FASN Inhibition Decreases MHC-I Degradation and Synergizes with PD-L1 Checkpoint Blockade in Hepatocellular Carcinoma.

Immune checkpoint inhibitors (ICI) transformed the treatment landscape of hepatocellular carcinoma (HCC). Unfortunately, patients with attenuated MHC-I expression remain refractory to ICIs, and druggable targets for upregulating MHC-I are limited. Here, we found that genetic or pharmacologic inhibition of fatty acid synthase (FASN) increased MHC-I levels in HCC cells, promoting antigen presentation and stimulating antigen-specific CD8+ T-cell cytotoxicity. Mechanistically, FASN inhibition reduced palmitoylation of MHC-I that led to its lysosomal degradation. The palmitoyltransferase DHHC3 directly bound MHC-I and negatively regulated MHC-I protein levels. In an orthotopic HCC mouse model, Fasn deficiency enhanced MHC-I levels and promoted cancer cell killing by tumor-infiltrating CD8+ T cells. Moreover, the combination of two different FASN inhibitors, orlistat and TVB-2640, with anti-PD-L1 antibody robustly suppressed tumor growth in vivo. Multiplex IHC of human HCC samples and bioinformatic analysis of The Cancer Genome Atlas data further illustrated that lower expression of FASN was correlated with a higher percentage of cytotoxic CD8+ T cells. The identification of FASN as a negative regulator of MHC-I provides the rationale for combining FASN inhibitors and immunotherapy for treating HCC. SIGNIFICANCE: Inhibition of FASN increases MHC-I protein levels by suppressing its palmitoylation and lysosomal degradation, which stimulates immune activity against hepatocellular carcinoma and enhances the efficacy of immune checkpoint inhibition.

Classification and molecular characteristics of tet(X)-carrying plasmids in Acinetobacter species.

The rapid dissemination of plasmid-mediated tet(X) genes in Acinetobacter species has compromised the clinical effectiveness of tigecycline, one of the last-resort antibiotics. However, the classification strategy and homology group of tet(X)-positive Acinetobacter spp. plasmids remain largely unknown. In this study, we classified them by genome-based replicon typing, followed by analyses of structural characteristics, transferability and in vivo effect. A total of 34 plasmids distributed in at least nine Acinetobacter species were collected, including three tet(X3)-positive plasmids and one tet(X6)-positive plasmid from our genome sequencing results. Among them, there were 28 plasmids carrying Rep_3 superfamily replicase genes and classified into six homology groups, consisting of GR31 (82.1%), GR26 (3.6%), GR41 (3.6%), GR59 (3.6%), and novel groups GR60 (3.6%) and GR61 (3.6%). Our tet(X3)-positive plasmids pYH16040-1, pYH16056-1, and pYH12068-1 belonged to the dominant GR31 group, whereas the tet(X6)-positive plasmid pYH12068-2 was unclassified. Structurally, all tet(X)-positive GR31 plasmids shared similar plasmid replication (repB), stability (parA and parB) and accessory modules [tet(X) and sul2], and 97.6% of plasmid-mediated tet(X) genes in Acinetobacter species were adjacent to ISCR2. Conjugation and susceptibility testing revealed pYH16040-1, pYH16056-1, and pYH12068-2, carrying plasmid transfer modules, were able to mediate the mobilization of multiple antibiotic resistance. Under the treatment of tigecycline, the mortality rate of Galleria mellonella infected by pYH16040-1-mediated tet(X3)-positive Acinetobacter spp. isolate significantly increased when compared with its plasmid-cured strain (p < 0.0001). The spread of such plasmids is of great clinical concern, more effects are needed and will facilitate the future analysis of tet(X)-positive Acinetobacter spp. plasmids.

Expression of the Campylobacter jejuni FliD protein and its reaction to chicken sera.

