Progress of polysaccharide-based tissue adhesives.

Recently, polymer-based tissue adhesives (TAs) have gained the attention of scientists and industries as alternatives to sutures for sealing and closing wounds or incisions because of their ease of use, low cost, minimal tissue damage, and short application time. However, poor mechanical properties and weak adhesion strength limit the application of TAs, although numerous studies have attempted to develop new TAs with enhanced performance. Therefore, next-generation TAs with improved multifunctional properties are required. In this review, we address the requirements of polymeric TAs, adhesive characteristics, adhesion strength assessment methods, adhesion mechanisms, applications, advantages and disadvantages, and commercial products of polysaccharide (PS)-based TAs, including chitosan (CS), alginate (AL), dextran (DE), and hyaluronic acid (HA). Additionally, future perspectives are discussed.

Development of a new broad-spectrum microencapsulation-based spray drying formulation of Bacillus thuringiensis subsp. kurstaki IMBL-B9 for the control of moths.

Certain Bacillus thuringiensis (Bt) strains such as Bt subsp. kurstaki and Bt subsp. aizawai have been widely used for pest management in agricultural practices. However, each strain only shows high specificity for pest control against a narrow range of lepidopteran species, and numerous lepidopteran pests have developed resistance to commercialized Bt strains. Therefore, there is a need for the development of novel Bt bioinsecticides which allow for potent and broad-spectrum insecticidal activity against lepidopteran species, including Spodoptera spp. (Noctuidae) and Plutella xylostealla (Plutellidae). In order to develop a novel bioinsecticide using Bt subsp. kurstaki IMBL-B9 (Btk IMBL-B9) that exhibits excellent insecticidal activity against three different lepidopteran species, we have developed a viable microencapsulation-based spray drying Btk IMBL-B9 formulation. The spore-crystal complex of Btk IMBL-B9 was microencapsulated using coating materials such as gum arabic, maltodextrin, and corn starch via spray drying. The encapsulated formulation of Btk IMBL-B9 presented an increased survival rate and storage stability at 54 ± 2°C for up to 6 weeks. The formulation showed similar insecticidal activity as the commercial bioinsecticide XenTari® against P. xylostella. Under controlled greenhouse conditions, the Btk IMBL-B9 formulation was more effective against Lepidoptera spp. S. frugiperda and P. xylostella, than XenTari®. These results suggest that the microencapsulation-based spray drying formulation of Btk IMBL-B9 can be used effectively for the control of a wide range of moths.

Transformation of Bacillus thuringiensis plasmid DNA by a new polyethylenimine polymeric nanoparticles method.

Although electroporation technique has been mostly used to transform Bacillus thuringiensis (Bt), this method is not readily applicable to strains other than the one for which it was optimized. Polyethylenimine (PEI) is a golden standard non-viral vector that interacts with plasmids to form compact polymeric nanoparticles (PNPs) via electrostatic interactions. This PNPs system is very attractive because they are easily prepared, able to carry large nucleic acid constructs, and show low toxicity. In this study, PEI/pBTdsSBV-VP1 PNPs were successfully prepared at various N/P ratios which is positively-chargeable polymer amine (N = nitrogen) groups to negatively-charged nucleic acid phosphate (P) groups, and the internalization of the complexes into Bt 4Q7 was confirmed by confocal laser scanning microscopy. The PEI-mediated transformation showed similar efficiency comparable to that of electroporation method, suggesting that the method of PNPs will be an effective alternative for transformation of Bt strains.

Isolation and molecular characterization of Bacillus thuringiensis subsp. kurstaki toxic to lepidopteran pests Spodoptera spp. and Plutella xylostella.

