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A multi-enzyme-like activity exhibiting nanozyme hydrogel with oxidase and catalase-like activity was designed for wound healing, which exhibited excellent antibacterial performance. The catalase-like activity of the nanozyme provides adequate O2 for hypoxia abatement.

A histidine (His)-tag is composed of six His residues and typically exerts little influence on the structure and solubility of expressed recombinant fusion proteins. Purification methods for recombinant proteins containing His-tags are relatively well-established, thus His-tags are widely used in protein recombination technology. We established a one-step enzyme-linked immunosorbent assay (ELISA) for His-tagged recombinant proteins. We analyzed variable heavy and light chains of the anti-His-tag monoclonal antibody 4C9 and used BLAST analyses to determine variable zones in light (VL) and heavy chains (VH). VH, VL, and alkaline phosphatase (ALP) regions were connected via a linker sequence and ligated into the pGEX-4T-1 expression vector. Different recombinant proteins with His tags were used to evaluate and detect ALP-scFv activity. Antigen and anti-His-scFv-ALP concentrations for direct ELISA were optimized using the checkerboard method. ZIKV-NS1, CHIKV-E2, SCRV-N, and other His-tag fusion proteins demonstrated specific reactions with anti-His-scFv-ALP, which were accurate and reproducible when the antigen concentration was 50 µg mL−1 and the antibody concentration was 6.25 µg mL−1. For competitive ELISA, we observed a good linear relationship when coating concentrations of recombinant human anti-Müllerian hormone (hAMH) were between 0.78 and 12.5 µg mL−1. Our direct ELISA method is simple, rapid, and accurate. The scFv antibody can be purified using a prokaryotic expression system, which provides uniform product quality and reduces variations between batches.

develop aptamer-regulated CDs for detection of FB1 by catalytic fluorescence. 2,2′-Dithiosalicylic acid was used to prepare carbon dots catalysing the fluorescence of TMB. Aptamer F10 can bind to FB1, restoring the catalytic capacity of CDs and increasing fluorescence.

Dengue fever is a classic mosquito viral disease. Dengue virus non-structural protein-1 as a membrane-associated homologous dimer anchored to the surface of infected cells and also secreted into the blood. The nonstructural protein-1 levels are related to disease severity, and the presence of nonstructural protein-1 secreted from cells to the serum of people infected with the dengue virus is an early marker of infection. Paired antibodies are key in the establishment of rapid detection technology. In this study, the prepared recombinant nonstructural protein-1 protein of dengue virus serotype 3 was purified by the prokaryotic expression, and prepared monoclonal antibodies by cell fusion. A method for paired antibody screening was established based on the N-hydroxy succinimide-nanobeads and the prepared monoclonal antibodies. A simple and rapid point-of-care system integrating the paired antibodies and lateral flow assay was established to verify the screened antibody pairs. The results confirmed that the antibody pair screening method based on N-hydroxy succinimide-nanobeads is feasible.

Escherichia coli, Staphylococcus aureus, Shigella, Pseudomonas aeruginosa, and Klebsiella pneumoniae are common foodborne pathogens. In this study, the light-induced PMAxx-coupled multiplex PCR (PM...

Because of diverse adverse effects of carbendazim (CBZ) upon the human body and environment, a simple method for the determination of this molecule is urgently needed. Therefore, a method based on ...

S-nitrosylation, the addition of nitric oxide (NO) moiety to a reactive cysteine thiol, to form an S-nitrosothiol (SNO), is emerging as a prototypic redox-based post-translational modification. S-nitrosoglutathione reductase (GSNOR) is thought to be the major regulator of total cellular SNO levels in plants. However, its role in excess nitrate stress has not been investigated in spinach. In this study, a spinach GSNOR gene (GenBank Accession No. KR381778) was amplified and designated as SoGSNOR. The transcript and protein level of SoGSNOR were reduced after excess nitrate treatment for 24 h. Addition of NO donor to the nitrate solution decreased the SoGSNOR expression, while supplementation inhibitor of nitrate reductase and nitric oxide synthase increased its expression. Overexpression of SoGSNOR in tobacco increased the germination rate of transgenic seeds, compared to the wild type (WT) under nitrate stress. Higher photosynthetic rate, transpiration rate, stomatal conductance, water use efficiency and expression level of some stress-related genes were detected in the transgenic seedlings than the WT under nitrate stress. The transgenic tobacco seedlings have lower malondialdehyde content, reactive oxygen species (ROS) fluorescence, and higher activities and transcript level of superoxide dismutase, catalase, peroxidase under nitrate stress. SoGSNOR transgenic tobacco plants have lower NR activity and protein level, higher GSNOR and non-symbiotic class 1 hemoglobin (nsHb) protein level than the WT plants, leading to lower NO accumulation and SNOs contents under nitrate stress. These results suggested that overexpression of SoGSNOR increased nitrate stress tolerance of tobacco by regulating ROS and RNS metabolism. A spinach S-nitrosoglutathione reductase (SoGSNOR) gene was isolated from spinach root. The expression level and activities of SoGSNOR were reduced by excess nitrate treatment. Overexpression of SoGSNOR in tobacco increased nitrate stress tolerance by regulating ROS and RNS metabolism.

