Visualization and real-time fluorescence RPA detection methods for enterohemorrhagic Escherichia coli O157:H7

ObjectiveTo detect enterohemorrhagic Escherichia coli （EHEC） O157：H7， visualization recombinant enzyme polymerase amplification （RPA） detection and real-time fluorescence RPA detection methods were established based on RPA technology.MethodsPrimers and probes were designed based on the rfbE gene of EHEC O157：H7， and specificity and sensitivity were determined. A real-time fluorescent RPA method was established. Combined with the color change characteristics of SYBR Green I in nucleic acid reactions， a visualization RPA method for the detection of EHEC O157：H7 was established.ResultsThe established real-time fluorescence RPA detection and visual RPA detection methods both detected EHEC O157：H7 in 25 min at a reaction temperature of 35 ℃. The designed primers and probes showed specific results only for EHEC O157：H7. The detection limits of the two methods were lower than 10-5 ng/μL.ConclusionThe visual RPA detection and real-time fluorescence RPA detection methods of EHEC O157：H7 were easy to operate with strong specificity and high sensitivity. This study provides novel methods for the rapid detection of EHEC O157：H7 in the field and laboratory

Heterologous Boost Following Mycobacterium bovis BCG Reduces the Late Persistent, Rather Than the Early Stage of Intranasal Tuberculosis Challenge Infection

Adults are the leading population affected by tuberculosis (TB) epidemic and death. Developing an effective vaccine against adult TB is urgently needed. Mycobacterium bovis Bacillus Calmette-Guerin (BCG) prime-heterologous boost strategy has been explored extensively to protect adults against primary TB infection, but the majority of experimental regimens have not improved the protection primed by the BCG vaccine. The reason attributed to the failure remains unknown. In this study, CTT3H-based vaccines, namely DMT adjuvanted CTT3H subunit or DNA vaccine (pCTT3H-DMT), and recombinant adenovirus rAdCTT3H were constructed. Protective efficacy and immunogenicity of BCG prime-CTT3H based boosters were compared in C57BL/c mice models of primary or late persistent TB infection. Similar protective efficacy against early intranasal infection was provided by different CTT3H-based vaccines alone in vaccinated mice, and their protection was inferior to that of the BCG vaccine. In addition, CTT3H-based heterologous boosters did not enhance the protection conferred by the BCG vaccine against primary infection. However, all of these three boosters provided stronger protection against late persistent TB infection than BCG alone, regardless of vaccine types. Although BCG prime-boosters elicited Th1-biased responses to the antigen CTT3H, the number of CTT3H-sepcific IFN-γ-expressing TEM (CD62LloCD44hi) and IL-2-expressing TCM (CD62LhiCD44hi) cells in the spleen was not improved before exposure to Mycobacterium tuberculosis infection. In contrast, IFN-γ+ TEM and IL-2+ TCM cells in spleens, especially in lungs were significantly increased in BCG prime-boosters after exposure vaccination. Our results indicate that BCG prime-boost strategy might be a promising measure for the prevention against late persistent TB infection by induction of IFN-γ+ TEM and IL-2+ TCM cells in the lung, which can be used as alternative biomarkers for guiding the clinical practice and future development of TB vaccine for adults

Immunogenicity of heparin-binding hemagglutinin expressed by Pichia pastoris GS115 strain

Objective(s): Heparin-binding hemagglutinin (HBHA), a mycobacterial cell surface protein, mediates adhesion to nonphagocytic cells and the dissemination of Mycobacterium tuberculosis (M. tuberculosis) from the site of primary infection. Superior expression systems are required to obtain abundant M. tuberculosis proteins for the purpose of diagnosing M. tuberculosis infection or for the immunization. Here,  HBHA was expressed by Pichia pastoris (P. pastoris) GS115 strain , and the  immunogenicity of  HBHA was evaluated. Materials and Methods: The HBHA gene of M. tuberculosis was cloned into the pPIC9K plasmid, which was good for electroporation into P. pastoris GS115 strain. Unlabeled HBHA protein was purified using a Sepharose CL-6B column, and its expression was confirmed using anti-HBHA polyclonal antibody from mouse serum. We injected C57BL/6 mice with HBHA/ dimethyldioctadecylammonium/trehalose 6,6′-dibehenate (HBHA/DDA/TDB) to investigate the immunogenicity of this potential vaccine. Results: The results demonstrated that HBHA/DDA/TDB has the ability to induce high levels of HBHA-specific IgG antibody and its subclasses, as well as interferon-gamma, compared with injection of phosphate-buffered saline, DDA/TDB alone and Bacillus Calmette-Guérin (BCG) controls (