BACKGROUND: Bacillus thuringiensis (Bt) has been widely used as a biological control agent for lepidopteran pests. However, resistance to Bt is a major concern associated with Spodoptera spp. (Noctuidae) and Plutella xylostella (Plutellidae). For efficient control of Noctuidae and Plutellidae, novel Bt strains which have high toxicity and a broad host range are needed. RESULTS: To develop novel Bt strains as used for bio-insecticides, the Bt IMBL-B9 with high toxicity against Spodoptera exigua, Spodoptera frugiperda and P. xylostella was isolated and characterized. The Bt kurstaki IMBL-B9 strain produced bipyramidal and cuboidal crystals consisting of cry toxins with molecular weights of 130 and 65 kDa, respectively. This strain harbors eight crystal protein genes in total, including cry1Ea and one vegetative insecticidal protein gene. The median lethal concentration (LC50 ) values of IMBL-B9 against S. exigua and S. frugiperda were 21.8- and 19.3-fold lower than those of the Bt kusrstaki strain, and 5.6- and 4.9-fold lower than those of Bt aizawai strain, respectively. To evaluate the insecticidal activity of Cry proteins from IMBL-B9, cry gene-sourced recombinant Bt strains were constructed. These strains have insecticidal activity and synergic action against lepidopteran pests. CONCLUSION: In this study, a novel Bt kurstaki IMBL-B9 strain was isolated and this could be useful for the development of new bio-insecticide or cry gene-based recombinant products as an alternative solution against lepidopterans, including Noctuidae and Plutellidae. © 2022 Society of Chemical Industry.

Convergence of Bar and Cry1Ac Mutant Genes in Soybean Confers Synergistic Resistance to Herbicide and Lepidopteran Insects.

Soybean is a globally important crop species, which is subject to pressure by insects and weeds causing severe substantially reduce yield and quality. Despite the success of transgenic soybean in terms of Bacillus thuringiensis (Bt) and herbicide tolerance, unforeseen mitigated performances have still been inspected due to climate changes that favor the emergence of insect resistance. Therefore, there is a need to develop a biotech soybean with elaborated gene stacking to improve insect and herbicide tolerance in the field. In this study, new gene stacking soybean events, such as bialaphos resistance (bar) and pesticidal crystal protein (cry)1Ac mutant 2 (M#2), are being developed in Vietnamese soybean under field condition. Five transgenic plants were extensively studied in the herbicide effects, gene expression patterns, and insect mortality across generations. The increase in the expression of the bar gene by 100% in the leaves of putative transgenic plants was a determinant of herbicide tolerance. In an insect bioassay, the cry1Ac-M#2 protein tested yielded higher than expected larval mortality (86%), reflecting larval weight gain and weight of leaf consumed were less in the T1 generation. Similarly, in the field tests, the expression of cry1Ac-M#2 in the transgenic soybean lines was relatively stable from T0 to T3 generations that corresponded to a large reduction in the rate of leaves and pods damage caused by Lamprosema indicata and Helicoverpa armigera. The transgenic lines converged two genes, producing a soybean phenotype that was resistant to herbicide and lepidopteran insects. Furthermore, the expression of cry1Ac-M#2 was dominant in the T1 generation leading to the exhibit of better phenotypic traits. These results underscored the great potential of combining bar and cry1Ac mutation genes in transgenic soybean as pursuant of ensuring resistance to herbicide and lepidopteran insects.

Suppression of Rice Stripe Virus Replication in Laodelphax striatellus Using Vector Insect-Derived Double-Stranded RNAs.

RNA interference (RNAi) has attracted attention as a promising approach to control plant viruses in their insect vectors. In the present study, to suppress replication of the rice stripe virus (RSV) in its vector, Laodelphax striatellus, using RNAi, dsRNAs against L. striatellus genes that are strongly upregulated upon RSV infection were delivered through a rice leaf-mediated method. RNAi-based silencing of peroxiredoxin, cathepsin B, and cytochrome P450 resulted in significant down regulation of the NS3 gene of RSV, achieving a transcriptional reduction greater than 73.6% at a concentration of 100 ng/µl and, possibly compromising viral replication. L. striatellus genes might play crucial roles in the transmission of RSV; transcriptional silencing of these genes could suppress viral replication in L. striatellus. These results suggest effective RNAi-based approaches for controlling RSV and provide insight into RSV-L. striatellus interactions.

Isolation and characterization of the insect growth regulatory substances from actinomycetes.

