Jingfu Qiu

A novel electrochemical immunosensor for highly sensitive detection of aflatoxin B1 in corn using single-walled carbon nanotubes/chitosan

A sensitive electrochemical immunosensor for aflatoxin B1 (AFB1) detection based on single-walled carbon nanotubes/chitosan was presented. The immunosensor was based on an indirect competitive binding to a fixed amount of anti-AFB1 between free AFB1 and AFB1-bovine serum albumin, which conjugate immobilized on covalently functionalized nanotubes/chitosan laid on the glass carbon electrode. Then, the anti-mouse immunoglobulin G secondary antibody labeled with alkaline phosphatase was bound to the electrode surface through reacting with primary antibody. Finally, alkaline phosphatase catalyzes the hydrolysis of the substrate α-naphthyl phosphate, which produced electrochemical signal. Compared with conventional methods, the established immunosensor was more sensitive and simple. Under optimal conditions, this method could quantitatively detect AFB1 from 0.01 to 100 ng mL(-1) with a detection limit of 3.5 pg mL(-1). Moreover, the immunosensor was successfully applied to assay AFB1 in corn powder, which showed good correlation with the results obtained from high performance liquid chromatography.

AphA is required for biofilm formation, motility, and virulence in pandemic Vibrio parahaemolyticus

AphA is a small PadR-family DNA-binding regulator in vibrios. AphA has been shown to be involved in transcriptional auto-repression, intestinal colonization and lethality in mice, biofilm formation, and quorum sensing in Vibrio cholerae. The AphA protein of Vibrio parahaemolyticus has 85% identity to that of V. cholerae with the same number of amino acids. In this work, the aphA null mutant was constructed from a wild-type pandemic strain of V. parahaemolyticus for characterization of the phenotypic changes. AphA is required for biofilm formation in V. parahaemolyticus, and a decreased production of biofilm exopolysaccharide matrix in the aphA mutant relative to the wild-type parent strain accounts for its reduced biofilm formation. AphA is also necessary for the optimal swimming and swarming motility of V. parahaemolyticus. In addition, AphA is essential for lethality in mice and cytotoxic activity, but the aphA deletion did not have effect on enterotoxicity.

Molecular Characterization of Direct Target Genes and cis-Acting Consensus Recognized by Quorum-Sensing Regulator AphA in Vibrio parahaemolyticus

Background AphA is the master quorum-sensing (QS) regulator operating at low cell density in vibrios. Molecular regulation of target genes by AphA has been characterized in Vibrio harveyi and V. cholerae, but it is still poorly understood in V. parahaemolyticus. Methodology/Principal Findings The AphA proteins are extremely conserved in V. parahaemolyticus, Vibrio sp. Ex25, Vibrio sp. EJY3, V. harveyi, V. vulnificus, V. splendidus, V. anguillarum, V. cholerae, and V. furnissii. The above nine AphA orthologs appear to recognize conserved cis-acting DNA signals which can be represented by two consensus constructs, a 20 bp box sequence and a position frequency matrix. V. parahaemolyticus AphA represses the transcription of ahpA, qrr4, and opaR through direct AphA-target promoter DNA association, while it inhibits the qrr2-3 transcription in an indirect manner. Translation and transcription starts, core promoter elements for sigma factor recognition, Shine-Dalgarno sequences for ribosome recognition, and AphA-binding sites (containing corresponding AphA box-like sequences) were determined for the three direct AphA targets ahpA, qrr4, and opaR in V. parahaemolyticus. Conclusions/Significance AphA-mediated repression of ahpA, qrr2-4, and opaR was characterized in V. parahaemolyticus by using multiple biochemical and molecular experiments. The computational promoter analysis indicated the conserved mechanism of transcriptional regulation of QS regulator-encoding genes ahpA, qrr4, and opaR in vibrios.

