistry on a tissue microarray. Upregulation of SCUBE2 was achieved by transient transfection in RKO cell lines, and the effects of SCUBE2 on tumor proliferation, invasion, migration and apoptosis were evaluated by a series of functional experiments. The results indicated that SCUBE2 expression was decreased at the transcriptional and translational levels in CRC tissues and significantly associated with clinical stage, the depth of tumor invasion, lymph­node metastasis, distant metastasis and histological grade. Patients with SCUBE2­positive tumors had a lower recurrence rate and better survival than patients with SCUBE2-negative tumors. Moreover, upregulation of SCUBE2 had a limited effect on cell apoptosis but significantly inhibited tumor cell proliferation, migration and invasion in vitro. In conclusion, SCUBE2 plays an important role in suppressing CRC progression and prognosis. Our findings suggested that SCUBE2 may serve as a novel tumor suppressor and a potential therapeutic target for CRC patients.

Characteristics of airborne Staphylococcus aureus (including MRSA) in Chinese public buildings.

The aim of this study was to evaluate the concentration and size distribution of airborne culturable Staphylococcus aureus (S. aureus) (including MRSA) in Chinese public buildings. Air samples were collected, using six-stage Andersen sampler from five different public buildings in one large Chinese community. The mean indoor concentrations of the total and respirable airborne S. aureus were 72 and 50 CFU/m3 in the general hospital, 72 and 49 CFU/m3 in the kindergarten, 76 and 52 CFU/m3 in the hotel, 84 and 57 CFU/m3 in the movie theater, and 55 and 40 CFU/m3 in the university classroom. Respirable S. aureus amounted to approximately 57-73 % of the total S. aureus concentrations. Mean total and respirable concentrations of airborne MRSA were 32 and 20 CFU/m3 in the general hospital, 20 and 13 CFU/m3 in the kindergarten, 23 and 16 CFU/m3 in the hotel, 33 and 20 CFU/m3 in the movie theater, and 24 and 17 CFU/m3 in the university classroom. Respirable MRSA amounted to approximately 61-72 % of the total MRSA concentrations. The ratios of indoor and outdoor concentration for airborne S. aureus and MRSA were more than 1.0 in all the investigated public buildings. The size distribution results showed relatively high collection rates on stage 4 (2.1-3.3 µm) for both airborne culturable S. aureus and MRSA regardless of the type of public buildings.

Global and local persistence of influenza A(H5N1) virus.

An understanding of the global migration dynamics of highly pathogenic avian influenza A(H5N1) virus is helpful for surveillance and disease prevention. To characterize the migration network of this virus, we used genetic analysis, which supported a global persistence model in which each of 9 regions acts to some extent as a source. Siberia is the major hub for the dispersal of the virus. Southeast Asia and Africa are major sources of genetically and antigenically novel strains. We found evidence of local persistence of the virus in Southeast Asia and Africa, which is rare for human influenza A viruses. The differences in migration dynamics between avian and human influenza viruses might help with the design of region-specific surveillance efforts and the selection of vaccine candidates.

Immunogenicity of oral vaccination with Lactococcus lactis derived vaccine candidate antigen (UreB) of Helicobacter pylori fused with the human interleukin 2 as adjuvant.

Helicobacter pylori (H. pylori) infection remains a significant global public health problem. Vaccine, especially edible vaccine, is considered to be effective in the management of H. pylori infections. By using recombinant technology, Lactococcus lactis (L. lactis) could serve as an antigen-delivering vehicle for the development of edible vaccine. The aim of this study was to produce edible UreB (urease B) vaccine derived from L. lactis against H. pylori. The UreB subunit is the most effective and common immunogen of all strains of H. pylori. The UreB was produced as a chimeric protein fused with IL-2 (human interleukin 2) as the mucosal adjuvant. Mucosal immunization of mice with recombinant L. lactis NZ9000 containing the UreB-IL-2 protein elicited more anti-UreB antibody that specifically bounded to the purified bacterial UreB protein and more cytokines such as IFN-γ, IL-4, and IL-17, and had a lower H. pylori burden and urease activity than control mice. These results suggest that the recombinant L. lactis expressing UreB-IL-2 can be potentially used as an edible vaccine for controlling H. pylori infection.