Insect growth regulators (IGRs) are attractive alternatives to chemical insecticides. Since it has been reported that secondary metabolites from actinomycetes show insecticidal activities against various insect pests, actinomycetes could be a potential source of novel IGR compounds. In the present study, insect juvenile hormone antagonists (JHANs) were identified from actinomycetes and their insect growth regulatory and insecticidal activities were investigated. A total of 363 actinomycetes were screened for their insect growth regulatory and insecticidal activities against Aedes albopictus and Plutella xylostella. Among them, Streptomyces sp. AN120537 showed the highest JHAN and insecticidal activities. Five antimycins were isolated as active compounds by assay-guided fractionation and showed high JHAN activities. These antimycins also exhibited significant insecticidal activities against A. albopictus, P. xylostella, F. occidentalis, and T. urticae. Moreover, dead larvae treated with these antimycins displayed morphological deformities that are similar to those of JH-based IGR-treated insects. This is the first report demonstrating that the insecticidal activities of antimycins resulted from their possible JHAN activity. Based on our results, it is expected that novel JHAN compounds potentially derived from actinomycetes could be efficiently applied as IGR insecticides with a broad insecticidal spectrum.

Baculovirus entire ORF1629 is not essential for viral replication.

It is generally accepted that ORF1629 is essential for baculovirus replication, which has enabled isolation of recombinant viruses in a baculovirus expression system using linearized viral DNA. ORF1629-defective viruses cannot replicate in insect cells; only recombinant virus with complete ORF1629 restoration by recombination can propagate, allowing for pure isolation and the development of bacmids for easy selection of recombinant viruses. We inadvertently found proliferation in insect cells of a bacmid lacking a complete ORF1629. PCR indicated no other viruses but a lack of complete ORF1629 in the proliferated bacmid, suggesting that the baculovirus propagated without a complete ORF1629. Lack of ORF1629 decreased the virus growth rate and yield; it also increased the occlusion body (OB) size but decreased its yield. These results were confirmed for Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Bombyx mori NPV (BmNPV). Thus, entire ORF1629 is not essential for viral replication, though it does affect the virus growth rate, yield, and size and OB production.

Plant-derived compounds regulate formation of the insect juvenile hormone receptor complex.

Insect growth regulators (IGRs) are attractive pest control agents due to their high target specificity and relative safety to the environment. Recently, plants have been shown to synthesize IGRs that affect the insect juvenile hormone (JH) as a part of their defense mechanisms. Using a yeast two-hybrid system transformed with the Aedes aegypti JH receptor as a reporter system, we identified several JH agonists (JHAs) and antagonists (JHANs) causing retardation in the ovarian development of female Asian tiger mosquito, Aedes albopictus, from plant essential oil compounds. While the JHAs increased the expression of a JH-induced gene, the JHANs caused a reduction in the expression of the same gene. The compounds identified in this study could provide insights into plant-insect interactions and may be useful for the development of novel IGR insecticides.

Cysteine Protease Profiles of the Medicinal Plant Calotropis procera R. Br. revealed by de novo transcriptome analysis.

Calotropis procera R. Br., a traditional medicinal plant in India, is a promising source of commercial proteases, because the cysteine proteases from the plant exhibit high thermo-stability, broad pH optima, and plasma-clotting activity. Though several proteases such as Procerain, Procerain B, CpCp-1, CpCp-2, and CpCp-3 have been isolated and characterized, the information of their transcripts is limited to cDNAs encoding their mature peptides. Due to this limitation, in this study, to determine the cDNA sequences encoding full open reading frame of these cysteine proteases, transcripts were sequenced with an Illumina Hiseq2000 sequencer. A total of 171,253,393 clean reads were assembled into 106,093 contigs with an average length of 1,614 bp and an N50 of 2,703 bp, and 70,797 contigs with an average length of 1,565 bp and N50 of 2,082 bp using Trinity and Velvet-Oases software, respectively. Among these contigs, we found 20 unigenes related to papain-like cysteine proteases by BLASTX analysis against a non-redundant NCBI protein database. Our expression analysis revealed that the cysteine protease contains an N-terminal pro-peptide domain (inhibitor region), which is necessary for correct folding and proteolytic activity. It was evident that expression yields using an inducible T7 expression system in Escherichia coli were considerably higher with the pro-peptide domain than without the domain, which could contribute to molecular cloning of the Calotropis procera protease as an active form with correct folding.