In the field of environmental science research, an important research direction is to find a suitable, effective, material and method for desulphurization of fossil fuels and prevent the release of sulfide in fuels into the environment. In this study, a novel catalyst consisting of Pd(CH3COO)2 and MIL-101(Cr) was used to study the sulfur oxidation of organic sulfides, which are difficult to treat, In simulated fuel oil. The optimum reaction conditions were discussed, including the loading of The optimum reaction conditions were discussed, including reaction temperature, the content of Pd in MIL-101(Cr), catalyst dosage, initial BT concentration and cycle times. The BT conversion efficiency of the catalyst in simulated fuel is 88.77%, It shows that the catalyst has good desulfurization performance.

An electro-enhanced membrane bioreactor (EMBR) was constructed with polyaniline-based carbon (PAC) separation membrane as the membrane-electrode, which could realize the in-situ electro-generation and activation of H

The catalysis of disulphide (SS) bonds is the most important characteristic of protein disulphide isomerase (PDI) family. Catalysis occurs in the endoplasmic reticulum, which contains many proteins, most of which are secretory in nature and that have at least one s-s bond. Protein disulphide isomerase A3 (PDIA3) is a member of the PDI family that acts as a chaperone. PDIA3 is highly expressed in response to cellular stress, and also intercept the apoptotic cellular death related to endoplasmic reticulum (ER) stress, and protein misfolding. PDIA3 expression is elevated in almost 70% of cancers and its expression has been linked with overall low cell invasiveness, survival and metastasis. Viral diseases present a significant public health threat. The presence of PDIA3 on the cell surface helps different viruses to enter the cells and also helps in replication. Therefore, inhibitors of PDIA3 have great potential to interfere with viral infections. In this review, we summarize what is known about the basic structure, functions and role of PDIA3 in viral infections. The review will inspire studies of pathogenic mechanisms and drug targeting to counter viral diseases.

Klebsiella pneumoniae belongs to Enterobacteriaceae, which is the commonest bacterium causing nosocomial respiratory tract infection. It ranks second in bacteremia and urinary tract infection in gram-negative bacteria. Therefore, the rapid and accurate identification of K. pneumoniae was of great significance for the guide of clinical medication, and timely treatment of patients. The purpose of this study was to establish a rapid and sensitive molecular detection method for K. pneumoniae based on loop-mediated isothermal amplification (LAMP) technology. Firstly, local BLAST and NCBI BLAST were used to analyze the genome of K. pneumoniae. According to the principle of interspecific and intraspecific specificity, CelB (GenBank ID 11847805) was selected as the specific gene. Then, the LAMP and PCR identification systems were established with this target gene. Thirty-six clinical isolates of K. pneumoniae and 50 non-K. pneumoniae were used for the specific evaluation, and both LAMP and PCR could specifically distinguish K. pneumoniae from non-K. pneumoniae. A 10-fold series diluted positive plasmids and simulated infected blood samples were used as the templates in the sensitivity assay, and the results showed that the sensitivity could reach 1 copy/reaction. In summary, a rapid, specific, and sensitive LAMP method was established to detect K. pneumoniae in clinics.

Aluminum (Al) toxicity and magnesium (Mg) deficiency often coexist in acidic soils. Nitric oxide (NO) is involved in diverse physiological processes and stress responses. Here, we investigated the role of NO in Mg-mediated root growth promotion and Al tolerance in Arabidopsis. Physiological and pharmacological methods together with molecular and genetic analyses were used. Al toxicity- and Mg deficiency-induced NO production contributed to inhibition of primary root growth. In contrast, Mg supply promoted root growth associated with decreasing NO production under Al stress and/or Mg deficiency conditions. Magnesium decreased the activities and expression of the genes related to NO biosynthesis enzymes in Col-0 roots. The NO-associated protein 1 mutant noa1 and the nitrate reductase mutant nia1nia2 with impaired NO production showed Al toxicity- and Mg deficiency-insensitive phenotypes, further confirming involvement of NO in Mg-mediated root growth promotion and alleviation of Al toxicity. Taken together, our results suggested that Mg-mediated enhancement of root growth and Al tolerance is associated with altering NO production in Arabidopsis.

