Individual transmembrane domains of SfABCC2 from Spodoptera frugiperda do not serve as functional Cry1F receptors.

The fall armyworm (Spodoptera frugiperda) is a major global pest causing severe damage to various crops, especially corn. Transgenic corn producing the Cry1F pesticidal protein from the bacterium Bacillus thuringiensis (Cry1F corn) showed effectiveness in controlling this pest until S. frugiperda populations at locations in North and South America evolved practical resistance. The mechanism for practical resistance involved disruptive mutations in an ATP binding cassette transporter subfamily C2 gene (SfABCC2), which serves as a functional Cry1F receptor in the midgut cells of susceptible S. frugiperda. The SfABCC2 protein contains two transmembrane domains (TMD1 and TMD2), each with a cytosolic nucleotide (ATP) binding domain (NBD1 and NBD2, respectively). Previous reports have demonstrated that disruptive mutations in TMD2 were linked with resistance to Cry1F, yet whether the complete SfABCC2 structure is needed for receptor functionality or if a single TMD-NBD protein can serve as functional Cry1F receptor remains unknown. In the present study, we separately expressed TMD1 and TMD2 with their corresponding NBDs in cultured insect cells and tested their Cry1F receptor functionality. Our results show that the complete SfABCC2 structure is required for Cry1F receptor functionality. Moreover, binding competition assays revealed that Cry1F specifically bound to SfABCC2, whereas neither SfTMD1-NBD1 nor SfTMD2-NBD2 exhibited any significant binding. These results provide insights into the molecular mechanism of Cry1F recognition by SfABCC2 in S. frugiperda, which could facilitate the development of more effective insecticidal proteins.

New roles for Bacillus thuringiensis in the removal of environmental pollutants.

For a long time, the well-known Gram-positive bacterium Bacillus thuringiensis (Bt) has been extensively studied and developed as a biological insecticide for Lepidoptera and Coleoptera pests due to its ability to secrete a large number of specific insecticidal proteins. In recent years, studies have found that Bt strains can also potentially biodegrade residual pollutants in the environment. Many researchers have isolated Bt strains from multiple sites polluted by exogenous compounds and characterized and identified their xenobiotic-degrading potential. Furthermore, its pathway for degradation was also investigated at molecular level, and a number of major genes/enzymes responsible for degradation have been explored. At present, a variety of xenobiotics involved in degradation in Bt have been reported, including inorganic pollutants (used in the field of heavy metal biosorption and recovery and precious metal recovery and regeneration), pesticides (chlorpyrifos, cypermethrin, 2,2-dichloropropionic acid, etc.), organic tin, petroleum and polycyclic aromatic hydrocarbons, reactive dyes (congo red, methyl orange, methyl blue, etc.), and ibuprofen, among others. In this paper, the biodegrading ability of Bt is reviewed according to the categories of related pollutants, so as to emphasize that Bt is a powerful agent for removing environmental pollutants.

Microbial degradation as a powerful weapon in the removal of sulfonylurea herbicides.

Sulfonylurea herbicides have been widely used worldwide and play a significant role in modern agricultural production. However, these herbicides have adverse biological effects that can damage the ecosystems and harm human health. As such, rapid and effective techniques that remove sulfonylurea residues from the environment are urgently required. Attempts have been made to remove sulfonylurea residues from environment using various techniques such as incineration, adsorption, photolysis, ozonation, and microbial degradation. Among them, biodegradation is regarded as a practical and environmentally responsible way to eliminate pesticide residues. Microbial strains such as Talaromyces flavus LZM1, Methylopila sp. SD-1, Ochrobactrum sp. ZWS16, Staphylococcus cohnii ZWS13, Enterobacter ludwigii sp. CE-1, Phlebia sp. 606, and Bacillus subtilis LXL-7 can almost completely degrade sulfonylureas. The degradation mechanism of the strains is such that sulfonylureas can be catalyzed by bridge hydrolysis to produce sulfonamides and heterocyclic compounds, which deactivate sulfonylureas. The molecular mechanisms associated with microbial degradation of sulfonylureas are relatively poorly studied, with hydrolase, oxidase, dehydrogenase and esterase currently known to play a pivotal role in the catabolic pathways of sulfonylureas. Till date, there are no reports specifically on the microbial degrading species and biochemical mechanisms of sulfonylureas. Hence, in this article, the degradation strains, metabolic pathways, and biochemical mechanisms of sulfonylurea biodegradation, along with its toxic effects on aquatic and terrestrial animals, are discussed in depth in order to provide new ideas for remediation of soil and sediments polluted by sulfonylurea herbicides.

