The bacterium produces several insecticidal proteins, such as the crystal proteins (Cry) and the vegetative insecticidal proteins (Vip). In this work, we report that a specific interaction between two toxins creates insecticidal synergism and unravel the molecular basis of this interaction. When applied together, the three-domain Cry toxin Cry9Aa and the Vip Vip3Aa exhibited high insecticidal activity against an important insect pest, the Asiatic rice borer (). We found that these two proteins bind specifically to brush border membrane vesicles of and that they do not share binding sites because no binding competition was observed between them. Binding assays revealed that the Cry9Aa and Vip3Aa proteins interacted with high affinity. We mapped their specific interacting regions by analyzing binding of Cry9Aa to overlapping fragments of Vip3Aa and by analyzing binding of Vip3Aa to individual domains of Cry9Aa. Binding to peptide arrays helped narrow the binding sites to domain II loop-3 of Cry9Aa and toTKKMKTL in Vip3Aa. Site-directed mutagenesis confirmed that these binding regions participate in binding that directly correlates with the synergism between the two proteins. In summary, we show that the Cry9Aa and Vip3Aa toxins display potent synergy based on a specific interaction between them. Our results further our understanding of the complex synergistic activities among toxins and are highly relevant to the development of toxin combinations for effective insect control and for delaying development of insect resistance.

Cry1Ac toxin‐binding proteins from Helicoverpa armigera brush border membrane vesicles were identified by an improved pull‐down method that involves coupling Cry1Ac to CNBr agarose combined with liquid chromatography–tandem mass spectrometry (LC‐MS/MS). According to the LC‐MS/MS results, Cry1Ac toxin could bind to six classes of aminopeptidase‐N, alkaline phosphatase, cadherin‐like protein, ATP‐binding cassette transporter subfamily C protein (ABCC2), actin, ATPase, polycalin, and some other proteins not previously characterized as Cry toxin‐binding molecules such as dipeptidyl peptidase or carboxyl/choline esterase and some serine proteases. This is the first report that suggests the direct binding of Cry1Ac toxin to ABCC2 in H. armigera.

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Bacillus thuringiensis Cry1Ah protein is highly toxic against Helicoverpa armigera but shows no toxicity against Bombyx mori larvae. In contrast, the closely related Cry1Ai toxin showed the opposite phenotype: high activity against B. mori but no toxicity against H. armigera. Analysis of binding of Cry1Ah to brush border membrane vesicle (BBMV) proteins from H. armigera and B. mori by surface plasmon resonance revealed association of toxin binding with insect specificity. Pulldown experiments identified aminopeptidase N1 (APN1) as a Cry1Ah binding protein that was not observed in the assays using B. mori BBMV proteins. The APN1 Cry1Ah binding region was narrowed to the region from A 548 to S 798 (fragment H3) by expressing four different APN1 fragments in Escherichia coli and analyzing Cry1Ah binding by ligand blot. Binding competition experiments of Cry1Ah to APN1 fragment H3 using synthetic peptides corresponding to four predicted domain II loop regions showed that loop 2 and loop 3 have additive effects on binding to APN1 fragment H3. Moreover, switching of loop 2 and loop 3 regions from Cry1Ah to Cry1Ai toxins showed that loop 2 and loop 3 are both involved in specificity and toxicity against H. armigera. IMPORTANCE Domain II loop regions have been shown to be involved in binding to larval gut proteins mediating insect specificity. The modification of loop regions is a direct and effective method to construct new Cry toxin variants to increase toxicity or modify specificity. Our results show that the exchange of loop regions from one toxin into another is a successful scheme for modification of B. thuringiensis Cry toxin specificity.

A chimeric antigen receptor (CAR) is a type of fusion protein that comprises an antigen-recognition domain and signaling domains. In the present study, a programmed death-ligand 1 (PD-L1)-specific CAR, comprised of a single-chain variable fragment (scFv) derived from a monoclonal antibody, co-stimulatory domains of cluster of differentiation (CD) 28 and 4-1BB and a T-cell-activation domain derived from CD3ζ, was designed. The construction was cloned and packaged into the lentiviral vector pLVX. Flow cytometry confirmed that peripheral blood mononuclear cells were efficiently transduced and that the CAR was successfully expressed on T cells. The cytotoxicity of transduced T cells was detected using PD-L1-positive NCI-H358 bronchioalveolar carcinoma cells and A549 lung adenocarcinoma cells (with a low expression of PD-L1, only in the A549 cells). The results demonstrated mild cytotoxicity at an effector-to-target ratio of 10:1. An ELISA revealed a significant increase in the level of interferon-γ released from T cells transduced with scFv-28Bz when the cells were co-cultured with PD-L1-positive NCI-H358 cells, while interkeukin-2 and tumor necrosis factor-α levels remained unchanged. These data indicated a potential method for the treatment of solid tumors.