Identification of plant compounds that disrupt the insect juvenile hormone receptor complex.

Insects impact human health through vector-borne diseases and cause major economic losses by damaging crops and stored agricultural products. Insect-specific growth regulators represent attractive control agents because of their safety to the environment and humans. We identified plant compounds that serve as juvenile hormone antagonists (PJHANs). Using the yeast two-hybrid system transformed with the mosquito JH receptor as a reporter system, we demonstrate that PJHANs affect the JH receptor, methoprene-tolerant (Met), by disrupting its complex with CYCLE or FISC, formation of which is required for mediating JH action. We isolated five diterpene secondary metabolites with JH antagonist activity from two plants: Lindera erythrocarpa and Solidago serotina. They are effective in causing mortality of mosquito larvae at relatively low LD50 values. Topical application of two diterpenes caused reduction in the expression of Met target genes and retardation of follicle development in mosquito ovaries. Hence, the newly discovered PJHANs may lead to development of a new class of safe and effective pesticides.

Bombyx mori nucleopolyhedrovirus bacmid enabling rapid generation of recombinant virus by in vitro transposition.

A novel recombinant bacmid, bEasyBm, that enables the easy and fast generation of pure recombinant baculovirus without any purification step was constructed. In bEasyBm, attR recombination sites were introduced to facilitate the generation of a recombinant viral genome by in vitro transposition. Moreover, the extracellular RNase gene from Bacillus amyloliquefaciens, barnase, was expressed under the control of the Cotesia plutellae bracovirus early promoter to negatively select against the nonrecombinant background. The bEasyBm bacmid could only replicate in host insect cells when the barnase gene was replaced with the gene of interest by in vitro transposition. When bEasyBm was transposed with pDualBac-EGFP, the resulting recombinant virus, EasyBm-EGFP, showed high levels of EGFP expression efficiency compared with that of non-purified recombinant virus BmGOZA-EGFP, which was constructed using the bBmGOZA system. In addition, nonrecombinant backgrounds were not detected in unpurified EasyBm-EGFP stocks. Based on these results, a high-throughput system for the generation of multiple recombinant viruses at a time was established.

Production of antibacterial Bombyx mori cecropin A in mealworm-pathogenic Beauveria bassiana ERL1170.

Efforts are underway to produce antimicrobial peptides in yellow mealworms (Tenebrio molitor), which can be developed as more effective and safer animal feed additives. In this work, we expressed Bombyx mori (Bm) cecropin-A in mealworms by the infection of transformed entomopathogenic Beauveria bassiana ERL1170. The active domain of Bm cecropin A gene was tagged with a signal sequence of B. bassiana for extracellular secretion, and the fragment was inserted into ERL1170 by the restriction enzyme-mediated integration method. Transformant D-6 showed antibacterial activity against Bacillus subtilis and Listeria monocytogenes. Against T. molitor larvae, D-6 had similar mortality to wild-type, and D6-infected mealworm suspension showed strong antibacterial activity against the two bacteria, but not in the wild-type-infected mealworms, thereby increasing the value of mealworms as animal feed additives.

Autographa californica multiple nucleopolyhedrovirus ORF11 is essential for budded-virus production and occlusion-derived-virus envelopment.