Effect of Additional Mass on Natural Frequencies of Weight-Sensing Structures.

The phenomena of variability and interference in the natural frequencies of weight-sensing structures applied in complex working conditions must solve the problem of reducing or eliminating resonance under low-frequency vibrations to maximize stability, accuracy and reliability. The influence laws of the additional mass with relevant characteristics on the natural frequencies, which include the components of mass, stiffness and center-of-mass distribution, etc. Firstly, the theoretical formulas of the mathematical model are given based on different characteristics of the weight-sensing structure, and various combinations of additional masses on the weight-sensing structures are adjusted in the X-, Y-, and Z-directions. The key factors to be specifically considered in the theoretical formulas are discussed through simulation analysis and experimental validation. Secondly, the locking strength of the fastening screws of some components was changed, and another component was placed on the experimental platform in the experiment. The results show that the mass, center-of-mass, stiffness distribution and other factors of the additional mass have different effects on the natural frequencies, which are important for the demand for high-precision, high-stability weighing measurement. The results of this research can provide an effective scientific evaluation basis for the reliable prediction of natural frequencies.

Insights into the microbial degradation and resistance mechanisms of glyphosate.

Glyphosate, as one of the broad-spectrum herbicides for controlling annual and perennial weeds, is widely distributed in various environments and seriously threatens the safety of human beings and ecology. Glyphosate is currently degraded by abiotic and biotic methods, such as adsorption, photolysis, ozone oxidation, and microbial degradation. Of these, microbial degradation has become the most promising method to treat glyphosate because of its high efficiency and environmental protection. Microorganisms are capable of using glyphosate as a phosphorus, nitrogen, or carbon source and subsequently degrade glyphosate into harmless products by cleaving C-N and C-P bonds, in which enzymes and functional genes related to glyphosate degradation play an indispensable role. There have been many studies on the abiotic and biotic treatment technologies, microbial degradation pathways and intermediate products of glyphosate, but the related enzymes and functional genes involved in the glyphosate degradation pathways have not been further discussed. There is little information on the resistance mechanisms of bacteria and fungi to glyphosate, and previous investigations of resistance mechanisms have mainly focused on how bacteria resist glyphosate damage. Therefore, this review explores the microorganisms, enzymes and functional genes related to the microbial degradation of glyphosate and discusses the pathways of microbial degradation and the resistance mechanisms of microorganisms to glyphosate. This review is expected to provide reference for the application and improvement of the microbial degradation of glyphosate in microbial remediation.

Anti-idiotypic single-chain variable fragment antibody partially mimic the functionally spatial structure of Cry2Aa toxin.

The anti-idiotypic antibody is widely used in the field of immunology to simulate structural features or even induce the biological activity of antigens. In this study, we obtained seven anti-idiotypic single-chain variable fragments (scFv) antibodies of Cry2Aa toxin from a phage-displayed mutant library constructed using error-prone PCR technique. A mutant designated 2-12B showed the best binding ability amongst all anti-idiotypic scFv isolates to Plutella xylostella brush border membrane vesicles (BBMVs). 2-12B and Cry2Aa toxin shared a potential receptor of polycalin in P. xylostella BBMVs. Homology modeling and molecular docking demonstrated that 2-12B and Cry2Aa toxin have seven common binding amino acid residues in polycalin. Insect bioassay results suggested that 2-12 had insecticidal efficacy against P. xylostella larvae. These results indicated that the Cry2Aa anti-idiotypic scFv antibody 2-12B partially mimicked the structure and function of Cry2Aa toxin. The anti-idiotypic scFv antibody provides the basic material for the future study of surrogate molecules or new insecticidal materials.

Identification of the association of CD28+ CD244+ Tc17/IFN-γ cells with chronic hepatitis C virus infection.

