Characterization of the individual domains of the Bacillus thuringiensis Cry2Aa implicates Domain I as a possible binding site to Helicoverpa armigera.

Bacillus thuringiensis (Bt) Cry2Aa is a member of the Cry pore-forming, 3-domain, toxin family with activity against both lepidopteran and dipteran insects. Although domains II and III of the Cry toxins are believed to represent the primary specificity determinant through specific binding to cell receptors, it has been proposed that the pore-forming domain I of Cry2Aa also has such a role. Thus, a greater understanding of the functions of Cry2Aa's different domains could potentially be helpful in the rational design of improved toxins. In this work, cry2Aa and its domain fragments (DI, DII, DIII, DI-II and DII-DIII) were subcloned into the vector pGEX-6P-1 and expressed in Escherichia coli. Each protein was recognized by anti-Cry2Aa antibodies and, except for the DII fragment, could block binding of the antibody to Cry2Aa. Cry2Aa and its DI and DI-II fragments bound to brush border membrane vesicles (BBMV) from H. armigera and also to a ca 150 kDa BBMV protein on a far western (ligand) blot. In contrast the DII, DIII and DII-III fragments bound to neither of these. None of the fragments were stable in H. armigera gut juice nor showed any toxicity towards this insect. Our results indicate that contrary to the general model of Cry toxin activity domain I plays a role in the binding of the toxin to the insect midgut.

PM2.5-induced premature senescence in HUVECs through the SIRT1/PGC-1α/SIRT3 pathway.

Vascular endothelial cell senescence plays a pivotal role in the development of atherosclerosis. Recent studies have demonstrated that ambient fine particulate matter (PM2.5) induces stress-induced premature senescence (SIPS) in vascular endothelial cells. However, the precise mechanisms underlying this process remain to be fully elucidated. Cellular senescence is closely associated with reactive oxygen species (ROS), and emerging research has established a strong connection between the SIRT1/PGC-1α/SIRT3 signaling pathway and the antioxidant system in vascular endothelial cells. In this study, we aimed to investigate the impact of PM2.5 on vascular endothelial cell senescence and to elucidate the underlying mechanisms. Our findings revealed that PM2.5 exposure led to an increase in senescence-associated ß-galactosidase (SA-ß-gal) activity and the expression of the cell cycle-blocking proteins P53/P21 and P16 in human umbilical vein endothelial cells (HUVECs). Flow cytometry analysis demonstrated an elevated proportion of cells arrested in the G0/G1 phase after PM2.5 exposure. In addition, PM2.5-induced cellular senescence was attributed to the disruption of the cellular antioxidative defense system through the SIRT1/PGC-1α/SIRT3 signaling pathway. The expression of cellular senescence markers was reduced after targeted scavenging of mitochondrial ROS using MitoQ. Moreover, treatment with SRT1720, a SIRT1-specific activator, upregulated the SIRT1/PGC-1α/SIRT3 signaling pathway, restored the antioxidant system, and attenuated the expression of cellular senescence markers. Taken together, our results suggest that PM2.5 downregulates the SIRT1/PGC-1α/SIRT3 signaling pathway, resulting in impaired antioxidant defenses in HUVECs. This, in turn, allows for the accumulation of ROS, leading to inhibition of endothelial cell cycle progression and the onset of stress-induced senescence in HUVECs.

Screening and Identification of Anti-Idiotypic Nanobody Capable of Broad-Spectrum Recognition of the Toxin Binding Region of Lepidopteran Cadherins and Mimicking Domain II of Cry2Aa Toxin.