Anti-proliferation effects of interferon-gamma on gastric cancer cells.

IFN-γ plays an indirect anti-cancer role through the immune system but may have direct negative effects on cancer cells. It regulates the viability of gastric cancer cells, so we examined whether it affects their proliferation and how that might be brought about. We exposed AGS, HGC-27 and GES-1 gastric cancer cell lines to IFN-γ and found significantly reduced colony formation ability. Flow cytometry revealed no effect of IFN-γ on apoptosis of cell lines and no effect on cell aging as assessed by ß-gal staining. Microarray assay revealed that IFN-γ changed the mRNA expression of genes related to the cell cycle and cell proliferation and migration, as well as chemokines and chemokine receptors, and immunity-related genes. Finally, flow cytometry revealed that IFN-γ arrested the cells in the G1/S phase. IFN-γ may slow proliferation of some gastric cancer cells by affecting the cell cycle to play a negative role in the development of gastric cancer.

[Characterization of human rhinovirus in children with acute respiratory infections in Gansu Province during 2011].

To study the epidemic characteristics of human rhinovirus (HRV) in children with acute respiratory infections in Gansu Province. 286 throat swabs were collected from children with acute respiratory in fections in Gansu Province during 2011. Multiplex reverse transcription-PCR (multiplex RT-PCR) assay was used to screen those specimens for detection of common respiratory tract pathogens. For HRV-positive samples, nested reverse transcription polymerase chain reaction (nested RT-PCR) was performed to amplify VP1 and VP4/VP2 gene fragments of HRV. The VP4/VP2 and VP1 regions of HRV-positive samples were sequenced and performed genotype analysis. Of 286 specimens fested, 27 were positive for HRV by multiplex RT-PCR and nested RT-PCR, of which 16 children were made (16/185), 8.64%) and 11 female (11/101,10.89%). The positive rate was 9.44% (27/286). The mean age of HRV-positive children was 3 years in this study, children less than one year old had the highest proportion 44.4% (12/ 27, 44.4%). The highest HRV positive rate fell on May, 2011 (6/27, 22.2%). Common cold accounted for the highest proportion, 12.24% (12/98) followed by pneumonia, 8.50% (13/153). The remaining 2 cases were bronchitis. Sequence analysis showed HRV A was the predominant genotype in Gansu Province in 2011, accounting for 84.62% (22/26) of positive cases, followed by HRV C (11.54%, 3/26) and only one HRV B was detected (3.85%, 1/26). HRV could be detected throughout the year in Gansu Province and primarily infected children under one year old. The group A was the epidemic genotype of HRV and move than one genotype existed in Gansu Province during 2011.

Evaluation of fetal skeletal malformations in deoxynivalenol-treated mice using microarray analysis.

Deoxynivalenol (DON [vomitoxin]), one of trichothecene mycotoxins produced by the fungus Fusarium, is commonly detected in cereal foods across the world. DON induces diverse toxic effects in humans and animals, including emesis and diarrhea, anorexia, and immunotoxicity, and impaired maternal reproduction and fetal development. Recently, the teratogenic potential of DON has been shown and has received much attention. DON can cause various skeletal deformities in fetuses, but the underlying mechanisms have not yet been fully examined. In this study, fetal skeletal malformations in DON-treated maternal mice were thoroughly investigated using microarray assay. The results showed that DON administration caused various skeletal defects in fetuses, including misaligned or fused sternebrae and vertebrae, divided or fused ribs and polydactyly, hemivertebrae, short toes, and tail anomalies. Microarray analysis showed that 282 genes, including 148 downregulated and 134 upregulated genes, were abnormally expressed in fetal vertebral bones after maternal DON exposure. These identified genes can be classified into several categories: skeletal development, carcinogenesis, nervous disorders, sperm development and embryogenesis, and inflammation. Of these, 6 genes, mostly related to bone development, were intentionally selected for further validation using real-time reverse transcription-Polymerase Chain Reaction (RT-PCR). It was confirmed that the mRNA expression of 4 genes, i.e., fibrillin-1, Col9A2, 3'-phosphoadenosine 5'-phosphosulfate synthase 2, and Pax1, was upregulated significantly by DON administration, whereas that of 2 other genes, Runx2 and parathyroid hormone-like hormone, was downregulated significantly. Taken together, the results of our study suggest that altered expression of these 6 genes plays a critical role in fetal skeletal deformities induced by DON and thus they are worthy of further investigation.