CD8+ T cells play multiple and complex immunological roles including antiviral, regulatory, and exhaustive effects in hepatitis C virus (HCV) infected patients. Some CD8+ T-cell subsets were confirmed to be closely related to HCV infection such as TCM , TEM , TEM RA, Tc17, and CD8+ Treg. Herein, we report a new subset of interleukin (IL)-17/interferon (IFN)-γ producing CD8+ T (Tc17/IFN-γ) cells that markedly correlate with CD28+ CD244+ cells, IL-17 levels, and HCV RNA in HCV patients. During early treatment with peg-IFN-a2a plus ribavirin, the imbalance of these Tc17/IFN-γ cells could be partially restored, together with normalized serum alanine aminotransferase but not aspartate transaminase. Also, we analyzed the dynamic change of the percentage of this T cells subset in patients with different outcome after 4-week course of treatment with peg-IFN-a2a plus ribavirin and found that the percentage of CD8+ CD28+ CD244+ T cells significantly decreased in recovered patients but not in nonrecovered patients. In vitro, CD28+ CD244+ T cells were the only CD8+ T-cell group that secreted both IL-17 and IFN-γ in this axis and blockade with anti-CD244 antibodies significantly reduced cytokine production. Taken together, this study demonstrates that the frequency and regulatory functions of CD28+ CD244+ Tc17/IFN-γ cells may play an important role in persistent HCV infection.

Construction and characterization of a class-specific single-chain variable fragment against pyrethroid metabolites.

Pyrethroids are insecticides that are widely used in rural and urban areas worldwide. After entering the environment, pyrethroids are rapidly metabolized or degraded by various biological or abiotic methods. In this study, a single-chain variable fragment (scFv) which could simultaneously detect three pyrethroid metabolites was constructed based on a hybridoma raised against 3-phenoxybenzoic acid (3-PBA). By molecular docking, it showed that there were hydrogen bonds, hydrophobic interactions, CH-π interaction, and cation-π interaction between 3-PBA and its scFv. All the contact residues contributing to hydrogen bonds are located in VH-CDR2 or its neighboring region, and two of them were mutants of the closest germline sequence. Based on competitive ELISA, the half maximal inhibitory concentration (IC50) of the scFv for 3-PBA, 3-phenoxybenzaldehyde (PBAld), and 3-phenoxybenzyl alcohol (PBAlc) were calculated to be 0.55, 0.59, and 0.63 µgmL-1, respectively. The scFv also showed 23.91%, 13.41%, 1.15%, 1.00%, and 0.56% cross-reactivity with phenothrin, deltamethrin, fenvalerate, beta-cypermethrin, and fenpropathrin. The broad specificity of the scFv may be due to its hapten design. The scFv could be employed in class-specific immunoassays for pyrethroid metabolites with phenoxybenzyl (PB) group. It is also potentially used for characterizing degradation of pyrethroids or detecting PBAlc (PBAld) alone, and the detection results should be confirmed by other selective methods. KEY POINTS: • A scFv which can simultaneously detect 3-PBA, PBAlc, and PBAld was constructed. • Antibody informatics and binding mode of the scFv were obtained. • The reason for its broad specificity was discussed. • It could be used to monitor single or multi-pyrethroid metabolites with PB group.

miR-324-5p protects against oxidative stress-induced endothelial progenitor cell injury by targeting Mtfr1.

Endothelial progenitor cells (EPCs) belong to bone marrow-derived myeloid progenitor cells that have strong proliferative ability. Dysregulation of miRNAs after acute myocardial infarction (AMI) can result in EPCs injury, thus we hypothesize that correction of miRNA expression may contribute to the tolerance of EPCs against oxidative stress. The peripheral blood of healthy volunteers and patients with ST-segment elevation myocardial infarction (STEMI) was clinically collected. EPCs derived from peripheral blood were transfected by miR-324-5p mimic and simultaneously handled with hydrogen peroxide (H2 O2 ) to inducing EPCs injury. At 24 hrs after the H2 O2 treatment, cell viability, the uptake capacity on DiI-Ac-LDL, and carrying ability on FITC-UEA-l and multiplication capacity were analyzed. The mechanism process was carefully researched by valued the characteristics of the mitochondrion morphology, membrane potential, ATP levels, and the expressing of apoptosis pathways. Small RNA sequencing indicated that the expression level of miR-324-5p in peripheral blood EPCs of patients with STEMI was significantly lower compared with the healthy volunteers. The Mtfr1 has been confirmed as a targeted gene of miR-324-5p through miRTarBase software and western blot. The miR-324-5p mimic units could be contributed for the improvement of viability, the uptake capacity on DiI-Ac-LDL and carrying ability on FITC-UEA-l and multiplication capacity on oxidative stress-injured EPCs. miR-324-5p could suppress mitochondrial fragmentation, promote membrane potential, and ATP levels, as well as protect against oxidative stress-induced EPCs apoptosis. Our results suggested that miR-324-5p protects against oxidative stress-induced EPCs injury by regulating Mtfr1.