Klebsiella pneumoniae is a common human pathogen that causes several nosocomial chronic infections. Many methods have been reported to detect Klebsiella pneumonia; however, a simple, rapid, and visual method remains to be developed. Here, we developed a rapid, visual detection method employing a nanoparticle immunosensor (NPIS) with a sensitivity of 8 CFU mL-1. Two monoclonal antibodies (mAbs) recognizing different antigenic determinants on the surface of Klebsiella pneumonia were screened by an indirect enzyme-linked immunosorbent assay (ELISA), Western blotting, and mass spectrometry assay. The mAb 1E6 was conjugated to magnetic nanoparticles (MNPs) to generate the immuno-magnetic nanoparticles (IMNPs), whereas the horseradish peroxidase (HRP)-mAb probe was synthesized by conjugating the mAb 2F1 with HRP. Thereafter, the NPIS was developed based on the IMNPs and the HRP-mAb probe, and the total assay time for the detection of Klebsiella pneumoniae was ∼60 min. A rapid, sensitive method was developed for screening Klebsiella pneumoniae in clinical samples. The NPIS developed in this study can detect Klebsiella pneumoniae without the enrichment of bacteria and the extraction of the genome; therefore, it can be used as a primary screening method to compliment other methods in the detection of Klebsiella pneumoniae.

With the panel data of 218 prefecture-level cities from 2003 to 2017, this paper empirically tested whether urban environmental legislation realized Porter Hypothesis(PH) in China. After a series of model estimation and robustness tests, the results show that urban environmental legislation increased the number of local green patents, which means that the weak Porter Hypothesis was established. However, the urban environmental legislation did not lead to an increase in green total factor productivity(GTFP). In other words, the strong version of PH did not hold. Further analysis shows that urban environmental legislation led to the decline of GTFP and the increase of green patents in the west of China, but not in the east and central cities. Besides, the legislation did not promote GTFP improvement through green innovation in the short term, which means it did not realize process compensation.

To improve fresh-cut produce quality and shelf life, 0.5% or 1.0% MicroGARD® 730 (MG) as a natural alternative to synthetic chemical preservatives, 2.5% NatureSeal® (NS) product (vitamin/mineral-based blends), 0.5% MG combined with 2.5% NS, and 1% MG combined with 2.5% NS were used to treat fresh-cut butternut squash (Cucurbita moschata). The 240 g samples were put into food grade bags and stored at 4 or 7 °C. Microbial population, including aerobic plate counts (APCs), yeast and molds, total coliforms, and quality parameters, including head space O2 /CO2 concentration in package, pH, soluble solids, color, and conductivity, were evaluated after 0, 3, 6, 9, 12, and 20 days of storage. Results showed that after 6 days of storage at 7 °C, APC of check and control samples reached to 2.6 × 108 and 1.5 × 107 CFU/g, respectively; while they were kept at 104 CFU/g (3 to 4 log reduction) in the squash samples treated with 0.5% or 1% MG combined with NS at 7 °C. Similar results were found on squash samples stored at 4 °C for 9 days. The cut squash treated with MG combined with NS had APC ≤ 107 CFU/g at 4 °C for about 20 days compared to 9 days in controls or 0.5% MG-treated samples, and 12 days in 1% MG-treated or NS-treated samples, respectively. Considering overall quality and extended shelf life, MG combined with NS was recommended to apply to cut squashes stored at 4 °C. PRACTICAL APPLICATION: This research provided useful information and practical treatment application for developing fresh-cut produce with good quality and extended shelf life up to 20 days at 4 °C.