[Construction of a recombinant eukaryotic expression vector containing HSV 2gD and its immune responses].

AIM: To construct and express pcDNA3.1-IL-18-HSV P6 recombinant DNA vaccine, and to observe the immune responses of pcDNA3.1-IL-18-HSV P6 genetic vaccine. METHODS: Genes of P6 and IL-18 were cloned into the eukaryotic expression vector pcDNA3.1(-) respectively, and confirmed by enzyme digestion and sequence analysis, the recombinant plasmid pcDNA3.1-IL-18-HSVP6 was transformed into CHO cells. The expressed protein was characterized by indirect immunofluorescence and Western blotting. The recombinant plasmid pcDNA3.1-IL-18-HSVP6 was used to inoculate BALB/c mice by intramuscular injection for three times, once a week. One week after the last vaccination, the levels of specific IgG antibody, IFN-γ and IL-18 were detected by ELISA; One month after the last vaccination, spleen cells of vaccinated mice were separated and proliferation of spleen lymphocytes were detected by MTT assay. RESULTS: Recombinant pcDNA3.1-IL-18-HSV P6 plasmid was confirmed by enzyme digestion and DNA sequencing. After inoculated by pcDNA3.1-IL-18-HSV P6 vaccine, the mice could produce higher level of splenocytes proliferation and secrected higher level of IFN-γ, IL-18 and specific antibody. CONCLUSION: DNA vaccine of pcDNA3.1-IL-18-HSV P6 has been successfully constructed, and it can effectively induce humoral and cellular immune responses, which provided a basis for constructing new type of DNA vaccine.

Identification of a lead small-molecule inhibitor of anthrax lethal toxin by using fluorescence-based high-throughput screening.

Inhalational anthrax is caused by B. anthracis, a virulent sporeforming bacterium which secretes anthrax toxins consisting of protective antigen (PA), lethal factor (LF) and edema factor (EF). LF is a Zn-dependent metalloprotease and is the main determinant in the pathogenesis of anthrax. Here we report the identification of a lead small-molecule inhibitor of anthrax lethal factor by screening an available synthetic small-molecule inhibitor library using fluorescence-based high-throughput screening (HTS) approach. Seven small molecules were found to have inhibitory effect against LF activity, among which SM157 had the highest inhibitory activity. All theses small molecule inhibitors inhibited LF in a noncompetitive inhibition mode. SM157 and SM167 are from the same family, both having an identical group complex, which is predicted to insert into S1' pocket of LF. More potent small-molecule inhibitors could be developed by modifying SM157 based on this identical group complex.

Virulence factor cytotoxin-associated gene A in Helicobacter pylori is downregulated by interferon-γ in vitro.

Helicobacter pylori-infected gastric mucosa is characterized by high levels of interferon-γ (IFN-γ), but whether the high level of IFN-γ regulates the virulence of H. pylori is unclear. Here, we characterized the response of H. pylori to IFN-γ and found by indirect immunofluorescence that IFN-γ can bind to H. pylori. The binding resulted in the altered expression of 14 proteins, including the virulence factor, cytotoxin-associated gene A (CagA), whose expression was downregulated. The transcription and translation of CagA downregulated by IFN-γ was further confirmed by reverse transcriptase-PCR and Western blot analysis. We co-cultured the human gastric cancer cell line AGS with H. pylori exposed to IFN-γ; both phosphorylated CagA and nonphosphorylated CagA in AGS cells were downregulated by IFN-γ, and the proportion of cells with the 'hummingbird' phenotype was also decreased. Thus, IFN-γ can help control H. pylori infection indirectly through the virulence factor CagA.

[Qingli shengjing pills decrease apoptosis of germ cells and expressions of fas and fasL in male mice infected with Escherichia coli].