UNLABELLED: ORF11 (ac11) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved gene with unknown function. To determine the role of ac11 in the baculovirus life cycle, an ac11 knockout mutant of AcMNPV, Ac11KO, was constructed. Northern blot and 5' rapid amplification of cDNA ends (RACE) analyses revealed that ac11 is an early gene in the life cycle. Microscopy, titration assays, and Western blot analysis revealed that budded viruses (BVs) were not produced in Ac11KO-transfected Sf9 cells. However, quantitative PCR (qPCR) analysis demonstrated that the deletion of ac11 did not affect viral DNA replication. Furthermore, electron microscopy revealed that there was no nucleocapsid in the cytoplasm or plasma membrane of Ac11KO-transfected cells, which demonstrates that the defect in BV production in Ac11KO-transfected cells is due to the inefficient egress of nucleocapsids from the nucleus to the cytoplasm. In addition, electron microscopy observations showed that the nucleocapsids in the nucleus were not enveloped to form occlusion-derived viruses (ODVs) and that their subsequent embedding into occlusion bodies (OBs) was also blocked in Ac11KO-transfected cells, demonstrating that ac11 is required for ODV envelopment. These results therefore demonstrate that ac11 is an early gene that is essential for BV production and ODV envelopment. IMPORTANCE: Baculoviruses have been extensively used not only as specific, environmentally benign insecticides but also as helper-independent protein expression vectors. Although the function of baculovirus genes in viral replication has been studied by using gene knockout technology, the functions of more than one-third of viral genes, which include some highly conserved genes, are still unknown. In this study, ac11 was proven to play a crucial role in BV production and ODV envelopment. These results will lead to a better understanding of baculovirus infection cycles.

Dual function of a bee (Apis cerana) inhibitor cysteine knot peptide that acts as an antifungal peptide and insecticidal venom toxin.

Inhibitor cysteine knot (ICK) peptides exhibit ion channel blocking, insecticidal, and antimicrobial activities, but currently, no functional roles for bee-derived ICK peptides have been identified. In this study, a bee (Apis cerana) ICK peptide (AcICK) that acts as an antifungal peptide and as an insecticidal venom toxin was identified. AcICK contains an ICK fold that is expressed in the epidermis, fat body, or venom gland and is present as a 6.6-kDa peptide in bee venom. Recombinant AcICK peptide (expressed in baculovirus-infected insect cells) bound directly to Beauveria bassiana and Fusarium graminearum, but not to Escherichia coli or Bacillus thuringiensis. Consistent with these findings, AcICK showed antifungal activity, indicating that AcICK acts as an antifungal peptide. Furthermore, AcICK expression is induced in the fat body and epidermis after injection with B. bassiana. These results provide insight into the role of AcICK during the innate immune response following fungal infection. Additionally, we show that AcICK has insecticidal activity. Our results demonstrate a functional role for AcICK in bees: AcICK acts as an antifungal peptide in innate immune reactions in the body and as an insecticidal toxin in venom. The finding that the AcICK peptide functions with different mechanisms of action in the body and in venom highlights the two-pronged strategy that is possible with the bee ICK peptide.

Biochemical and toxicological properties of two acetylcholinesterases from the common bed bug, Cimex lectularius.

We examined the molecular and enzymatic properties of two acetylcholinesterases (AChEs; ClAChE1 and ClAChE2) from the common bed bug, Cimex lectularius. Native polyacrylamide gel electrophoresis followed by activity staining and Western blotting revealed that ClAChE1 is the main catalytic enzyme and is abundantly expressed in various tissues. Both ClAChEs existed in dimeric form connected by a disulfide bridge and were attached to the membrane via a glycophosphatidylinositol anchor. To determine their kinetic and inhibitory properties, both ClAChE1 and ClAChE2 were in vitro expressed in Sf9 cells using a baculovirus expression system. ClAChE1 showed higher catalytic efficiency toward acetylcholine, supporting the hypothesis that ClAChE1 plays a major role in postsynaptic transmission. An inhibition assay revealed that ClAChE1 is generally more sensitive to organophosphates and carbamates examined although ClAChE2 was >4000-fold more sensitive to malaoxon than ClAChE1. The relatively higher correlation between the in vitro ClAChE1 inhibition and the in vivo toxicity suggested that ClAChE1 is the more relevant toxicological target for organophosphates and carbamates. Although the physiological function of ClAChE2 remains to be elucidated, ClAChE2 also appears to have neuronal functions, as judged by its tissue distribution and molecular and kinetic properties. Our findings help expand our knowledge on insect AChEs and their toxicological properties.

Antimicrobial activity of a honeybee (Apis cerana) venom Kazal-type serine protease inhibitor.