The widespread use of Bacillus thuringiensis toxins as insecticides has brought about resistance problems. Anti-idiotypic nanobody approaches provide new strategies for resistance management and toxin evolution. In this study, the monoclonal antibody generated against the receptor binding region Domain II of Cry2Aa toxin was used as a target to screen materials with insecticidal activity. After four rounds of screening, anti-idiotypic nanobody 1C12 was obtained from the natural alpaca nanobody phage display library. To better analyze the activity of 1C12, soluble 1C12 was expressed by the Escherichia coli BL21 (DE3). The results showed that 1C12 not only binds the midgut brush border membrane vesicles (BBMV) of two lepidopteran species and cadherin CR9-CR11 of three lepidopteran species but also inhibits Cry2Aa toxins from binding to CR9-CR11. The insect bioassay showed that soluble 1C12 caused 25.65% and 23.61% larvae mortality of Helicoverpa armigera and Plutella xylostella, respectively. Although 1C12 has low insecticidal activity, soluble 1C12 possesses the ability to screen a broad-spectrum recognition of the toxin binding region of lepidopteran cadherins and can be used for the identification of the toxin binding region of other lepidopteran cadherins and the subsequent evolution of Cry2Aa toxin. The present study demonstrates a new strategy to screen for the production of novel insecticides.

Establishment of novel receptor-antibody sandwich assays to broadly detect Bacillus thuringiensis Cry1 and Cry2 toxins.

Bacillus thuringiensis (Bt) Cry toxins have been widely used in the development of genetically modified organisms (GMOs) for pest control. This work aimed to establish more cost effective and broader detection methods for commonly used Cry toxins. Using ligand blot and bio-layer interferometry, we confirmed that a recombinant toxin-binding fragments derived from Helicoverpa armigera cadherin-like protein (HaCad-TBR) could broadly bind Cry1Ab, Cry1Ac, Cry2Aa, and Cry2Ab with the affinity of 0.149, 0.402, 120, and 4.12 nM, respectively. Based on the affinity results, a novel receptor-antibody sandwich assay broadly detecting Cry1A and Cry2 toxins was developed by using HaCad-TBR as capture molecules, and anti-Cry1A/Cry2A polyclonal antibodies (pAbs) as the detection antibodies. The detection limit (LOD) for Cry1Ab, Cry1Ab, Cry2Aa, and Cry2Ab were 5.30, 5.75, 30.83 and 13.70 ng/mL. To distinguish Cry1A and Cry2A toxins in a singular test, anti-Cry1A pAbs and anti-Cry2A pAbs were labelled with different quantum dots (QDs). The LOD for the four toxins by receptor-QDs-pAbs sandwich assay were calculated to be 1.36, 4.71, 17.48, and 7.54 ng/mL, respectively. The two developed methods were validated by spiked rice and corn samples, suggesting they may potentially be used in monitoring and quantifying Cry toxins in food and environment.

Generation of anti-idiotypic antibodies mimicking Cry2Aa toxin from an immunized mouse phage display library as potential insecticidal agents against Plutella xylostella.

This study tried to generate anti-idiotypic antibodies (Ab2s) which mimic Cry2Aa toxin using a phage-display antibody library (2.8 × 107 CFU/mL). The latter was constructed from a mouse immunized with F (ab')2 fragments digested from anti-Cry2Aa polyclonal antibodies. The F (ab')2 fragments and Plutella xylostella (P. xylostella) brush border membrane vesicles (BBMV) were utilized as targets for selection. Eight mouse phage-display single-chain variable fragments (scFvs) were isolated and identified by enzyme-linked immunoassay (ELISA), PCR and DNA sequencing after four rounds of biopanning. Among them, M3 exhibited the highest binding affinity with F (ab')2, while M4 bound the best with the toxin binding region of cadherin of P. xylostella (PxCad-TBR). Both of these two fragments were chosen for prokaryotic expression. The expressed M3 and M4 proteins with molecular weights of 30 kDa were purified. The M4 showed a binding affinity of 29.9 ± 2.4 nM with the PxCad-TBR and resulted in 27.8 ± 4.3 % larvae mortality against P. xylostella. Computer-assisted molecular modeling and docking analysis showed that mouse scFv M4 mimicked some Cry2Aa toxin binding sites when interacting with PxCad-TBR. Therefore, anti-idiotypic antibodies generated by BBMV-based screening could be useful for the development of new bio-insecticides as an alternative to Cry2Aa toxin for pest control.