System-Level Power Estimation with On-Chip Bus Performance Monitoring Units in PKU-DSPII SoC

Runtime system-level power estimation is essential for dynamic power adaptation in integrated circuits. Indirect power estimation using a CPU performance monitoring unit (PMU) is widely used in modern microprocessors for its low cost. However, the existing CPU PMUs only monitor the activities of core and cache, resulting in accuracy limitation in power estimation for systems containing heterogeneous devices. In this paper, an on-chip bus PMU (OCB PMU) is proposed for PKU-DSPII SOC to achieve accurate system-level power estimation by taking range of peripheral devices into account. By monitoring the on-chip bus signals which indicate almost all the information of the system behavior, an OCB PMU is able to recognize different types of devices. By imitating the internal behavior of every device triggered by OCB signals using an energy state machine (ESM), an OCB PMU is capable of accurate power estimation of heterogeneous devices with complicated behavior. The proposed PMUs can also flexibly adapt to different SoC architectures. ? 2012 IEEE.EI

Immunogenicity and Protective Efficacy of a Novel Recombinant BCG Strain Overexpressing Antigens Ag85A and Ag85B

Recombinant Bacillus Calmette-Guérin (rBCG) strain is the promising vaccine candidate for tuberculosis (TB) prevention, which aims at providing more enduring and enhanced protection than the parental BCG vaccine. In this study, three rBCG strains overexpressing immunodominant antigens Ag85B (rBCG::85B), Ag85A (rBCG::85A), or both (rBCG::AB) of Mycobacterium tuberculosis were constructed, respectively. rBCG strains showed higher level of overexpression of Ag85A and/or Ag85B proteins than BCG containing empty vector pMV261(rBCG::261), which had low levels of endogenous expression of both proteins as expected. rBCG::AB strain could provide the strongest short-term and long-term protection in the lung against intravenous infection with virulent M. tuberculosis than rBCG::261 control and other two rBCG strains overexpressing single antigen. The stronger and longer-lasting protection provided by rBCG::AB than rBCG::261 was correlated with systemic in vitro antigen-specific IFN-γ responses. Therefore, our results indicate that rBCG::AB could be a very promising TB vaccine candidate and should be further evaluated for the preclinical test

Chromosome-level genome assembly of Przevalski’s partridge (Alectoris magna)

Abstract Przevalski’s partridge (Alectoris magna) is one of the birds in the genus Alectoris endemic to China. The distribution of A. magna was narrow, and it was only found in parts of the Qinghai, Gansu, and Ningxia provinces. A. magna was considered a monotypic species until it was distinguished into two subspecies. However, external morphological characteristics, rather than genetic differences or evolutionary relationships, are now commonly used as evidence of subspecies differentiation. In this study, a chromosome-level reference genome of A. magna has been constructed by combining Illumina, PacBio and Hi-C sequencing data. The 1135.01 Mb A. magna genome was ultimately assembled. The genome showed 96.9% completeness (BUSCO), with a contig N50 length of 23.34 Mb. The contigs were clustered and oriented on 20 chromosomes, covering approximately 99.96% of the genome assembly. Additionally, altogether 19,103 protein-coding genes were predicted, of which 95.10% were functionally annotated. This high-quality genome assembly could serve as a valuable genomic resource for future research on the functional genomics, genetic protection, and interspecific hybridization of A. magna

Bacterial organ load and pathological changes in the lung.