OBJECTIVE: To study the effects of Qingli Shengjing Pills (QSP) on the apoptosis of germ cells and expressions of Fas and FasL in male mice infected with Escherichia coli (E. coli), and to clarify the molecular mechanism of QSP in the treatment of male infertility induced by E. coli infection. METHODS: Fifty male mice were injected with E. coli via the bladder to make infection models, and at 15 dpi equally randomized into five groups: an untreated, a high-dose QSP (22.5 g/ml), a medium-dose QSP (13.5 g/ml), a low-dose QSP (4.50 g/ml) and a Furadantin treatment group, which were coded as MN, MTa, MTb, MTc and MTd, respectively. Another 10 mice were injected with saline and included in the control group coded as CT. After 10 days of oral medication, the apoptosis of germ cells in the testis of the mice was detected by flow cytometry, the expressions of Fas and FasL determined by immunohistochemistry and the histopathological changes observed simultaneously. RESULTS: After the treatment, the apoptosis of germ cells was observed in all the infected groups, and the apoptosis level in MN (57.44%) was significantly higher than that in CT (28.54%), MTb (28.59%) or MTa (30.11%) (P < 0.01) but had no significant difference from that in MTc (46.54%) or MTd (43.41%) (P > 0.05). The expressions of Fas and FasL proteins were significantly higher in MN than in CT, MTa, MTb, MTc and MTd (P < 0.01). Histopathological changes of the testis tissue were observed in MN, but not in other groups. CONCLUSION: E. coli infection could increase the apoptosis rate of germ cells and the expressions of Fas and FasL proteins. Qingli Shengjing Pills, capable of enhancing reproductivity by reducing the expressions of Fas and FasL and apoptosis of germ cells, can be used as one of the effective drugs for infertility induced by E. coli infection.

Degenerate primers based RT-PCR for rapid detection and differentiation of airborne chicken Newcastle disease virus in chicken houses.

Airborne Newcastle disease (ND) viruses in the air of five chicken houses were detected and differentiated by reverse transcriptase polymerase chain reaction (RT-PCR) using degenerate primers. Fifteen air samples were collected with All Glass Impinger-30 (AGI-30) air samplers in each house. Airborne ND viruses were also isolated and virulence identified by in vivo tests. Avirulent viruses were detected both in air samples and swab samples in four houses by degenerate primers based RT-PCR. Virulent viruses were detected only in the air samples by degenerate primers based RT-PCR in two houses. Seven strains viruses were isolated from the RT-PCR positive air samples. Of the seven strains, three strains were virulent viruses and four strains were avirulent viruses identified by in vivo tests. The results showed that it was feasible to detect and differentiate NDV in the air samples using degenerate primers based RT-PCR. This technique could decrease the time it required identify NDV infected flocks while distinguishing between virulent and avirulent viruses. It will help effectively to control Newcastle disease.

[Clinical observation on effect of combination of zhuanyindan and hormone in treating male infertility with positive antisperm antibody].

OBJECTIVE: To explore the therapeutic effect of combination of Zhuanyindan (ZYD, a self-made Chinese herbal preparation) and hormone in treating male infertility with positive antisperm antibody and its influence on nitric oxide (NO) level. METHODS: Eighty-two patients were randomly divided (according to the digital list) into the WM group (n = 20, treated with prednisone), the TCM group (n = 28, treated with ZYD) and the ICWM group (n = 34, treated with prednisone plus ZYD). The clinical effect, negative converting rate of antisperm antibody, changes of NO level in semen and various parameters of sperm motion before and after treatment were observed. RESULTS: The total effective rate in the ICWM group was 88.2%, that in the TCM group 75.0% and in the WM group 65.0%. Significant difference was seen in the ICWM and TCM group before and after treatment in NO level, sperm motion parameters, including linear motion speed, linearity, propulsion, whip frequency, sperm vitality and mean moving angle, and quality of semen (P < 0.05 or P < 0.01). In the WM group, significant difference in comparison before and after treatment was seen in NO level, propulsion, whip frequency, mean moving angle and quality of semen, including vitality and survival rate (P < 0.01). CONCLUSION: Combination of Chinese herbs and hormone could lower the NO level in semen and improve the quality of sperm.