Deoxynivalenol (DON) is a mycotoxin commonly found in cereals and animal feeds, which also is a carcinogen easily soluble in water. So it has drawn considerable interest in food safety. In this study, a specific aptamer named DON-A16 was developed from an immobilized ssDNA library by affinity chromatography column-based the SELEX. The complementary chain competition method for DON detection was established, and its limit of detection (LOD) was 488 ng/mL. Graphene oxide-based fluorescence quenching was developed using DON-A16 with fluorescein (FAM) label; its LOD was 200 ng/mL, and the detection time was 3 h. The recovery rate of fluorescence quenching was also detected in different samples (oat, wheat flour, corn), which showed the commercial DON ELISA kit led to an increase in repeatability, satisfactory recovery and consistency. The three-dimensional structure and the main binding site of of DON-A16 was ultimately obtained by molecular docking simulation.

Background: Shigella is among the most important human pathogenic microorganisms, infecting both humans and nonhuman and causing clinically severe diarrhea. Shigella must be enriched before detection, which is time-consuming. Objectives: To develop a sensitive, rapid, and specific method for Shigella detection. Materials and Methods: Shigella was used as an antigen to generate monoclonal antibodies (mAbs). mAbs were screened via indirect enzyme-linked immunosorbent assay (ELISA) and western blot, and two mAbs were selected. The mAb A3 showed high affinity and specificity and was used to develop immune magnetic beads (IMBs) for Shigella enrichment. An immunocapture (IC)-PCR primer was designed from the ipaH gene, and IC-PCR was developed based on the IMBs and PCR. Results: This system shortened the Shigella detection time to 70 min. The sensitivity of the IC-PCR was 9 colony-forming units.mL-1 in artificial milk. The accuracy of the IC-PCR was confirmed using 46 clinical samples collected from monkeys. The IC-PCR results were consistent with the serological and biochemical assays. Conclusion: The IC-PCR described herein accurately detected Shigella from milk samples, monkeys and can thus be used to complement classical detection methods.

Objective To prepare human anti-Mullerian hormone (AMH) immunomagnetic beads and HRP-labeled antibodies and establish a rapid double-antibody sandwich ELISA based on nanometer magnetic beads. Methods The expression vector of human AMH protein was constructed, and the recombinant AMH protein was expressed and purified. BALB/c mice were immunized with the recombinant protein to prepare the polyclonal antibody. Spleen cells were fused with myeloma Sp2/0 cells by PEG. Hybridoma cell lines which could stably secret monoclonal antibodies against AMH were screened out by ELISA. Monoclonal antibodies were produced from the ascites fluid of mice injected intraperitoneally with hybridoma cells and evaluated by Western blotting. Polyclonal antibodies purified from protein A were coupled to nano-magnetic' beads and used as capture antibodies, while HRP-labeled monoclonal antibody was prepared by sodium periodate method and used as probe antibody. A double antibody sandwich ELISA based on the nano-magnetic beads was established and optimized. Results A monoclonal antibody with good specificity for AMH was obtained,' and its subtype was IgG2b. The titers of purified polyclonal antibodies and monoclonal antibodies were up to 1:51 200. The capture antibody coupled with magnetic beads and the probe antibody labeled with HRP kept their good activity. The established method could detect AMH antigen within 1 hour and the detection limit was 50 ng/mL. Conclusion The prepared AMH immunomagnetic beads can be used for the fast and visualized detection of recombinant AMH.

NAC (for NAM, ATAF1-2, and CUC2) proteins are one of the largest transcription factor families in plants. They have various functions and are closely related to developmental processes of fruits. Tomato (Solanum lycopersicum) is a model plant for studies of fruit growth patterns. In this study, the functional characteristics and action mechanisms of a new NAC-type transcription factor, SlNAC3 (SGN-U568609), were examined to determine its role in tomato development and ripening. The SlNAC3 protein was produced by prokaryotic expression and used to immunize New Zealand white rabbits to obtain a specific polyclonal antibody against SlNAC3. By co-immunoprecipitation and MALDI-TOF-MS assays, we showed that there was an interaction between the SlNAC3 protein and Polygalacturonase-2 (PG-2), which is related to the ripening and softening of fruit. Chromatin immunoprecipitation assays revealed the genome of the highly stress-tolerant Solanum pennellii chromosome 10 (sequence ID, HG975449.1), further demonstrating that SlNAC3 is a negative regulator of drought and salinity stress resistance in tomato, consistent with previous reports. These results indicate that SlNAC3 is not only involved in abiotic stress, but also plays a necessary role in mediating tomato ripening.