Campylobacter is a leading causative pathogen of acute bacterial gastroenteritis among humans. Contaminated chicken products are regarded as major sources of human infection. The flagellar capping protein (FliD), which plays important roles in colonization and adhesion to the mucosal surface of chicken ceca, is conserved among Campylobacter jejuni strains. In this study, the recombinant C. jejuni FliD protein was expressed, purified and used as a coated protein to examine the prevalence of C. jejuni antibodies in chickens. The anti-FliD antibody was prevalent among chicken serum samples taken from different farms in the diverse regions of Jiangsu province by using enzyme-linked immunosorbent assay. The Campylobacter antibody was present in culture-negative chickens. No strong dose-response relationships were observed between serum FliD antibody levels and Campylobacter cultural status. These results provide a basis for further evaluating FliD as a vaccine candidate for broiler chickens or for examining host-C. jejuni interactions, with implications for improving food safety.

Responses to Crizotinib therapy in five patients with non-small-cell lung cancer who tested FISH negative and Ventana immunohistochemistry positive for ALK fusions.

AIM: Although immunohistochemistry (IHC) and reverse transcription-PCR can detect ALK rearrangements, the ALK break-apart FISH assay is currently considered the standard method. MATERIALS & METHODS: Five patients with advanced non-small-cell lung cancer, who had an ALK-negative FISH result that was later confirmed as positive by the Ventana IHC assay, were studied. Four had previously received chemotherapy or radiotherapy. All five were subsequently treated with Crizoitinib 250 mg twice daily. RESULTS & CONCLUSION: Four patients had a partial response to Crizotinib and one had stable disease. IHC is an efficient technique for diagnosing ALK rearrangements in patients with non-small-cell lung cancer, and may serve as an alternative to FISH in clinical practice.

BTB/POZ domain-containing protein 7: epithelial-mesenchymal transition promoter and prognostic biomarker of hepatocellular carcinoma.

UNLABELLED: Epithelial-mesenchymal transition (EMT) is a critical step in the metastasis of hepatocellular carcinoma (HCC). BTB/POZ domain-containing protein 7 (BTBD7) regulates EMT-associated proteins implicated in HCC progression. However, the role(s) of BTBD7 in HCC have not been identified. Using highly metastatic HCC HCCLM3 cells, immortalized L02 hepatocytes, metastatic HCC animal models, and three independent cohorts of HCC patient specimens, we aimed to determine the involvement of BTBD7 in HCC metastasis. We show that BTBD7 messenger RNA and protein was highly expressed in HCC cells and tumor tissues, with such expression being associated with: enhanced cell motility, venous invasion, and poor prognosis. BTBD7 promoted HCC angiogenesis and metastasis in vitro and in vivo, but did not influence cell proliferation or colony formation. BTBD7 enhancement of HCC invasion and EMT phenotype occurred through activation of a RhoC-Rock2-FAK-signaling pathway, resulting in matrix metalloproteinase-2/9 production and microvessel formation. Applying a predictive risk score model, Cox regression analysis revealed that high BTBD7 expression integrated with high microvessel density was a powerful independent predictive factor of HCC clinical outcome. CONCLUSION: The present study identifies BTBD7 as a novel candidate prognostic factor and a potential therapeutic target of HCC. (HEPATOLOGY 2013; 57:2326-2337).

[Preparation and characterization of monoclonal antibodies specific for CjaA protein of Campylobacter jejuni].

AIM: Expression, purification of Campylobacter jejuni CjaA protein and development of monoclonal antibodies (mAbs) against this protein. METHODS: The C. jejuni cjaA gene was amplified and inserted into the expression plasmids, pGEX-6p-1 and pET30a (+). The purified rGST-CjaA protein was used as an immunogen in 8-week-old BALB/c mice, and injected subcutaneously. The purified rHis-CjaA protein used as a detecting antigen for screening mAbs against CjaA was prepared. The specificity of mAbs was characterized by Dot-ELISA and Western blot assays. RESULTS: The recombinant expression plasmids, pGEX-6p-1-cjaA and pET30a(+)-cjaA were obtained. The sizes of the recombinant proteins, rGST-CjaA and rHis-CjaA, were consistent with their predicted size. Specific reaction was found between CjaA positive serum and expressed protein by Western blot assay, confirming its identification as a Campylobacter jejuni immunogen. Three hybridoma cell lines, designated 2B6, 3C2 and 4F11, secreting mAbs against CjaA were obtained. Their immunoglobulin subclasses were all IgG1. The ELISA titers of the ascites fluid were 1:1×10(5);, 1:2×10(5); and 1:4×10(5);, respectively. Western blot analysis confirmed that the three mAbs reacted with the rHis-CjaA fusion protein but not the His tag. The Dot-ELISA results demonstrated that the three mAbs only with CjaA and not the tags for the expression vectors. CONCLUSION: The successful preparation of three mAbs specific for the CjaA protein lays the foundation for further study regarding the biological characteristics of CjaA and the pathogenesis of C. jejuni.