PIK3CD induces cell growth and invasion by activating AKT/GSK-3ß/ß-catenin signaling in colorectal cancer.

The catalytic subunit p110δ of phosphoinositide 3-kinase (PI3K) encoded by PIK3CD has been implicated in some human solid tumors. However, its roles in colorectal cancer (CRC) remain largely unknown. Here we found that PIK3CD was overexpressed in colon cancer tissues and CRC cell lines and was an independent predictor for overall survival (OS) of patients with colon cancer. The ectopic overexpression of PIK3CD significantly promoted CRC cell growth, migration and invasion in vitro and tumor growth in vivo. In contrast, inhibition of PIK3CD by specific small-interfering RNA or idelalisib dramatically suppressed CRC cell growth, migration and invasion in vitro and tumor growth in vivo. Moreover, PIK3CD overexpression increased AKT activity, nuclear translocation of ß-catenin and T-cell factor/lymphoid enhancer factor (TCF/LEF) transcriptional activity and decreased glycogen synthase kinase 3ß (GSK-3ß) activity, whereas PIK3CD inhibition exhibited the opposite effects. Furthermore, PIK3CD-mediated cell growth, migration and invasion were reversed by blockade of AKT signaling or depletion of ß-catenin. In addition, PIK3CD expression in colon cancer tissues positively correlated with ß-catenin abnormal expression, which was an independent predictor for OS of colon cancer patients. Taken together, our findings demonstrate that PIK3CD is an independent prognostic factor in CRC and that PIK3CD induces CRC cell growth, migration and invasion by activating AKT/GSK-3ß/ß-catenin signaling, suggesting that PIK3CD might be a novel prognostic biomarker and a potential therapeutic target for CRC.

High-performance optical coherence velocimeter: theory and applications.

We proposed a high-performance optical coherence velocimeter (OCV) based on broadband optical interference which achieves spatial resolution from interference cancellation or enhancement of different components of the broadband light. There is a challengeable issue for OCV that the interference fringes become blurred when the velocity of detected object is relatively large, hindering the pace of OCV application in high-velocity field. To resolve this, the relationship between blurry coefficient and OCV system parameters (e.g., exposure time, central wavelength, bandwidth of source) was derived. It was found that blurry coefficient changed with oscillatory decay form and reached the minimum at each order blurry velocity. It showed that maximum measurable velocity of OCV systems could reach 10th order blurry velocity. The measurement of vibration of the loudspeaker driven by a function signal generator was employed to experimentally verify the velocity measurement performance of the system. The experiment demonstrated that the developed OCV can provide large velocity measurement ranges from static to 25.2 mm/s with nanometer-level precision and maximum measurable vibration frequency of up to 50 kHz. However, in theory, the theoretical maximum measurable velocity can be up to 1.06 m/s for current OCV configuration. The OCV has high precision, large dynamic range, and high-velocity measurement capability, making it attractive for applications in mechanical structure vibration monitoring and acoustic measurement.

Genome-Wide Association and Functional Studies Identify SCML4 and THSD7A as Novel Susceptibility Genes for Coronary Artery Disease.