Siniperca chuatsi rhabdovirus (SCRV) is a rod- or bullet-shaped negative-stranded RNA virus and a threat to mandarin fish farming. This virus naturally infects fish, such as S. chuatsi and Micropterus salmoides, among others. SCRV-infected fish have a 90% mortality rate. In this study, a recombinant SCRV-N protein and a polyclonal antibody were prepared by gene cloning, constructing the vector of the SCRV nucleoprotein gene, purifying the protein, and immunizing mice with the recombinant nucleoprotein. A SCRV-N competitive enzyme-linked immunosorbent assay was established based on the recombinant protein and polyclonal antibody and verified by a large number of experimental fish infected with SCRV. The results confirmed that the method established in this study can be used to confirm whether fish are infected with SCRV with high sensitivity and speed. This rapid detection test is suitable for large-scale applications and will provide support for diagnosing infection with the mandarin fish rhabdovirus.

Background: Zika Virus (ZIKV) is a new type of flaviviruses transmitted by mosquitoes to cause severe diseases including Guillain-Barre syndrome and congenital malformations during recent outbreaks. The early diagnosis of ZIKV infection and efficient vaccines would be effective in controlling epidemics and timely treatment. However, the native nonstructural protein 1 (NS1) mainly acquired from cells infected with ZIKV, which largely limits its application. Objectives: This study aimed to express and purify the recombinant NS1 protein and prepare its antibody that efficiently reacted with the native NS1 protein secreted in the supernatant of ZIKV infected host-cells. Methods: We constructed a prokaryotic expression vector containing the full length of the NS1 gene. Thus, the recombinant NS1 protein was efficiently expressed and purified. The purified NS1 protein was used to prepare an antibody. The Enzyme-linked Immunosorbent Assay (ELISA), Western blot, and Indirect Immunofluorescent Assay (IFA) were used to assess the reactivity and specificity of the antibody. Results: The recombinant NS1 protein from a prokaryotic expression vector had good immunogenicity and the prepared antibody could specifically react with the native NS1 produced in Vero, BHK-21 cells infected with ZIKV. Besides, the ELISA and Western blot showed that the native ZIKV-NS1 protein was secreted extracellularly and could play the role of an early diagnostic biomarker for ZIKV infection. Conclusions: The prepared antibody against the recombinant NS1 protein is a reliable biological tool that enables the rapid and sensitive detection of secreted NS1 from host cells infected with ZIKV. The recombinant NS1 protein and its antibody can contribute to developing vaccines and antibodies targeting the NS1 protein to prevent flavivirus disease progression.

Salmonella, a common foodborne pathogen, causes many cases of foodborne illness and poses a threat to public health worldwide. Immunological detection systems can be combined with nanoparticles to develop sensitive and portable detection technologies for timely screening of Salmonella infections. Here, we developed an antibody-probe-based immuno-N-hydroxysuccinimide (NHS) bead (AIB) system to detect Salmonella. After adding the antibody probe, Salmonella accumulated in the samples on the surfaces of the immuno-NHS beads (INBs), forming a sandwich structure (INB&ndash, Salmonella&ndash, probes). We demonstrated the utility of our AIB diagnostic system for detecting Salmonella in water, milk, and eggs, with a sensitivity of 9 CFU mL&minus, 1 in less than 50 min. The AIB diagnostic system exhibits highly specific detection and no cross-reaction with other similar microbial strains. With no specialized equipment or technical requirements, the AIB diagnostic method can be used for visual, rapid, and point-of-care detection of Salmonella.

Background: Klebsiella pneumoniae is an important human pathogen that causes severe diseases including urinary tract infection, pneumonia, and bacteremia. However, a rapid and sensitive detection method remains to be developed. Objectives: This study aimed to develop a rapid, real-time, and visual detection method for K. pneumoniae. This is a primary screening method to improve the diagnosis of K. pneumoniae infection and save much precious time for clinical practice. Methods: Klebsiella pneumoniae was used as an antigen to produce a monoclonal antibodies (mAb). The mAb 1E6 recognizing outer membrane protein C on the surface of K. pneumoniae was screened by an indirect enzyme-linked immunosorbent assay (ELISA), Western Blot, and mass spectrometry assay, and then conjugated with the protein A/G coated magnetic beads to generate the immune magnetic beads (IMBs). Thereafter, the IMBs were used to capture K. pneumoniae, and the complex (beads-mAb-K. pneumoniae) was used for loop-mediated isothermal amplification (LAMP) assay. Results: We developed a rapid, real-time, and visual detection method employing an immunocapture loop-mediated isothermal amplification (IC-LAMP). The sensitivity of the IC-LAMP was 4 CFU mL-1. The process of K. pneumoniae detection lasted ~ 60 minutes and had no cross-reaction to other microbial strains. Besides, the accuracy of IC-LAMP was further verified by examining 39 clinical isolates. Conclusions: Without the need for enrichment of bacteria and extraction of its genome, the IC-LAMP developed here could be used as a primary screening method supplementary to traditional detection methods to improve the diagnosis of K. pneumoniae infection for clinical practice.