<p>Three weeks after the last immunization, C57BL/6 mice (n = 5) were challenged i.v. with 1.2 × 10⁶ CFU of the <i>M</i>. <i>tuberculosis</i> H37Rv strain. Four weeks after the challenge, spleens and lungs were harvested and the CFU numbers per organ were counted. The bacterial load in the lung (A) and spleen (B) of the different groups is represented as the mean (±SEM) log10 CFU/organ (n = 5). Lung pathological scores (C) and representative lung pathological changes from the different groups (HE, scale bar, 400 μm) are also shown (D). AF staining (scale bar, 50 μm) of lung tissue section also supported the hierarchy of lung bacterial load in the different groups (D) and arrows indicate AF-positive bacteria in the lung tissue. Challenge experiments were repeated twice with similar results.</p

Construction, purification, and identification of A1D4 polyprotein and multistage antigens.

<p>A) Construction of the recombinant prokaryotic expression plasmid pET30b-A1D4 linking five genes of <i>M</i>. <i>tuberculosis</i>, <i>Rv1813</i>, <i>Rv2660c</i>, <i>Ag85B</i>, <i>Rv2623</i>, and <i>HspX</i> in tandem. B) Recombinant plasmid pET30b-A1D4 was characterized by restriction enzyme digestion. C) Successful expression and purification of the A1D4 polyprotein were confirmed by 15% SDS-PAGE. Lane M, protein marker (kDa); lane 1, recombinant <i>E</i>. <i>coli</i> BL21 (DE3) strain without IPTG; lane 2, recombinant <i>E</i>. <i>coli</i> BL21 (DE3) strain induced with IPTG for 4 h; lane 3, lysed <i>E</i>. <i>coli</i> pellets after sonication; lane 4, effluent after binding to the Ni-NTA column; lane 5, fraction after washing with denaturing binding buffer containing 8 M urea; lane 6–9, eluted with elution buffer containing 100 mM imidazole. D) Identification of purified A1D4 polyprotein and multistage antigens. E) Identification of purified A1D4 polyprotein and multistage antigens by western blotting with anti-His tag mcAb and F) with anti-A1D4 mouse sera.</p

Protection against <i>Mycobacterium tuberculosis</i> Infection Offered by a New Multistage Subunit Vaccine Correlates with Increased Number of IFN-γ⁺IL-2⁺ CD4⁺ and IFN-γ⁺ CD8⁺ T Cells

<div><p>Protein subunit vaccines present a compelling new area of research for control of tuberculosis (TB). Based on the interaction between <i>Mycobacterium tuberculosis</i> and its host, five stage-specific antigens of <i>M</i>. <i>tuberculosis</i> that participate in TB pathogenesis—Rv1813, Rv2660c, Ag85B, Rv2623, and HspX—were selected. These antigens were verified to be recognized by T cells from a total of 42 whole blood samples obtained from active TB patients, patients with latent TB infections (LTBIs), and healthy control donors. The multistage polyprotein A1D4 was developed using the selected five antigens as a potentially more effective novel subunit vaccine. The immunogenicity and protective efficacy of A1D4 emulsified in the adjuvant MTO [monophosphoryl lipid A (MPL), trehalose-6,6′-dibehenate (TDB), components of MF59] was compared with Bacillus Calmette-Guerin (BCG) in C57BL/6 mice. Our results demonstrated that A1D4/MTO could provide more significant protection against <i>M</i>. <i>tuberculosis</i> infection than the PBS control or MTO adjuvant alone judging from the A1D4-specific Th1-type immune response; however, its efficacy was inferior to BCG as demonstrated by the bacterial load in the lung and spleen, and by the pathological changes in the lung. Antigen-specific single IL-2-secreting cells and different combinations with IL-2-secreting CD4⁺ T cells were beneficial and correlated with BCG vaccine-induced protection against TB. Antigen-specific IFN-γ⁺IL-2⁺ CD4⁺ T cells were the only effective biomarker significantly induced by A1D4/MTO. Among all groups, A1D4/MTO immunization also conferred the highest number of antigen-specific single IFN-γ⁺ and IFN-γ⁺TNF-α⁺ CD4⁺ T cells, which might be related to the antigen load <i>in vivo</i>, and single IFN-γ⁺ CD8⁺ T cells by mimicking the immune patterns of LTBIs or curable TB patients. Our strategy seems promising for the development of a TB vaccine based on multistage antigens, and subunit antigen A1D4 suspended in MTO adjuvant warrants preclinical evaluation in animal models of latent infection and may boost BCG vaccination.</p></div