[Prokaryotic expression of recombinant bovine IL-4 and development of monoclonal antibodies against bovine IL-4].

AIM: To express recombinant bovine IL-4 (rBoIL-4) in Escherichia coli and prepare monoclonal antibody (mAb) against rBoIL-4. METHODS: The IL-4 gene without coding signal peptides was amplified from pSP73-BoIL-4 by PCR, then inserted into prokaryotic expression vector pGEX-6p-1 and pET-30a(+). The recombinant plasmids pGEX-6p-1-BoIL-4 and pET-BoIL-4 were transformed into DH5α for sequencing. After sequencing confirmation, the two recombinant plasmids were transformed into expression bacteria BL21 and BL21(DE3) respectively. BALB/c mice were immunized with the purified protein rHis-BoIL-4. With the purified rGST-BoIL-4 as detecting antigen, mAb-produced hybridoma cells against BoIL-4 were screened by indirect ELISA. The specificity of the mAbs was characterized by indirect ELlSA, Dot-ELlSA and Western blot. RESULTS: The recombinant bacteria BL21(pGEX-6p-1-BoIL-4) and BL21(DE3)(pET-BoIL-4) were developed. After induced by IPTG, SDS-PAGE analysis showed that the expression products of rGST-BoIL-4 and rHis-BoIL-4 had a molecular weight of 39 kD and 19 kD respectively, and expressed in inclusion body form. Seven hybridoma cell lines secreting mAbs against BoIL-4, named 2B8, 4A10, 5D6, 5D8, 7G10, 8B7 and 10F8 were obtained. The immunoglobulin subclasses were IgG1. The ascitic titers of these mAbs were 5 000, 16 0000, 10 000, 640 000, 5 000, 40 000 and 40 000, respectively. In Dot-ELISA, all mAbs could only react to the immunogen and the detecting antigen. Western-blot analysis confirmed that mAbs could only react to the corresponding recombinant proteins. The mAbs also reacted to the standard recombinant boIL-4 with biological activity. CONCLUSION: Seven mAbs specific to rBoIL-4 protein are obtained, which may have important application value in further study on diagnosis and pathogenesis of cattle diseases.

[The adjuvant effect of chimeric flagellin fliC/esat].

AIM: To determine the role of ESAT-6 chimeric flagellin in TB immunology. METHODS: The coding sequences of flagellin of Salmonella typhimurium and ESAT-6 of Mycobacterium tuberculosis were cloned by PCR and identified by sequencing, respectively. Chimeric flagellin gene fliC/esat was constructed by overlap PCR technique. The ESAT-6 coding fragment was inserted to the hypervariable region of Salmonella flagellin gene fliCi. And then prokaryotic exprssion plasmids of pET-fliC/esat, pET-fliC and pBCX-esat were constructed and transformed into E.coli BL21(DE3), followed by induction of IPTG. The expressed proteins fliC/esat and ESAT-6 were identified by Western-blot assay using specific monoclonal antibody (mAb) HYB076-08. Bone marrow dendritic cells (BMDCs) were in vitro stimulated by fliC/esat and ESAT-6 proteins, and analyzed for the expression levels of CD40, CD80, CD86 and CD54 molecules. The secreted IL-12p70 was determined by ELISA. Moreover, C57BL/6 mice were immunized intravenously with fliC/esat or ESAT-6 protein. The specific IFN-γ-secreting cells and IL-4-secreting cells from the immunized mice were detected by ELISPOT assay using an ESAT-6 peptide as a stimulus. RESULTS: The results showed that the proteins of fliC/esat and ESAT-6 were expressed solubly, with the sizes of 64 kD and 39 kD respectively. Western blot analysis showed that both proteins reacted with the specific mAb against ESAT-6. BMDCs maturation was triggered by the chimeric flagellin fliC/esat. In contrast, ESAT-6 protein alone didn't activate BMDCs. IL-12p70 was also detected in the supernatants of BMDCs. The results showed that the chimeric flagellin fliC/esat induced significantly higher level of the secreted IL-12p70 than that of ESAT-6 protein. Furthermore, the chimeric flagellin fliC/esat significantly enhanced the Th1-biased immune responses against ESAT-6 in the immunized C57BL/6 mice. CONCLUSION: The chimeric flagellin we generated exerts Th1 type adjuvant activity for ESAT-6 protein.