Borax is considered highly toxic and a strong carcinogen, but it is widely used as a toughening and brightening additive in food processing. At present, there is no definitive standard assay for the direct identification of borax in foods in most countries. In this study, borax-specific aptamers with high affinity and specificity were selected using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) with high throughput sequencing. Next, a colorimetric detection method was proved to be feasible by nuclear magnetic resonance (NMR), using a selected aptamer, Bor-A01, with gold nanoparticles (AuNPs) for the direct detection of borax. Due to the matrix effect of real foods, the actual results of most detection methods may have large deviation or error. After optimizing detection conditions and pretreating food samples, the colorimetric detection assay allowed the specific recognition of borax within 1 h, with the visual limit of detection (LOD) range for borax of 0.30−0.50 μg/mL in different food samples. Overall, this colorimetric method based on aptamer-AuNPs with high sensitivity, simple pre-treatment, short operation time, and low cost will be applicable as a novel detection strategy to prohibit the use of borax in foods.

Background: The outbreak of Zika virus (ZIKV) in many countries caused alarming numbers of babies born with microcephalus and severe neurologic disorders, however, the methods for the detection of the ZIKV are limited at present. Objectives: The aim of this study was to produce polyclonal antibody against full length envelop protein of ZIKV (ZIKV-E protein) in prokaryotic system and to evaluate its efficacy to capture ZIKV virion. Methods: The recombinant full length ZIKV-E protein was purified and then used as an immunogen to vaccinate BALB/c mice to produce polyclonal antibody. Protein A/G coated magnetic beads were coated with the polyclonal antibody to capture the ZIKV virion in the culture medium of Vero cells infected with ZIKV. The products of immunoprecipitation were further used for Western Blot, Loop-mediated isothermal amplification (LAMP), and PCR assay. Results: Western Blot and indirect immunofluorescence analysis showed that the mouse polyclonal antibody could react specifically with the native E protein in Vero cells infected with ZIKV. The results of Western Blot, Immunocaptured-LAMP (IC-LAMP), and Immunocaptured-PCR (IC-PCR) showed that polyclonal antibody of ZIKV-E recombinant protein can capture ZIKV virion specifically, however, the polyclonal antibody against the ZIKV-NS1, Shigella, and Salmonella without such a function. Conclusions: These findings may provide the basis for the development of the rapid diagnostic kits such as, IC-PCR, IC-LAMP, and sandwich ELISA. The serum virion capture activity elicited by prokaryotic expressed recombinant ZIKV-E protein may also provide a basis for further preparation of neutralizing antibody and protein subunit vaccine candidate against ZIKV.

Background: Brucella spp. are Gram-negative bacteria that cause a zoonotic disease called brucellosis in humans as well as many animals. Brucella suis (B.suis) is one of the greatest threats to the human health and food safety. Studying macrophage and B. suis interaction is critical for understanding the chronic infection mechanism. However, the interaction mechanisms, especially for molecular events triggered by B. suis infected macrophage, such as biological pathways, are still obscure. Objectives: We will use gene set enrichment analysis (GSEA) to microarray in an attempt to find critical pathways in the interaction of macrophage and B. suis. Methods: We applied a standardized microarray preprocessing and GSEA to 2 independent macrophage and B. suis interaction studies including smooth virulent B. suis strain 1330 (S1330) data sets and rough attenuated B. suis strain VTRS1 (VTRS1) data sets. Integrative analysis was used to find critical pathways for 2 independent macrophage and B. suis interaction data sets. Results: The results demonstrated that for S1330 data sets, 8 and 13 common up- and down- regulated pathways were found in 4 interaction stages including 4h, 8h, 24h, and 48h post macrophage infected S1330 B. suis, and for VTRS1 data sets, we found 30 and 19 common up- and down- regulated pathways. Comparing the results of S1330 and VTRS1 data sets, 6 and 8 common up- and down- regulated pathways were identified. Conclusions: The study of macrophage and B. suis interaction through pathway analysis highlighted genes weakly connected to the phenotype, and discovered common critical pathways in the process of macrophages and different phenotypes of B. suis interaction. The identified pathways will shed light on the understanding of the functional events within macrophage post infected B. suis.