The cry1-type genes of Bacillus thuringiensis represent the largest cry gene family, which contains 50 distinct holotypes. It is becoming more and more difficult to identify cry1-type genes using current methods because of the increasing number of cry1-type genes. In the present study, an improved PCR-restriction fragment length polymorphism (PCR-RFLP) method which can distinguish 41 holotypes of cry1-type genes was developed. This improved method was used to identify cry1-type genes in 20 B. thuringiensis strains that are toxic to lepidoptera. The results showed that the improved method can efficiently identify single and clustered cry1-type genes and can be used to evaluate cry1-type genes in novel strain collections of B. thuringiensis. Among the detected cry1-type genes, we identified four novel genes, cry1Ai, cry1Bb, cry1Ja, and cry1La. The bioassay results from the expressed products of the four novel cry genes showed that Cry1Ai2, Cry1Bb2, and Cry1Ja2 were highly toxic against Plutella xylostella, whereas Cry1La2 exhibited no activity. Moreover, Cry1Ai2 had good lethal activity against Ostrinia furnacalis, Hyphantria cunea, Chilo suppressalis, and Bombyx mori larvae and considerable weight loss activity against Helicoverpa armigera. Bacillus thuringiensis, which is a Gram-positive bacterium, is known for its specific toxicity toward insect pests (1). This toxicity is largely attributed to the insecticidal crystal proteins encoded by the cry genes (2-4). The cry1-type genes of B. thuringiensis are highly toxic to lepidopteran pests, and some genes have been used to develop plants with resistance to insect pests (5-7). Because of their potential application and commercial value, much research has focused on the discovery of novel cry1 genes; to date, approximately 258 cry1 genes have been cloned and named, and 50 distinct holotypes have been classified (http://www.lifesci .sussex.ac.uk/home/Neil_Crickmore/Bt/). Previous research has revealed that cry1 genes are typically found in clusters; for example, B. thuringiensis strains HD12 and HD525 contain at least four different cry1-type genes (8).PCR is a simple and convenient investigative method and has been widely used to identify the vast variety of cry genes by the use of different primers (9-12). PCR-restriction fragment length polymorphism (PCR-RFLP) is a modified PCR technique that is generally used for the identification of known and unknown cry1-type genes (except for cry1I-type genes), and of parts of the cry7-type and cry9-type genes, according to the fragment lengths of digested PCR-amplified products described by Kuo and Chak (8). Some primers have been designed for the identification of cry1-type genes (13) and cry8-type genes (14) based on the PCR-RFLP method. However, it is becoming difficult to identify novel cry1-type genes using the PCR-RFLP method (8) because of the increase in the numbers of cry1-type genes.To resolve this problem, an improved PCR-RFLP method was designed to directly identify the fourth class of cry1-type genes by dividing...

Owing to their antitumor and major histocompatibility complex (MHC)-independent capacities, γδ T cells have gained popularity in adoptive T-cell immunotherapy in recent years. However, many unknowns still exist regarding γδ T cells, and few clinical data have been collected. Therefore, this review aims to describe all the main features of the applications of γδ T cells and provide a systematic view of current γδ T-cell immunotherapy. Specifically, this review will focus on how γδ T cells performed in treating cancers in clinics, on the γδ T-cell clinical trials that have been conducted to date, and the role of γδ T cells in the pharmaceutical industry.

The novel cry1Ai gene cloned from Bacillus thuringiensis strain SC6H8 encoded a protein exhibiting strong toxicity against Plutella xylostella and Chilo suppressalis in our previous study. Using the available information for the active fragments of other Cry toxins, eight truncated fragments were constructed to identify the minimal active fragment of Cry1Ai. All truncated fragments were expressed in Escherichia coli strain BL21 (DE3), and the insecticidal activity against 2 nd-instar P. xylostella larvae was assessed using full-length Cry1Ai as a positive control. The results indicate that the minimal active fragment of the Cry1Ai toxin against P. xylostella is located between amino acid residues 36 I and 605 I , which is smaller than the regions previously reported for Cry1A. The first two amino acids (34 T and 35 P) on Helix α-1 and whole Helix α-2 of Domain I and sheet β-32 of Domain III are necessary for Cry1Ai toxin to keep its toxicity against P. xylostella.