Interferon-Inducible Transmembrane Protein 3 Genetic Variant rs12252 and Influenza Susceptibility and Severity: A Meta-Analysis

Background The pandemic influenza A (H1N1) pdm09 virus, avian influenza A (H5N1) virus, and influenza A (H7N9) virus induced severe morbidity and mortality throughout the world. Previous studies suggested a close association between the interferon-induced transmembrane protein-3 (IFITM3) genetic variant rs12252 and influenza. Here, we explored the correlation between the rs12252 and influenza susceptibility and severity using meta-analysis. Methods Relevant studies published before May 22, 2014 were retrieved from PubMed, ISI web of knowledge, EBSCO, and Cochrane central register of controlled trials databases. Association between rs12252 and influenza susceptibility and severity were determined using statistical analysis of odds ratios (ORs). Results A total of four studies consisting of 445 cases and 4180 controls were included in our analysis. Generally, there is increased risk of influenza in subjects carrying rs12252 in the recessive model (CC vs. CT+TT: OR = 2.35, 95% CI: 1.49-3.70, P<0.001), the dominant model (CC+CT vs. TT: OR=1.60, 95% CI: 1.18–2.22, P=0.003), the homozygote comparison (CC vs. TT: OR=4.11, 95% CI: 2.15–7.84, P<0.001), and the allele contrast (C vs. T: OR=1.67, 95% CI: 1.32–2.13, P<0.001). Stratification analysis of ethnicity and severity revealed a significant increase in influenza susceptibility by IFITM3-SNP rs12252 among both Asian and Caucasian population. SNP rs12252 shows significant impact on severe infections (P<0.05), but not on mild influenza. Besides, our result also associated rs12252 with influenza severity (severe vs. mild: OR=2.37, 95% CI: 1.32–4.25, P=0.004), (severe vs. control: OR=2.70, 95% CI: 1.85–3.94, P<0.001). Conclusion Our meta-analysis suggests a significant association between a minor IFITM3 allele (SNP rs12252-C) with severe influenza susceptibility, but not in mild influenza subjects, in both UK Caucasians and Han Chinese population. The rs12252-C allele causes a 23.7% higher chance of infection and also constitutes a risk factor for more severe influenza.

Electrochemical aptasensor for thrombin using co-catalysis of hemin/G-quadruplex DNAzyme and octahedral Cu2O-Au nanocomposites for signal amplification

In this work, novel octahedral Cu2O-Au nanocomposites were synthesized and first applied in an electrochemical aptasensor to detect thrombin (TB) with the aid of a DNAzyme for signal amplification. The octahedral Cu2O-Au nanocomposites have not only simultaneously served as signal amplifying molecules but have also been utilized as an ideal loading platform to immobilize a large number of electroactive substances and recognition probes. Gold nanoparticles (AuNPs) were grown directly on the surface of the octahedral Cu2O nanocrystals, and the Cu2O-Au nanocomposites obtained had the advantages of large surface areas and excellent biocompatibilities. The hemin/G-quadruplex, which was formed by intercalating hemin into the amino terminated thrombin binding aptamer (NH2-TBA), and the electroactive toluidine blue (Tb) were immobilized onto the Cu2O-Au nanocomposite surfaces through a stable Au-N bond. AuNPs, Cu2O and hemin/G-quadruplex co-catalyse the H2O2 in the working buffer to promote the electron transfer of Tb as a multiple signal amplification strategy in order to improve the performance of the electrochemical aptasensor. Under optimal conditions, the designed aptasensor exhibited sensitive detection of TB from 100 fM to 20nM with a lower detection limit of 23fM. This proposed aptasensor exhibited good sensitivity, high specificity and acceptable reproducibility and could be widely applied in bioassay analysis.

Bronchopulmonary Infection of Lophomonas blattarum : A Case and Literature Review

Human infections with Lophomonas blattarum are rare. However, the majority of the infections occurred in China, 94.4% (136 cases) of all cases in the world. This infection is difficult to differentiate from other pulmonary infections with similar symptoms. Here we reported a case of L. blattarum infection confirmed by bronchoalveolar lavage fluid smear on the microscopic observations. The patient was a 21-year-old female college student. The previous case which occurred in Chongqing was 20 years ago. We briefly reviewed on this infection reported in the world during the recent 20 years. The epidemiological characteristics, possible diagnostic basis, and treatment of this disease is discussed in order to provide a better understanding of recognition, diagnosis, and treatment of L. blattarum infection.