Self-expansion full information optimization strategy: Convenient and efficient method for near infrared spectrum auto-analysis.

An essential step in the application of near infrared spectroscopy technology is the spectrum preprocessing. A reasonable implementation of it ensures that the effective spectral information is correctly extracted and, also that the model's accuracy is increased. However, some analysts' research still uses the manual approach of trial and error, particularly those less skilled ones. Previous papers have provided preprocessing optimization algorithms for NIR, but there are still some problems that need to be resolved, such as the unwieldy sequence determination of preprocessing method or, the fluctuated optimization outcomes or, lack of sufficient statistical information. This research suggests a spectrum auto-analysis methodology named self-expansion full information optimization strategy, a new powerful open-source technique for concurrently addressing all of these above issues simultaneously. For the first time in the field of chemometrics, this algorithm offers a reliable and effective automatic near infrared auto-modelling method based on the statistical informatics. With the aid of its built-in modules, such as information generators, spectrum processors, etc., it is able to fully search the common preprocessing techniques, which is determined by Monte Carlo cross validation. Then the final ensemble calibration model is built by employing the optimized preprocessing schemes, along with the wavelength variables screening algorithm. The optimization strategy can offer the user objective useful statistics information created throughout the modeling process to further examine the model's effectiveness. The results demonstrate that the suggested method can easily and successfully extract spectrum information and develop calibration models by putting it to the test on two groups of actual near-infrared spectral data. Additionally, this optimization strategy can also be applied to other spectrum analysis areas, such Raman spectroscopy or infrared spectroscopy, by changing a few of its parameters, and has extraordinary application value.

Establishment of monoclonal antibody and scFv immuno-based assay for Cry2Aa toxin in spiked grain samples.

Bacillus thuringiensis (Bt) Cry toxins have been widely used in the development of genetically modified organisms (GMOs) for pest control. This work aimed to establish more cost effective methods for used Cry2Aa toxins. Three immunoassay methods (IC-ELISA, DAS-ELISA, and CLEIA) were successfully developed in this work. The mAb was used as the detecting antibody, for the IC-ELISA, the range of IC20 to IC80 was 1.11 µg/mL - 60.70 µg/mL, and an IC50 of 10.65 µg/mL. For the DAS-ELISA, the limit of detection (LOD) and limit of quantitation (LOQ) were 10.76 ng/mL and 20.70 ng/mL, respectively. For the CLEIA, the LOD and LOQ were 6.17 ng/mL and 7.40 ng/mL, respectively. The scFv-based detections were the most sensitive for detecting Cry2Aa. The LOD and LOQ for the DAS-ELISA were 118.75 ng/mL and 633.48 ng/mL, respectively. The LOD and LOQ for the CLEIA, read as 37.47 ng/mL and 70.23 ng/mL, respectively. The fact that Cry2Aa toxin was recovered in spiked grain samples further demonstrated that the approaches might be used to identify field samples. These methods provided good sensitivity, stability, and applicability for detecting Cry2Aa toxin, promising ultrasensitive monitoring and references for Cry toxins risk assessment.

Development of a micro-Raman system for in vivo studying the mechanism of laser biological effects.

The laser irradiation on organism will produce a series of biological effects, which can be used for basic medical research, diagnosis and treatments of diseases. However, the mechanism of this biological effects is still unclear. As a sensitive molecular monitoring technique, Raman spectroscopy has became a very popular detection method in biomedical research especially in vivo study. In this paper, we present a compact and flexible micro-Raman system for in vivo studying the mechanism of laser biological effects. The system has the two functions of laser induction and Raman measurement, which can realize the micro-area radiation of laser and simultaneously collect the corresponding Raman spectra in vivo. The detection method provided by this home-built system is able to deepen the understanding of laser biological effects mechanism at molecular level, so it is expected that the system is significant for the treatments and diagnosis of diseases in the near future.