Shigella is an important human food-borne zoonosis bacterial pathogen, and can cause clinically severe diarrhea. There is an urgent need to develop a specific, sensitive, and rapid methodology for detection of this pathogen. In this study, loop-mediated isothermal amplification (LAMP) combined with magnetic immunocapture assay (IC-LAMP) was first developed for the detection of Shigella in pure culture, artificial milk, and clinical stool samples. This method exhibited a detection limit of 8.7 CFU/mL. Compared with polymerase chain reaction, IC-LAMP is sensitive, specific, and reliable for monitoring Shigella. Additionally, IC-LAMP is more convenient, efficient, and rapid than ordinary LAMP, as it is more efficiently enriches pathogen cells without extraction of genomic DNA. Under isothermal conditions, the amplification curves and the green fluorescence were detected within 30 min in the presence of genomic DNA template. The overall analysis time was approximately 1 h, including the enrichment and lysis of the bacterial cells, a significantly short detection time. Therefore, the IC-LAMP methodology described here is potentially useful for the efficient detection of Shigella in various samples.

Background: Rabies is a zoonotic infectious disease that infects the human and animal central nervous system. Worldwide, especially in low-income countries, this disease is still a burden for public health. Among the rabies virus proteins, phosphoprotein plays a very important role in viral infection, and this research found that immunization of rabies virus vaccines could widely induce antibody responses against phosphoprotein, therefore rabies virus phosphoprotein may be a useful target for development of rapid and low cost serological diagnosis test, and therapeutic drugs. Objectives: The aim of this study was to prepare anti-rabies virus phosphoprotein monoclonal antibody, which is used for rapid detection and diagnosis of rabies virus infection. Methods: The phosphoprotein gene was amplified by the polymerase chain reaction (PCR), and sub-cloned in a his-tagged prokaryotic expression vector to generate a recombinant plasmid named pET-32a-RABV-P. Recombinant RABV-P was induced by isopropyl-β-D-Thiogalactopyranoside (IPTG), and then purified by the Ni-NTA purification system. Immunization of BALB/c mice with the recombinant protein was performed, and the spleen cells of the immunized mice and SP2/0 myeloma cells were fused together to obtain the monoclonal cell strains, and then identification of the characteristics of the antibody by the enzyme linked immunosorbent assay (ELISA), western blotting, and indirect immunofluorescence assay was done. Results: The prokaryotic expression vector of pET-32a-RABV-P was successfully constructed. The fusion protein was expressed in Escherichia coli Rosetta and purified. After cell fusion, a hybridoma cell line 1A4 was successfully obtained. The antibody titer of the anti-RABV-P ascites reached 256,000. The results of western blotting and indirect immunofluorescence assay indicated that 1A4 hybridoma cell line was able to produce specific antibodies against rabies virus phosphoprotein. Conclusions: Recombinant rabies virus phosphoprotein could be successfully expressed in E. coli. A specific monoclonal antibody against rabies virus phosphoprotein has been successfully prepared.

Background and Aims Aluminium (Al) toxicity is a limiting factor for plant growth and crop production in acidic soils. Citrate exudation and activation of the plasma membrane H+-ATPase are involved in soybean responses to Al stress. Auxin has crucial functions in plant growth and stress responses. However, little is known about possible interactions between auxin and citrate exudation under Al stress. In this study, we elucidated the regulatory roles of IAA in Al-induced citrate exudation in soybean roots. Methods We measured IAA content, Al concentration, citrate exudation, plasma membrane H+-ATPase activity, expression of the relevant genes and phosphorylation of the plasma membrane H+-ATPase by integrating physiological characterization and molecular analysis using hydroponically grown soybean. Key Results The concentration of IAA was increased by 25 and 50 μm Al, but decreased to the control level at 200 μm Al. External addition of 50 μm IAA to the root medium containing 25, 50 or 200 μm Al decreased root Al concentration and stimulated Al-induced citrate exudation and the plasma membrane H+-ATPase activity. Reverse transcription-PCR analysis showed that exogenous IAA enhanced the expression of citrate exudation transporter (GmMATE) but not the plasma membrane H+-ATPase gene. The western blot results suggested that IAA enhanced phosphorylation of the plasma membrane H+-ATPase under Al stress. Conclusions Auxin enhanced Al-induced citrate exudation through upregulation of GmMATE and an increase in phosphorylation of the plasma membrane H+-ATPase in soybean roots.