A novel PCR-based genotyping scheme for clinical Klebsiella pneumoniae

AIM To establish a PCR-based genotyping method for clinical Klebsiella pneumoniae. MATERIALS & METHODS The prevalence of six serotype markers, 41 large variably presented gene clusters, and seven additional virulence markers were screened by PCR in 327 clinical K. pneumoniae strains from China. RESULTS Detection of serotype markers enabled the identification of capsular serotypes K1, K2, K5, K20, K54 and K57. K. pneumoniae isolates of different origins gave distinct profiles of virulence loci, allowing us to gain a full overview of virulence gene distribution of the strains tested. A novel genotyping scheme was established to group clinical K. pneumoniae strains into distinct complexes based on the profiles of large variably presented gene clusters and virulence markers. CONCLUSION This PCR-based genotyping method would be useful to not only characterize genetic diversity and virulence gene distribution, but also for genotyping, origin tracing and risk estimation of K. pneumoniae.

Reciprocal regulation of pH 6 antigen gene loci by PhoP and RovA in Yersinia pestis biovar Microtus

AIM To explore the transcriptional regulation of the psaEF and psaABC loci by the RovA and PhoP regulators in Yersinia pestis. MATERIALS & METHODS Primer extension, LacZ fusion, gel mobility shift and DNase I footprinting assays were conducted in combination for this gene regulation study. RESULTS It was determined that PhoP and RovA recognized the promoter-proximal regions of psaEF and psaABC in order to repress and stimulate their transcription, respectively. The translation/transcription start sites, Shine-Dalgarno sequences (ribosomal binding site), core promoter -10 and -35 elements, PhoP and RovA sites and PhoP/RovA consensus-like sequences were identified to determine the structural organization of PhoP/RovA-dependent promoters of psaEF and psaABC. CONCLUSION RovA stimulated psaEF and psaABC, while PhoP repressed these two operons involving the direct association between RovA/PhoP and target promoter regions. The reciprocal regulation of psa genes by PhoP and RovA could contribute to the tightly controlled expression of the pH 6 antigen during infection.

Quorum sensing modulates transcription of cpsQ-mfpABC and mfpABC in Vibrio parahaemolyticus

Vibrio parahaemolyticus AphA and OpaR are the two master regulators of quorum sensing (QS) that are abundantly produced and operate at low cell density (LCD) and high cell density (HCD), respectively, with an outcome of reciprocally gradient production of these two proteins with transition between LCD and HCD. The cpsQ-mfpABC gene cluster is transcribed as two operons cpsQ-mfpABC and mfpABC in V. parahaemolyticus. MfpABC is a putative membrane fusion transporter that contributes to biofilm development. CpsQ is a c-di-GMP-binding regulator that activates the expression of capsular polysaccharide genes and mfpABC and, thus, induces biofilm development. As shown in this study, OpaR and AphA bind to the promoter region of mfpABC to enhance and repress its transcription, respectively. In contrast, the positive and negative regulation of cpsQ-mfpABC by AphA and OpaR, respectively, achieves probably through acting of AphA or OpaR on additional unknown regulator(s) of cpsQ-mfpABC. The transcriptional levels of cpsQ-mfpABC and mfpABC enhance gradually with transition from LCD to HCD due to the above reciprocal regulatory action of OpaR and AphA. Data presented here present a novel paradigm of combined action of the two master QS regulators in controlling expression of the QS regulon members.

Development and evaluation of an up-converting phosphor technology-based lateral flow assay for rapid and quantitative detection of aflatoxin B1 in crops

Contamination of grains and other crops by aflatoxin B1 (AFB1), a highly toxic aflatoxin produced by Aspergillus flavus and Aspergillus parasiticus, poses a serious threat to human health and is an important food safety issue. In this study, a competitive up-converting phosphor technology-based lateral flow (AFB1-UPT-LF) assay was developed for rapid detection of AFB1. Detection sensitivity of the proposed assay can reach 0.03ngmL-1 for standard AFB1 solutions, with the coefficients of variation (CV) less than 10% (from 1.0 to 9.4%). A good linearity (r=0.9889) was observed for quantification of AFB1 from 0.03 to 1000ngmL-1. Except for aflatoxin M1, no cross-reactivity was found with the abrin, ricin, ochratoxin A, botulinum toxin, shiga toxin 1, shiga toxin 2, and staphylococcal enterotoxin B, even at high concentrations of 100 or 1000ngmL-1. After optimizing the extraction of AFB1, the assay showed good tolerance to various crop samples, with the detection limit (from 0.1 to 5ngg-1) lower than the corresponding maximum residue level (MRL) set in China. The AFB1-UPT-LF assay provides a promising tool for rapid on-site detection of AFB1 because of its high sensitivity, specificity, and sample tolerance.