Improved sensitivity of the anti-microcystin-LR ELISA using phage-displayed alpha-type anti-idiotypic nanobody.

Anti-idiotypic antibodies (Ab2) are valuable tools that can be used for a better understanding of molecular mimicry and the immunological network. In this work, we showed a new application of a phage-displayed alpha-type Ab2 (Ab2α) to improve the sensitivity of an enzyme-linked immunosorbent assay (ELISA) detecting cyanobacterial toxin microcystin-LR (MC-LR). A monoclonal antibody (mAb) against MC-LR was used as an antigen to isolate binders in a camelid nanobody library. After three rounds of panning, three unique clones with strong binding against anti-MC-LR mAbs were isolated. These clones could specifically bind to anti-MC-LR mAbs without influencing mAbs binding with MC-LR, meaning these clones were Ab2αs. Based on the signal amplification effect of phage coat proteins and the non-competitive nature of Ab2α, a novel competitive ELISA method for MC-LR was established with a phage-displayed Ab2α. It showed that the phage-displayed Ab2α greatly enhanced the ELISA signal and sensitivity of the method was improved 3.5-fold to the conventional one. Combining with the optimization of pre-incubation time, the optimized ELISA decreased its limit of detection (LOD) from 4.5 ng/mL to 0.8 ng/mL (5.6-fold improvement). This new application of Ab2α may potentially be employed to improve the sensitivity of immunoassays for other environmental pollutants.

Identifying the Epitopes of Bacillus thuringiensis Cry2Aa Toxin Involved in Cadherin Interaction by a Monoclonal Antibody.

Antibodies are a useful tool for assistance to map the binding epitopes in Bacillus thuringiensis Cry toxins and their receptors, and even determine how receptors promote toxicity. In this work, a monoclonal antibody (mAb-1D2) was produced by the hybridoma cell line raised against Cry2Aa toxins, with a half inhibition concentration (IC50) of 9.16 µg/mL. The affinity constant of two recombinant toxin-binding fragments derived from Helicoverpa armigera and Plutella xylostella cadherin-like protein (HaCad-TBR or PxCad-TBR) to Cry2Aa toxin was measured to be 1.21 µM and 1.24 µM, respectively. Competitive ELISA showed that mAb-1D2 competed with HaCad-TBR or PxCad-TBR binding to Cry2Aa. Meanwhile, the toxicity of the Cry2Aa toxin to the H. armigera and P. xylostella larvae were greatly reduced when the toxin was mixed with mAb-1D2, which indicated that cadherin may play an important functional role in the toxicity of Cry2Aa. After transforming mAb-1D2 to a single-chain variable fragment (scFv), the hot spot residues of Cry2Aa with 1D2-scFv, PxCad-TBR, and HaCad-TBR were analyzed by molecular docking. It was demonstrated that the hot spot residues of Cry2Aa involving with 1D2-scFv interaction were mainly in Domain II, and some residues in Domain I. Moreover, mAb-1D2 and the two cadherin fragments shared the common hot spot residues on Cry2Aa, which could explain mAb-1D2 inhibited Cry2Aa binding with cadherin fragments. This monoclonal antibody could be a useful tool for identifying the binding epitopes between Cry2Aa and cadherin, and even assist to analyze the roles of cadherin in Cry2Aa toxicity.

Generation of Human Domain Antibody Fragments as Potential Insecticidal Agents against Helicoverpa armigera by Cadherin-Based Screening.