OBJECTIVE: The genetic contribution to coronary artery disease (CAD) remains largely unclear. We combined genetic screening with functional characterizations to identify novel loci and candidate genes for CAD. APPROACH AND RESULTS: We performed genome-wide screening followed by multicenter validation in 8 cohorts consisting of 21 828 participants of Han ethnicity and identified 3 novel intragenic SNPs (single nucleotide polymorphisms), rs9486729 (SCML4 [Scm polycomb group protein-like 4]; odds ratio, 1.25; 95% CI, 1.17-1.34; P=3.51×10-11), rs17165136 (THSD7A [thrombospondin type 1 domain-containing 7A]; odds ratio 1.28; 95% CI, 1.21-1.35; P<1.00×10-25), and rs852787 (DAB1 [disabled-1]; odds ratio, 1.29; 95% CI, 1.21-1.38; P=2.02×10-14), associated with CAD with genome-wide significance. The risk allele of rs9486729 and protective allele of rs17165136 were associated with the decreased expression of their host genes, SCML4 and THSD7A, respectively, whereas rs852787 did not have transcriptional effects on any gene. Knockdown of SCML4 activated endothelial cells by increasing the expression of IL-6, E-selectin, and ICAM and weakened their antiapoptotic activity, whereas the knockdown of THSD7A had little effect on these endothelial cell functions but attenuated monocyte adhesion via decreasing the expression of ICAM, L-selectin, and ITGB2. We further showed that inhibiting the expression of SCML4 exacerbated endothelial dysfunction and vascular remodeling in a rat model with partial carotid ligation. CONCLUSIONS: We identify 3 novel loci associated with CAD and show that 2 genes, SCML4 and THSD7A, make functional contributions to atherosclerosis. How rs852787 and its host gene DAB1 are linked to CAD needs further studies.

Serum miR-146a and miR-150 as Potential New Biomarkers for Hip Fracture-Induced Acute Lung Injury.

BACKGROUND: Acute lung injury (ALI) and subsequent pulmonary infection are the most severe and usually fatal complications for elderly hip fracture patients. It is necessary to find some biomarkers for early diagnosis and prognosis of it. OBJECTIVE: This study is aimed at examining the differential expression of miR-146a, miR-150, and cytokines (IL-6 and IL-10) between younger and elderly rats suffering from hip fracture and investigating the possible meaning of them in early diagnosis and prognosis of ALI after hip fracture. METHODS AND SUBJECTS: Elderly rats and younger rats were randomly divided into sham group and fracture group, respectively. Two fracture groups received hip fracture operations. The damage degree of ALI was evaluated by histological observation and pathological score. Cytokines were measured by ELISA; miR-146a and miR-150 were analysed by qRT-PCR. RESULTS: After treatment, compared with the corresponding sham groups, the pulmonary histological score, the serum miR-146a concentrations, and the cytokine (IL-6 and IL-10) levels in serum and BALF were significantly higher (the miR-150 were lower) in the fracture groups (with the exception of IL-6 of the younger fracture group at 72 h, all P < 0.05). Meanwhile, compared with the younger fracture group, the aforementioned variables were significantly higher (the miR-150 levels were lower) in the elderly fracture group (with the exception of serum IL-10 and pulmonary histological score at 8 h, all P < 0.05). The results of linear regression analysis showed that serum miR-146a and miR-150 were significantly associated with pulmonary histological score. CONCLUSION: Hip fracture can result in significant systemic inflammation and ALI in the rats. Compared to the younger rats, the elderly rats suffered a more remarkable ALI after hip fracture. It may be related to the abnormal expression of miR-146a and miR-150. Serum miR-146a and miR-150 are potential biomarkers for diagnosis and prognosis of ALI after hip fracture.

Establishment of a sensitive time-resolved fluoroimmunoassay for detection of Bacillus thuringiensis Cry1Ie toxin based nanobody from a phage display library.

Cry1Ie toxin was an insect-resistant protein used in genetically modified crops (GMC). In this study, a large human VH gene nanobodies phage displayed library was employed to select anti-Cry1Ie toxin antibody by affinity panning. After 5 rounds of panning, total 12 positive monoclonal phage particles were obtained. One of the identified positive phage nanobody was expressed in E.coli BL21 and the purified protein was indicated as a molecular mass of approximately 20 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then a sensitive indirect competitive time-resolved fluoroimmunoassay (IC-TRFIA) was established for detection of Cry1Ie toxin by the purified protein. The working range of detection for Cry1Ie toxin standards in the IC-TRFIA were 0.08-6.44 ng mL-1 and the medium inhibition of control (IC50) was 0.73 ng mL-1. It showed a weak cross-reactivity with Cry1Ab toxin (at 5.6%), but did not recognize Cry1B, Cry1C, Cry1F, and Cry2A toxins (were <0.1%). The average recoveries of Cry1Ie toxin from respectively spiked in rice, corn and soil samples were in the range of 83.5%-96.6% and with a coefficient of variation (CV) among 2.0%-8.6%. These results showed the IC-TRFIA was promising for detection of Cry1Ie toxin in agricultural and environmental samples.