Intranasal immunization with chitosan/pCAGGS-flaA nanoparticles inhibits Campylobacter jejuni in a White Leghorn model.

Campylobacter jejuni is the most common zoonotic bacterium associated with human diarrhea, and chickens are considered to be one of the most important sources for human infection, with no effective prophylactic treatment available. We describe here a prophylactic strategy using chitosan-DNA intranasal immunization to induce specific immune responses. The chitosan used for intranasal administration is a natural mucus absorption enhancer, which results in transgenic DNA expression in chicken nasopharynx. Chickens immunized with chitosan-DNA nanoparticles, which carried a gene for the major structural protein FlaA, produced significantly increased levels of serum anti-Campylobacter jejuni IgG and intestinal mucosal antibody (IgA), compared to those treated with chitosan-DNA (pCAGGS). Chitosan-pCAGGS-flaA intranasal immunization induced reductions of bacterial expellation by 2-3 log(10) and 2 log(10) in large intestine and cecum of chickens, respectively, when administered with the isolated C. jejuni strain. This study demonstrated that intranasal delivery of chitosan-DNA vaccine successfully induced effective immune response and might be a promising vaccine candidate against C. jejuni infection.

A reusable capacitive immunosensor for detection of Salmonella spp. based on grafted ethylene diamine and self-assembled gold nanoparticle monolayers.

Fabrication of a novel capacitive immunosensor based on grafted ethylene diamine and self-assembled gold nanoparticle monolayer on glassy carbon electrode for the detection of Salmonella spp. is described for the first time. In the present study, the Salmonella spp. monoclonal antibodies (denoted as McAbs) was immobilized on gold nanoparticles. Interaction of McAbs and Salmonella spp. was detected directly using the electrochemical impedance spectroscopy (EIS) technique. The experimental results showed that the concentration of antigen was measured through the relative change in capacitance in the corresponding specific binding of Salmonella spp. and McAbs. Under the optimized conditions, the relative changes in capacitance were proportional to the logarithmic values of Salmonella spp. concentrations in the range of 1.0 x 10(2) to 1.0 x 10(5) CFU mL(-1) (r = 0.991) with the detection limit of 1.0 x 10(2) CFU mL(-1). The stability of proposed immunosensor could be estimated by determining the relative change in capacitance, which remained almost the same in two months and decreased gradually to 85.3% of initial value after four months' storage. The used immunosensor could be regenerated repeatedly by immersing in glycine-HCl buffer solution (pH 2.8). Finally, the proposed immunosensor was successfully used for the detection of Salmonella spp. in lab-processed commercial pork samples.

Predictive model of Vibrio parahaemolyticus growth and survival on salmon meat as a function of temperature.

The growth and survival curves of a strain of pandemic Vibrio parahaemolyticus TGqx01 (serotype O3:K6) on salmon meat at different storage temperatures (range from 0 degrees C to 35 degrees C) were determined. In order to model the growth or inactivation kinetics of this pathogen during storage, the modified Gompertz and Weibull equations were chosen to regress growth and survival curves, respectively, and both equations produced good fit to the observed data (the average R2 value equals to 0.990 for modified Gompertz and 0.920 for Weibull equation). The effect of storage temperature on the specific growth rate (mu) was modeled by square root type equation, and the relationship between mu and lag time (lambda) was described by a rule of mu x lambda = constant. The shape factor (n) and scale factor (b) values of the Weibull equations versus the temperature (degrees C) were plotted and the temperature effects on these parameters were described by two linear empirical equations. The predicted growth and survival curves from the model were compared to real enumeration results, using the correlation coefficient (R2), bias factor (Bf) and accuracy factor (Af), to assess the performance of the established model. The results showed that the overall predictions for V. parahaemolyticus TGqx01 growth or inactivation on salmon at tested temperatures agreed well with observed plate counts, and the average R2, Bf and Af values were 0.958, 1.019 and 1.035, respectively.