New insecticidal genes and approaches for pest control are a hot research area. In the present study, we explored a novel strategy for the generation of insecticidal proteins. The midgut cadherin of Helicoverpa armigera (H. armigera) was used as a target to screen materials that have insecticidal activity. After three rounds of panning, the phage-displayed human domain antibody B1F6, which not only binds to the H. armigera cadherin CR9-CR11 but also significantly inhibits Cry1Ac toxins from binding to CR9-CR11, was obtained from a phage-displayed human domain antibody (DAb) library. To better analyze the relevant activity of B1F6, soluble B1F6 protein was expressed by Escherichia coli BL21 (DE3). The cytotoxicity assays demonstrated that soluble B1F6 induced Sf9 cell death when expressing H. armigera cadherin on the cell membrane. The insect bioassay results showed that soluble B1F6 protein (90 µg/cm2) caused 49.5 ± 3.3% H. armigera larvae mortality. The midgut histological results showed that soluble B1F6 caused damage to the midgut epithelium of H. armigera larvae. The present study explored a new strategy and provided a basic material for the generation of new insecticidal materials.

A powerful tool for near-infrared spectroscopy: Synergy adaptive moving window algorithm based on the immune support vector machine.

Traditional trial-and-error methods are time-consuming and inefficient, especially very unfriendly to inexperienced analysts, and are sometimes still used to select preprocessing methods or wavelength variables in near-infrared spectroscopy (NIR). To deal with this problem, a new optimization algorithm called synergy adaptive moving window algorithm based on the immune support vector machine (SA-MW-ISVM) is proposed in this paper. Following the principle of SA-MW-ISVM, the original problem of calibration model optimization is transformed into a mathematical optimization problem that can be processed by the proposed immune support vector machine regression algorithm. The main objective of this optimization problem is the calibration model performance; meanwhile, the constraint conditions include a reasonable spectral data value, spectral data preprocessing method, and calibration model parameters. A unique antibody structure and specific coding and decoding method are used to achieve collaborative optimization in NIR spectroscopy. The tests on four actual near-infrared datasets, including a group of gasoline and three groups of diesel fuels, have shown that the proposed SA-MW-ISVM algorithm can significantly improve the calibration performance and thus achieve accurate prediction results. In the case of gasoline, the SA-MW-ISVM algorithm can decrease the prediction error by 44.09% compared with the common benchmark partial least square (PLS). Meanwhile, in the case of diesel fuels, the SA-MW-ISVM algorithm can decrease the prediction error of cetane number, freezing temperature, and viscosity by 9.99%, 28.69%, and 43.85%, respectively, compared with the PLS. The powerful prediction performance of the SA-MW-ISVM algorithm makes it an ideal tool for modeling near-infrared spectral data or other related application fields.

Laser tweezers Raman spectroscopy combined with machine learning for diagnosis of Alzheimer's disease.

Alzheimer's disease (AD) is a common nervous system disease to affect mostly elderly people over the age of 65 years. However, the diagnosis of AD is mainly depend on the imaging examination, clinical assessments and neuropsychological tests, which may get error diagnosis results and are not able to detect early AD. Here, a rapid, non-invasive, and high accuracy diagnostic method for AD especially early AD is provided based on the laser tweezers Raman spectroscopy (LTRS) combined with machine learning algorithms. AD platelets from different 3xTg-AD transgenic rats at different stages of disease are captured to collect high signal-to-noise ratio Raman signals without contact by LTRS, which is then combined with partial least squares discriminant analysis (PLS-DA), support vector machine (SVM) and principal component analysis (PCA)-canonical discriminate function (CDA) for classification. The results show that the normal and diseased platelets at 3-, 6- and 12-month AD are successfully distinguished and the accuracy is 91%, 68% and 97% respectively, which demonstrates the suggested method can provide a precise detection for AD diagnosis at early, middle and advanced stages.

Screening and identification of vancomycin anti-idiotypic antibodies for against Staphylococcus aureus from a human phage display domain antibody library.