Simultaneous production of monoclonal antibodies against Bacillus thuringiensis (Bt) Cry1 toxins using a mixture immunization.

The detections of Cry1 toxins are mainly dependent on immunoassays based on specific monoclonal antibodies (mAb). In the present study, a mixture immunization with seven Cry1 toxins was administered. The results showed that five mAbs with different characteristics, especially one mAb named 5-E8 which could recognize all the seven Cry1 toxins were obtained. Based on the 5-E8 mAb, a double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) which can specifically detect the seven Cry1 toxins without cross-reactivity to Cry2A and vip3 was developed with the limit of detection (LOD) and limit of quantification (LOQ) of 6.37-11.35 ng mL-1 and 17.36-24.48 ng mL-1, respectively. The recovery tests showed that the recoveries ranged from 78% to 110% within the quantitation range (LOQ-100 ng mL-1). The established DAS-ELISA can be a useful tool for monitoring the Cry1 toxins in agricultural products. Mixture immunization opens a new path for producing diverse mAbs simultaneously in a single immunization circle.

Development of competitive ELISA for the detection of bovine serum albumin using single-chain variable fragments.

Soluble anti-bovine serum albumin (BSA) single-chain variable fragments (scFvs) were expressed in E. Coli. HB2151. The antigen-binding equilibrium dissociation constant of the scFvs was determined to be 2.9 × 10-8 M by surface plasmon resonance analysis. A competitive ELISA for the detection of BSA was developed using the antibody fragment above. The limits of detection (I10) and I50 were 0.002 and 0.74 µg/ml respectively, with a recovery between 87.8 and 119.2% in spiked milk samples. The assay has the potential to be used to detect concentration of BSA in milk or other matrix instead of the ELISA based on traditional antibodies.

Establishment of a sandwich enzyme-linked immunosorbent assay for specific detection of Bacillus thuringiensis (Bt) Cry1Ab toxin utilizing a monoclonal antibody produced with a novel hapten designed with molecular model.

Cry1Ab toxin is commonly expressed in genetically modified crops in order to control chewing pests. At present, the detection method with enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody cannot specifically detect Cry1Ab toxin for Cry1Ab's amino acid sequence and spatial structure are highly similar to Cry1Ac toxin. In this study, based on molecular design, a novel hapten polypeptide was synthesized and conjugated to keyhole limpet hemocyanin (KLH). Then, through animal immunization with this antigen, a monoclonal antibody named 2C12, showing high affinity to Cry1Ab and having no cross reaction with Cry1Ac, was produced. The equilibrium dissociation constant (K D) value of Cry1Ab toxin with MAb 2C12 was 1.947 × 10-8 M. Based on this specific monoclonal antibody, a sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for the specific determination of Cry1Ab toxin and the LOD and LOQ values were determined as 0.47 ± 0.11 and 2.43 ± 0.19 ng mL-1, respectively. The average recoveries of Cry1Ab from spiked rice leaf and rice flour samples ranged from 75 to 115%, with coefficient of variation (CV) less than 8.6% within the quantitation range (2.5-100 ng mL-1), showing good accuracy for the quantitative detection of Cry1Ab toxin in agricultural samples. In conclusion, this study provides a new approach for the production of high specific antibody and the newly developed DAS-ELISA is a useful method for Cry1Ab monitoring in agriculture products. Graphical Abstract Establishment of a DAS-ELISA for the specific detecting of Bacillus thuringiensis (Bt) Cry1Ab toxin.

Production and Characterization of Monoclonal Antibody Broadly Recognizing Cry1 Toxins by Use of Designed Polypeptide as Hapten.