[Perparation and characterization of the monoclonal antibodies against listeriolysin O].

AIM: To prepare the monoclonal antibodies (mAbs) against listeriolysin O (LLO), which is the major virulence factor of Listeria monocytogenes. METHODS: The BALB/c mice were immunized with the SDS-PAGE product of BL21(pGEX-6p-1-hly). The purified LLO-GST protein was used as antigen for detection. mAbs against LLO were prepared by using the lymphocyte hybridoma technique. The specificity of mAbs was characterized by Dot-ELISA and Western blot. RESULTS: Three hybridoma cell lines named 3B6, 4D1 and 5D10 secreting mAbs against LLO were obtained. The immunoglobulin subclasses of the mAbs were IgG1. The ELISA titer of the ascitic fluids of 3B6, 4D1 and 5D10 was 1:200,000, 1:200,000 and 1:100,000, respectively. Western blot analysis confirmed the three mAbs reacted on fusion protein LLO-GST but didn't react on protein GST. Dot-ELISA proved the three mAbs only react on the bacteria expressing LLO. CONCLUSION: The successful preparation of three mAbs specific to protein LLO lays a foundation for further study of the biological characteristics of LLO and the pathogenesis of Listeria monocytogenes.

[Construction and immunogenicity of attenuated Salmonella typhimurium stably harbouring DNA vaccine against Newcastle disease virus].

The fusion protein (F) gene of Newcastle disease virus was amplified by polymerase chain reaction (PCR) from the recombinant plasmid pVAX1-F, and subcloned into eukaryotic expression vector pmcDNA3. 1+. The F gene was identified by sequencing. The recombinant plasmid was transformed into attenuated Salmonella typhimurium SL7207, and the recombinant was designated as SL7207 (pmcDNA3. 1-F). In vitro and in vivo experiments showed that the plasmid stability of pmcDNA3. 1-F was apparently higher than that of pcDNA3. 1-F in SL7207. In order to compare the immune response induced by these two re combinant bacteria, BALB/c mice were immunized orally with them at the dosage of 2 x 10(9) CFU respectively. Both SL7207(pcDNA3. 1-F) and SL7207(pmcDNA3. 1-F) initiated F-specific serum and mucosal antibodies in immunized mice. Furthermore, 4-day-old SPF chickens were immunized with SL7207(pcDNA3. 1-F) and SL7207(pmcDNA3. 1-F) at the dosage of 5 x 10(9) CFU and boosted two weeks later with the same dosage. Humoral and intestinal mucosal immune responses were observed and their levels were significantly higher than that of negative and positive controls. The result of protective efficacy showed that the chickens immunized with SL7207(pmcDNA3. 1-F) had the protective rate of 70.0%, higher than that of the SL7207 (pcDNA3. 1-F) with 50.0%. In summary, the DNA vaccine delivered by attenuated Salmonella typhimurium has good immunogenicity. A novel mucosal DNA vaccine has been developed and could be useful for controlling the infection and epidemic of Newcastle disease in the poultry.

[Preparation and characterization of monoclonal antibodies against chicken interferon-gamma].

AIM: To prepare monoclonal antibodies (mAb) against chicken interferon-gamma (ChIFN-gamma). METHODS: By using lymphocyte hybridoma technique, the inclusion body of the recombined bacteria, BL21(DE3) (pET-ChIFN-gamma), was harvested and used to immunize BALB/c mice. With the purified GST-ChIFN-gamma as detecting antigen, mAbs against ChIFN-gamma were prepared, and positive hybridoma clones were screened by indirect ELISA. The specificity of the mAb was characterized by indirect ELISA, Dot-ELISA and Western blot. RESULTS: Two hybridoma cell lines secreting mAbs against ChIFN-gamma named 1G5, 5E3 were obtained. The immunoglobulin subclasses of both 2 mAbs were IgG2a, and the ELISA titers of 2 mAbs ascitic fluids were 1:160,000, 1:12,000 respectively. In Dot-ELISA test, the 2 mAbs could only react with BL21 (DE3) (pET-ChIFN-gamma), BL21 (pGEX-6P-1-ChIFN-gamma), which expressed His-ChIFN-gamma, GST-ChIFN-gamma, respectively. Western blot analysis confirmed that the 2 mAbs could only react with GST-ChIFN-gamma and His-ChIFN-gamma proteins. CONCLUSION: Two mAbs specific to the protein of chicken interferon gamma are obtained, which may have important application value in further studies on immune detection, the functions of immune cells and immune regulation.