Staphylococcus aureus is a common food-borne pathogenic microorganism that poses a serious threat to food quality and safety, and can do harm to human health. In the past, researchers relied on antibiotics to control Staphylococcus aureus, though very effective, yet it was also worrying in the aspect of bio-safety. In fact, anti-idiotypic antibody (Anti-Id) shows its potential to mimic some of the structural and biological functions of antigens. Therefore, in this study, based on Anti-Id theory and technology, we expect to obtain the vancomycin Anti-Id which can mimic vancomycin against Staphylococcus aureus from a human phage display domain antibody library. After four rounds of bio-panning, a total of 18 positive Anti-Ids were obtained. Among them, two Anti-Ids named Anti-Id-2C12 and Anti-Id-1F5 were identified as "ß" type Anti-Ids, and afterwards they were selected for gene cloning and protein expression in prokaryotic expression system. As a result, a concentration of purified proteins with 568.6 µg/mL (Anti-Id-2C12) and 602.3 µg/mL (Anti-Id-1F5) were successfully obtained, and their minimum inhibitory concentration (MIC) values for Staphylococcus aureus were 125 and 200 µg/mL, respectively. As they are human heavy-chain domain antibodies, which were theoretically harmless to humans, they have the potential application value as preservatives in food and edible agricultural products.

Identification of single domain antibodies with insect cytotoxicity using phage-display antibody library screening and Plutella xylostella ATP-binding cassette transporter subfamily C member 2 (ABCC2) -based insect cell expression system.

It is extremely imminent to study a new strategy to manage agricultural pest like Plutella xylostella (P. xylostella) which is currently resistant to most of pesticides, including three domain-Cry toxins from Bacillus thuringiensis (Bt). In this study, we reported a phage displayed single domain antibody screening from human domain antibody (DAb) library targeted on Spodoptera frugiperda 9 (Sf9) cells expressed Cry1Ac toxin receptor, ATP-dependent binding cassette transporter C2 in P. xylostella (PxABCC2). After three rounds of panning, three cytotoxic antibodies (1D2, 2B7, 3C4) were obtained from thirty-eight antibodies and displayed high binding ability towards PxABCC2-expressed Sf9 cells. Through homology modeling and molecular docking, the interaction mode indicated that the most cytotoxic 1D2 of the three antibodies presented the lowest binding free energy required and had the most hydrogen bond formed with PxABCC2 in molecular docking analysis. Functional assay of key regions in 1D2 via Alanine replacement indicated that complementarity-determining region (CDR) 3 played a crucial role in antibody exerts binding activity and cytotoxicity. This study provides the first trial for discovering of potential cytotoxic antibodies from the human antibody library via specific receptor-expressed insect cell system biopanning.

LINC00518 affects the proliferation, invasion and migration of cutaneous malignant melanoma cells via miR-526b-3p/EIF5A2 axis.

OBJECTIVE: To investigate the mechanism of LINC00518 affecting the proliferation, invasion, and migration of cutaneous malignant melanoma (CMM) cells via miR-526b-3p/EIF5A2 axis. METHODS: qRT-PCR was performed to measure the expression of LINC00518, miR-526b-3p, and EIF5A2 in CMM tissues from 40 patients. Si-LINC00518, pcDNA-LINC00518, miR-526b-3p mimic, miR-526b-3p inhibitor, si-EIF5A2, and their corresponding negative controls were transfected alone or co-transfected into CMM cells A375 and A2058. The expression of LINC00518, miR-526b-3p and EIF5A2 in A375 and A2058 cells was measured. Cell proliferation was tested by CCK-8 assay and EdU assay. Cell invasion and migration were detected by Transwell and scratch tests, respectively. The binding between LINC00518 and miR-526b-3p, and the binding between miR-526b-3p and EIF5A2 were verified by dual-luciferase reporter and RNA pull-down assays. RESULTS: LINC00518 and EIF5A2 were up-regulated and miR-526b-3p was down-regulated in CMM tissues and cells. CMM patients with highly expressed LINC00518 showed decreased survival time than those with lowly expressed LINC00518. Transfection of si-LINC00518, miR-526b-5p mimic or si-EIF5A2 weakened the proliferative, migratory, and invasive abilities of melanoma cells, while transfection of miR-526b-5p inhibitor or pcDNA-LINC00518 enhanced the progression of melanoma cells. Moreover, the proliferative, migratory, and invasive potentials of melanoma cells were decreased after co-transfection of si-EIF5A2 and pcDNA-LINC00518 compared with cells transfected with pcDNA-LINC00518 alone. LINC00518 bound to miR-526b-3p and miR-526b-3p targeted EIF5A2. LINC00518 negatively regulated miR-526b-3p expression but positively regulated EIF5A2. Furthermore, EIF5A2 expression was negatively associated with miR-526b-3p expression. CONCLUSION: LINC00518 encourages CMM through the miR-526b-3p/EIF5A2 axis in terms of cell proliferation, invasion, and migration.