Background Herpes B virus (BV) is a zoonotic disease caused by double-stranded enveloped DNA virus with cercopithecidae as its natural host. The mortality rate of infected people could be up to 70% with fatal encephalitis and encephalomyelitis. Up to now, there are no effective treatments for BV infection. Among the various proteins encoded by monkey B virus, gD, a conserved structural protein, harbors important application value for serological diagnosis of frequent variations of the monkey B virus. Objectives This study aimed to expressed the gD protein of BV in Escherichia coli by a recombinant vector, and prepare specific monoclonal antibodies against gD of BV to pave the way for effective and quick diagnosis reagent research. Materials and Methods The gD gene of BV was optimized by OptimWiz to improve codon usage bias and synthesis, and the recombinant plasmid, pET32a/gD, was constructed and expressed in E. coli Rosetta (DE3). The expressed fusion protein, His-gD, was purified and the BALB/c mice were immunized by this protein. Spleen cells from the immunized mice and SP2/0 myeloma cells were fused together, and the monoclonal cell strains were obtained by indirect enzyme-linked immunosorbent assay (ELISA) screening, followed by preparation of monoclonal antibody ascetic fluid. Results The optimized gD protein was highly expressed in E. coli and successfully purified. Five monoclonal antibodies (mAbs) against BV were obtained and named as 4E3, 3F8, 3E7, 1H3 and 4B6, and with ascetic fluid titers of 2 × 106, 2 × 105, 2 × 105, 2 × 103 and 2 × 102, respectively. The 1H3 and 4E3 belonged to the IgG2b subclass, while 3E7, 3F8 and 4B6 belonged to the IgG1 subclass. Conclusions The cell lines obtained in this work secreted potent, stable and specific anti-BV mAbs, which were suitable for the development of herpes B virus diagnosis reagents.

Annexins have been suggested to play pivotal roles in stress resistance and plant development. However, related studies on fruit-bearing plants, especially on fruit development, are very limited. In the present study, we provide a comprehensive overview of the annexin family in tomato, describing the gene structure, promoter cis-regulatory elements, organ expression profile, and gene expression patterns under hormone and stress treatments. Bioinformatic analysis revealed that the nine tomato annexins were structurally different from their animal counterparts, but highly conserved annexin domains were still found in most of them. Cis-regulatory element prediction showed that there were important elements in the 2kb upstream promoter regions, including stress- and hormone-responsive-related elements. The expression patterns of these genes were investigated, and the results revealed that they were regulated under developmental processes and environmental stimuli. Among them, AnnSl1.1 and AnnSl2 were highly expressed in most of the tested organs. Genes preferentially or specifically expressed in organs, such as stigma or ovary (AnnSl6), stamen (AnnSl8), and fruit pericarp (AnnSl1.2 and AnnSl9), were identified. Some annexin genes were induced by plant hormones including abscisic acid (AnnSl3, AnnSl6, AnnSl8, and AnnSl9) and gibberellic acid (AnnSl1.1, AnnSl1.2, AnnSl4, and AnnSl7). Most of these annexin genes were induced by salt, drought, wounding, and heat or cold stresses. The present study provides significant information for understanding the diverse roles of annexins in tomato growth and development.

NAC (for NAM, ATAF1, 2, and CUC2) family genes have been found to play an important role in diversified developmental processes and environmental responses. A new NAC-type transcription factor SlNAC3 was primarily identified and isolated from the cDNA libraries of tomato cultivar Ailsa Craig. It contains three exons and two introns within genomic DNA sequence and encodes a polypeptide of 329 amino acids. A plant-specific and conserved NAC domain is located in the N-terminus of SlNAC3. The protein SlNAC3 is subcellularly localized in the nucleus of onion epidemical cells and it has a transcriptional activation domain in the C-terminal region which shows extremely divergent among NACs. Phylogenetic analysis showed that SlNAC3 belonged to the OsNAC3 subgroup of the NAC protein family. Tissue expression profile analysis revealed that SlNAC3 was expressed mainly in flower, fruit and root. The transcription expression of SlNAC3 was inhibited by salt, drought stress and ABA treatment. These data demonstrate that SlNAC3 might interact with environmental and endogenous stimuli and probably function when plants response to salt and drought stresses through ABA signaling pathways as a transcriptional activator.