[Safety and efficacy of attenuated Salmonella typhimurium harbouring DNA vaccine against Newcastle disease virus].

A pair of primers were designed and synthesized according to the previously published sequence of fusion protein (F) gene of Newcastle disease virus (NDV) and used to amplify F gene by reverse-transcription polymerase chain reaction (RT-PCR) from the genomic RNA of a NDV strain JS5 isolated from goose. The PCR product was identified by sequencing. Then recombinant eukaryotic expression vector pVAX1-F was constructed through inserting F gene into MCS of pVAX1. The recombinant plasmid pVAX1-F was transfected in COS-7 cells, and identified for the transient expression of F gene by indirect immunofluorescent assay. Finally, the recombinant plasmid was transformed into attenuated Salmonella typhimurium SL7207, and the recombinant was screened and designated as SL7207 (pVAX1-F). It was verified that SL7207 (pVAX1-F) as the oral NDV DNA vaccine was safe for chickens after oral immunization at dosage of 10(10) CFU or below. 1-day-old commercial ISA brown chickens were immunized orally with SL7207 (pVAX1-F) at two different dosages (10(9) CFU and 10(8) CFU) on day 1, 14 and 28. On day 7 after the last immunization, no significant difference was observed in the body weight between these two groups (p > 0.05), and also no significant difference between those two groups and negative control group (p > 0.05). Since there were maternal antibodies, high ELISA titers of serum antibodies against NDV were detected in the chickens of all groups on day 14. However, the levels of serum antibodies were decreased in the chickens of all groups on day 28, but the anti-NDV antibody response detected in the sera of chickens immunized with SL7207 (pVAX1-F) at the dosage of 10(9) CFU were increased and significantly higher than the response induced by immunization with SL7207 (pVAX1) on day 35 (p < 0.05). Intestinal mucosal immune response was observed in chickens immunized with SL7207 (pVAX1-F) at the dosage of 10(9) CFU or 10(8) CFU. The high ELISA titers of antibodies against NDV in small intestinal mucosal samples from immunized chickens were on day 28 and 35. After challenged intranasally with virulent NDV strain F48E8, the chickens immunized with SL7207 (pVAX1-F) at the dosage of 10(9) CFU could be protected with the protective rate of 77.27%, significantly higher than those with SL7207 (pVAX1) (p < 0.05). In summary, the DNA vaccine delivered by attenuated Salmonella typhimurium was safe and has good immunogenicity for chickens. A novel mucosal DNA vaccine was developed and could be useful for controlling the infection and epidemic of ND in the poultry.

[Preparation and characterization of monoclonal antibodies against the hemagglutinin of H9 subtype of avian influenza virus].

AIM: To prepare monoclonal antibodies (mAb) against the hemagglutinin(HA) of H9 subtype of avian influenza virus (AIV). METHODS: 8 week-old female BALB/c mice were immunized with the inactivated vaccine of H9 subtype of AIV. Splenocytes from the immunized mice were fused with Sp2/0 myeloma cells, and positive hybridoma clones were screened by indirect ELISA and hemagglutination inhibition test (HI). The specificity of the mAb was characterized by ELISA, HI test, indirect immunofluorescence (IF) staining and Western blot. RESULTS: Three hybridoma cell lines named 2H1, 2A3 and 1C8 against HA of AIV H9 were obtained. The HI titers of 3 mAbs were 1 x 2(8)-1 x 2(13), and the ELISA titers were 1 x 10(-7), 1 x 10(-5) and 5 x 10(-6), respectively. The immunoglobulin subclass of all 3 mAbs was IgG1. Western blot analysis confirmed that mAb 2H1 could recognize HA and reacted to 31 out of 32 isolates of H9 subtype of AIV. CONCLUSION: Three mAbs recognizing HA of H9 subtype of AIV were obtained, which may provide an useful tool for the antigenic analysis, the serological diagnosis, the epidemiological survey and the evaluation of AIV vaccine.