Epitopes prediction for microcystin-LR by molecular docking.

Microcystin-LR (MC-LR) is one of the most worldwide harmful cyanobacterial toxins. A lots of antibodies against MC-LR have been generated and characterized. However, the knowledge about the epitopes of MC-LR was still limited. The objective of this study was to analyze the epitopes of MC-LR and demonstrate the binding mode of MC-LR with its antibody. The variable genes of a mouse hybridoma cell line (Mab5H1-3B3) raised against MC-LR have been cloned and assembled in a single chain variable fragment (scFv), and then soluble expressed in E.coli BL21. Based on the scFv, the IC50 and IC10 for MC-LR were determined to be 7.45 nM and 0.30 nM by competitive ELISA. And the scFv also showed 115% and 112% cross-reactivities to MC-RR and MC-YR, and 59% to MC-LA. By molecular docking, the binding mode between MC-LR and its scFv was demonstrated. A hydrogen bond interaction was observed between the carbonyl group of Adda5 residue of MC-LR and its scFv, and the guanidyl group of Arg4 residue and phenyl group of Adda5 residue of MC-LR were also involved in the interaction. These predicted epitopes were supported by antibody cross-reactivity data. By comparing the antibody informatics of MC-LR scFv with its predicted paratopes, VH-CDR1 was crucial for MC-LR binding, and its specificity could be tuned by engineering in Vκ-CDR1 and Vκ-CDR3. These information would be useful for the hapten design for microcystins or improving the properties of MC-LR scFv in vitro.

Docking-based generation of antibodies mimicking Cry1A/1B protein binding sites as potential insecticidal agents against diamondback moth (Plutella xylostella).

BACKGROUND: Broad use of insecticidal Cry proteins from Bacillus thuringiensis in biopesticides and transgenic crops has resulted in cases of practical field resistance, highlighting the need for novel approaches to insect control. Previously we described an anti-Cry1Ab idiotypic-antibody (B12-scFv) displaying toxicity against rice leafroller (Cnaphalocrocis medinalis) larvae, supporting the potential of antibodies for pest control. The goal of the present study was to generate insecticidal antibodies against diamondback moth (Plutella xylostella) larvae. RESULTS: Four genetically engineered antibodies (GEAbs) were designed in silico from B12-scFv using three-dimensional (3D) structure and docking predictions to alkaline phosphatase (ALP) as a Cry1Ac receptor in P. xylostella. Among these GEAbs, the GEAb-dVL antibody consisting of two light chains had overlapping binding sites with Cry1A and Cry1B proteins and displayed high binding affinity to P. xylostella midgut brush border membrane (BBM) proteins. Proteins in BBM identified by pull-down assays as binding to GEAb-dVL included an ABC transporter and V-ATPase subunit A protein. Despite lacking the α-helical structures in Cry1A that are responsible for pore formation, ingestion of GEAb-dVL disrupted the P. xylostella larval midgut epithelium and resulted in toxicity. Apoptotic genes were activated in gut cells upon treatment with GEAb-dVL . CONCLUSION: This study describes the first insecticidal GEAb targeting P. xylostella by mimicking Cry proteins. Data support that GEAb-dVL toxicity is associated to activation of intracellular cell death pathways, in contrast to pore-formation associated toxicity of Cry proteins. This work provides a foundation for the design of novel insecticidal antibodies for insect control. © 2021 Society of Chemical Industry.

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.

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.