In this study, by use of synthesized polypeptides as haptens, a monoclonal antibody with broad recognition against seven major Cry1 toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1E, and Cry1F) has been produced and characterized. First, by comparing the three-dimensional structures of seven Cry1 toxins, analyzing the conserved sequences, and considering the antigenicity and hydrophilicity, three polypeptides (T1, T2, and T3) have been chosen and coupled to keyhole limpet hemocyanin as immunogens for the generic monoclonal antibody (Mab) generation. Thereafter, a double antibody sandwich enzyme-linked immunosorbent assay method (DAS-ELISA) was developed for simultaneous determination of seven Cry1 toxins. The results revealed that the haptens T1, T2, and T3 had different effects in the production of antibodies. Among them, the obtained Mab (strain 2D3) generated by T2 can recognize seven Cry1 toxins simultaneously. Equilibrium dissociation constant (KD) values for seven Cry1 toxins with Mab 2D3 were 1.198 × 10(-8) M for Cry1Aa, 2.197 × 10(-8) M for Cry1Ab, 1.367 × 10(-8) M for Cry1Ac, 2.092 × 10(-8) M for Cry1B, 5.177 × 10(-8) M for Cry1C, 4.016 × 10(-8) M for Cry1E, and 3.497 × 10(-8) M for Cry1F. For 2D3-based DAS-ELISA, the limits of detection (LOD) and limits of quantification (LOQ) can reach 15 and 30 ng·mL(-1) for each Cry1 toxin, respectively. Our study is the first report of a broadly specific immunoassay for multidetermination of seven major Cry1 toxins, and it will provide a new idea and technical routes for development of multidetermination immunoassays.

Motor recovery at 6 months after admission is related to structural and functional reorganization of the spine and brain in patients with spinal cord injury.

This study aimed to explore structural and functional reorganization of the brain in the early stages of spinal cord injury (SCI) and identify brain areas that contribute to motor recovery. We studied 25 patients with SCI, including 10 with good motor recovery and 15 with poor motor recovery, along with 25 matched healthy controls. The mean period post-SCI was 9.2 ± 3.5 weeks in good recoverers and 8.8 ± 2.6 weeks in poor recoverers. All participants underwent structural and functional MRI on a 3-T magnetic resonance system. We evaluated differences in cross-sectional spinal cord area at the C2/C3 level, brain cortical thickness, white matter microstructure, and functional connectivity during the resting state among the three groups. We also evaluated associations between structural and functional reorganization and the rate of motor recovery. After SCI, compared with good recoverers, poor recoverers had a significantly decreased cross-sectional spinal cord area, cortical thickness in the right supplementary motor area and premotor cortex, and fractional anisotropy (FA) in the right primary motor cortex and posterior limb of the internal capsule. Meanwhile, poor recoverers showed decreased functional connectivity between the primary motor cortex and higher order motor areas (supplementary motor area and premotor cortex), while good recoverers showed increased functional connectivity among these regions. The structural and functional reorganization of the spine and brain was associated with motor recovery rate in all SCI patients. In conclusion, structural and functional reorganization of the spine and brain directly affected the motor recovery of SCI. Less structural atrophy and enhanced functional connectivity are associated with good motor recovery in patients with SCI. Multimodal imaging has the potential to predict motor recovery in the early stage of SCI. Hum Brain Mapp 37:2195-2209, 2016. © 2016 Wiley Periodicals, Inc.

Selection and application of broad-specificity human domain antibody for simultaneous detection of Bt Cry toxins.

Bt Cry toxin is a kind of bio-toxins that used for genetically modified crops (GMC) transformation widely. In this study, total 15 positive clones could bind the Bt Cry toxins which isolated from a human domain antibody library by 5 rounds affinity selection. According to analyzing of PCR amplification and enzyme-linked immunosorbent assay (ELISA), the most positive phage domain antibody (named F5) gene was cloned into the pET26b vector and expressed in E. coli BL21. The purified antibody was used to develop an indirect competitive ELISA (IC-ELISA) for Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F toxins, respectively. The working range of detection for standard curves in IC-ELISA were 0.258-1.407 µg/mL, the medium inhibition concentration (IC50) were 0.727-0.892 µg/mL and detection limit (IC10) were 0.029-0.074 µg/mL for those Bt Cry toxins. The affinity of F5 domain antibody with Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F toxins were 1.21-5.94 × 10(7) M(-1). The average recoveries of the 5 kinds of Bt Cry toxins from spiked wheat samples were ranged from 81.2%-100.8% with a CV at 2.5%-9.4%. The results showed that we successfully obtained the broad-specificity human domain antibody for simultaneous detection of Bt Cry toxins in agricultural product samples.