Insect-derived Kazal-type serine protease inhibitors exhibit thrombin, elastase, plasmin, proteinase K, or subtilisin A inhibition activity, but so far, no functional roles for bee-derived Kazal-type serine protease inhibitors have been identified. In this study, a bee (Apis cerana) venom Kazal-type serine protease inhibitor (AcKTSPI) that acts as a microbial serine protease inhibitor was identified. AcKTSPI contained a single Kazal domain that displayed six conserved cysteine residues and a P1 threonine residue. AcKTSPI was expressed in the venom gland and was present as a 10-kDa peptide in bee venom. Recombinant AcKTSPI Kazal domain (AcKTSPI-Kd) expressed in baculovirus-infected insect cells demonstrated inhibitory activity against subtilisin A (Ki 67.03 nM) and proteinase K (Ki 91.53 nM), but not against α-chymotrypsin or trypsin, which implies a role for AcKTSPI as a microbial serine protease inhibitor. However, AcKTSPI-Kd exhibited no detectable inhibitory effects on factor Xa, thrombin, tissue plasminogen activator, or elastase. Additionally, AcKTSPI-Kd bound directly to Bacillus subtilis, Bacillus thuringiensis, Beauveria bassiana, and Fusarium graminearum but not to Escherichia coli. Consistent with these findings, AcKTSPI-Kd showed antibacterial activity against Gram-positive bacteria and antifungal activity against both plant-pathogenic and entomopathogenic fungi. These findings constitute molecular evidence that AcKTSPI acts as an inhibitor of microbial serine proteases. This paper provides a novel view of the antimicrobial functions of a bee venom Kazal-type serine protease inhibitor.

Functional characterization of Autographa californica multiple nucleopolyhedrovirus ORF43 and phenotypic changes of ORF43-knockout mutant.

ORF43 (ac43) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved baculovirus gene of unknown function. To investigate the role of ac43 in the baculovirus lifecycle, we constructed an ac43-deleted mutant AcMNPV, Ac43KO. After transfection into Spodoptera frugiperda cells, Ac43KO produced polyhedra much larger in size than those of wild-type AcMNPV. Interestingly, some of the nucleocapsids were singly enveloped in the polyhedrin matrix while the nucleocapsids of AcMNPV are known to be multiply enveloped. Furthermore, Ac43KO led to a defect in the transcription and expression of polyhedrin, which resulted in reduced occlusion body production. However, Ac43KO did not affect production of budded virus as there was no remarkable difference in budded virus titer. These results suggest that ac43 plays an important role in the expression of polyhedrin, the morphogenesis of occlusion body, and the assembly of virions occluded in occlusion bodies.

Existence of lysogenic bacteriophages in Bacillus thuringiensis type strains.

We screened the existence of bacteriophages in 67 Bacillus thuringiensis type strains by phage DNA extraction and PCR using phage terminase small subunit (TerS)-specific primers to the supernatants and the precipitated pellets of Bt cultures, and by transmission electron microscopy. The various bacteriophages were observed from the supernatants of 22 type strains. Ten type strains showed the extracted phage DNAs and the amplified fragment by TerS PCR but 12 type strains showed only the phage DNAs. Their morphological characteristic suggests that they belong to Family Siphoviridae which had a long tail and symmetrical head.

The Autographa californica multiple nucleopolyhedrovirus ORF78 is essential for budded virus production and general occlusion body formation.

ORF78 (ac78) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a baculovirus core gene of unknown function. To determine the role of ac78 in the baculovirus life cycle, an AcMNPV mutant with ac78 deleted, Ac78KO, was constructed. Quantitative PCR analysis revealed that ac78 is a late gene in the viral life cycle. After transfection into Spodoptera frugiperda cells, Ac78KO produced a single-cell infection phenotype, indicating that no infectious budded viruses (BVs) were produced. The defect in BV production was also confirmed by both viral titration and Western blotting. However, viral DNA replication was unaffected, and occlusion bodies were formed. An analysis of BVs and occlusion-derived viruses (ODVs) revealed that AC78 is associated with both forms of the virions and is an envelope structural protein. Electron microscopy revealed that AC78 also plays an important role in the embedding of ODV into the occlusion body. The results of this study demonstrate that AC78 is a late virion-associated protein and is essential for the viral life cycle.