[Combination of direct-ELISA and PCR for the rapid detection and identification of Salmonella spp].

OBJECTIVE: To develop a protocol for the rapid detection of Salmonellae. METHODS: A mono-antibody-based direct-ELISA and PCR methods for the detection of Salmonella were developed previously. This study assessed the accuracy of both direct-ELISA and PCR methods for the rapid detection of Salmonella and set up a new detection protocol. RESULTS: The sensitivity of the PCR method was higher than that of direct-ELISA method. In the 2002 spring physical examination for employees, 1 546 human fecal samples were examined by the combination of direct-ELISA and PCR method. Compared with the results of national standard method, the sensitivity and specificity of direct ELISA was 100% and 97.14%, respectively, while those of PCR method reached both 100%. It also indicated that combination use of two methods could give positive report within 40 hrs, and also achieve high sensitivity and specificity. CONCLUSIONS: Based on the results obtained, a protocol for the rapid detection of Salmonella was developed. The first step is to us direct-ELISA method to screen the large number of samples, and then use PCR method to validate the ELISA positive samples, and the final step is, if needed, is to use the national standard method to determine the serotypes of Salmonellae.

Fundamental electronic properties and applications of single-walled carbon nanotubes.

Recent scanning tunneling microscopy studies of the intrinsic electronic properties of single-walled carbon nanotubes (SWNTs) are overviewed in this Account. A brief theoretical treatment of the electronic properties of SWNTs is developed, and then the effects of finite curvature and broken symmetry on electronic properties, the unique one-dimensional energy dispersion in nanotubes, the interaction between local spins and carriers in metallic nanotubes systems, and the atomic structure and electronic properties of intramolecular junctions are described. The implications of these studies for understanding fundamental one-dimensional physics and future nanotube device applications are also discussed.

Single-walled carbon nanotubes: from fundamental studies to new device concepts.

Single-walled carbon nanotubes (SWNTs) are ideal systems for investigating fundamental properties in one-dimensional electronic systems and have the potential to revolutionize many aspects of nano/molecular electronics. Scanning tunneling microscopy (STM) has been used to characterize the atomic structure and tunneling density of states of individual SWNTs. Detailed spectroscopic measurements showed one-dimensional singularities in the SWNT density of states for both metallic and semiconducting nanotubes. The results obtained were compared to and agree well with theoretical predictions and tight-binding calculations. SWNTs were also shortened using the STM to explore the role of finite size, which might be exploited for device applications. Segments less than 10 nm exhibited discrete peaks in their tunneling spectra, which correspond to quantized energy levels, and whose spacing scales inversely with length. Finally, the interaction between magnetic impurities and electrons confined to one dimension was studied by spatially resolving the local electronic density of states of small cobalt clusters on metallic SWNTs. Spectroscopic measurements performed on and near these clusters exhibited a narrow peak near the Fermi level that has been identified as a Kondo resonance. In addition, spectroscopic studies of ultrasmall magnetic nanostructures, consisting of small cobalt clusters on short nanotube pieces, exhibited features characteristic of the bulk Kondo resonance, but also new features due to their finite size.

Scanning tunneling microscopy studies of the one-dimensional electronic properties of single-walled carbon nanotubes.

Recent developments in scanning tunneling microscopy studies of the electronic properties of single-walled carbon nanotubes are reviewed. A broad range of topics focused on the unique electronic properties of nanotubes are discussed, including (a) the underlying theoretical description of the electronic properties of nanotubes; (b) the roles of finite curvature and broken symmetries in perturbing electronic properties; (c) the unique one-dimensional energy dispersion in nanotubes; (d) the nature of end states; (e) quantum size effects in short tubes; (f) the interactions between local spins and carriers in metallic systems (the Kondo effect); and (g) the atomic structure and electronic properties of intramolecular junctions. The implications of these studies for understanding fundamental one-dimensional physics and future nanotube device applications are discussed.