ABC transporter subfamily B1 as a susceptibility determinant of Bombyx mori larvae to Cry1Ba, Cry1Ia and Cry9Da toxins.

ATP binding cassette (ABC) transporters are a diverse family of transmembrane proteins. Specific subfamily members expressed in the lepidopteran midgut can act as susceptibility determinants for several insecticidal Bt Cry proteins. However, the susceptibility determinants to many Cry toxins still remain unclear. Therefore, we knocked out a series of ABC transporters that are highly expressed in the midgut of Bombyx mori larvae by transcription activator-like effector nuclease (TALEN)-mediated gene editing, and the lineages that became resistant to Cry toxins were searched by toxin overlay bioassay. As a result, the B. mori ABC transporter subfamily B1 (BmABCB1) knockout lineage showed 19.17-fold resistance to Cry1Ba, 876.2-fold resistance to Cry1Ia, and 29.1-fold resistance to Cry9Da, suggesting that BmABCB1 is the determinant of susceptibility to these toxins. BmABCC2 and BmABCC3 have been shown to be susceptibility determinants based on their function as receptors. Therefore, we next heterologously expressed these ABC transporters in HEK293T cells and performed a cell swelling assay to examine whether these molecules could exert receptor functions. As a result, BmABCB1-expressing cells showed swelling response to Cry1Ia and Cry9Da, and cells expressing PxABCB1, which is the Plutella xylostella ortholog of BmABCB1, showed swelling for Cry1Ba, suggesting that ABCB1 is a susceptibility determinant by functioning as a receptor to these toxins. Furthermore, in order to clarify how high binding affinity is based on receptor function, we performed surface plasmon resonance analysis and found that each KD of Cry1Ba, Cry1Ia, and Cry9Da to BmABCB1 were 7.69 × 10-8 M, 2.19 × 10-9 M, and 4.17 × 10-6 M respectively.

ATP-binding cassette transporter subfamily C members 2, 3 and cadherin protein are susceptibility-determining factors in Bombyx mori for multiple Bacillus thuringiensis Cry1 toxins.

Field-evolved resistance of insect pests to Bacillus thuringiensis (Bt) toxins (Cry toxins) is a threat to the efficacy of Bt-based bio-insecticides and transgenic crops. Recent reports have suggested that ATP-binding cassette transporter subfamily C2 (ABCC2) and cadherin-like receptor play important roles in conferring susceptibility to Cry1 toxins. However, the receptors involved in Bt susceptibility in each insect remain unclear. To determine the receptors that are involved in the susceptibility of Bombyx mori to Cry1 toxins (1Ab, 1Ac and 1Fa), we conducted diet overlay bioassay using B. mori strains disrupted with one or two receptor (s) among BmABCC2, BmABCC3, and cadherin-like receptor (BtR175) generated by transcription activator-like effector nuclease (TALEN)-mediated gene editing. The single-knockout strains for BmABCC2 showed resistance to Cry1Ab and Cry1Ac, whereas only strains with double knockout of BmABCC2 and BmABCC3 exhibited high resistance to Cry1Fa. Progeny populations generated from the crossing of heterozygotes for BtR175 knockout allele included 25% theoretical homozygotes for the BtR175 knockout allele and they showed resistance to Cry1Ab and Cry1Ac. Then, through a cell swelling assay using Sf9 cells ectopically expressing the receptor, we analyzed the mechanisms underlying the different contributions of BmABCC2, BmABCC3, and BtR175 to larval susceptibility. The receptor activity of BmABCC2 for Cry1Ab and Cry1Ac was far higher than that of BmABCC3, and BtR175 synergistically enhanced the receptor activity of BmABCC2. This result well explained the important involvement of BmABCC2 and BtR175 in the larval susceptibility to Cry1A toxins. By contrast, the receptor activities of BmABCC2 and BmABCC3 for Cry1Fa were observed at a similar level and synergistic effect of BtR175 was small. This finding explains the equal importance of BmABCC2 and BmABCC3 and very small contribution of BtR175 on larval susceptibility to Cry1Fa. Thus, we demonstrated the different importance of BmABCC2, BmABCC3, and BtR175 to various Cry1 toxins as susceptibility-determining factors in B. mori larvae and the underlying basis for the observed differences. Furthermore, a weak correlation was indicated between the binding affinity and receptor activities of BmABCC2 and BmABCC3 to Cry1 toxins.

ATP-Binding Cassette Subfamily A Member 2 is a Functional Receptor for Bacillus thuringiensis Cry2A Toxins in Bombyx mori, but not for Cry1A, Cry1C, Cry1D, Cry1F, or Cry9A Toxins.

: Cry toxins are insecticidal proteins produced by Bacillus thuringiensis (Bt). They are used commercially to control insect pests since they are very active in specific insects and are harmless to the environment and human health. The gene encoding ATP-binding cassette subfamily A member 2 (ABCA2) was identified in an analysis of Cry2A toxin resistance genes. However, we do not have direct evidence for the role of ABCA2 for Cry2A toxins or why Cry2A toxin resistance does not cross to other Cry toxins. Therefore, we performed two experiments. First, we edited the ABCA2 sequence in Bombyx mori using transcription activator-like effector-nucleases (TALENs) and confirmed the susceptibility-determining ability in a diet overlay bioassay. Strains with C-terminal half-deleted BmABCA2 showed strong and specific resistance to Cry2A toxins; even strains carrying a deletion of 1 to 3 amino acids showed resistance. However, the C-terminal half-deleted strains did not show cross-resistance to other toxins. Second, we conducted a cell swelling assay and confirmed the specific ability of BmABCA2 to Cry2A toxins in HEK239 cells. Those demonstrated that BmABCA2 is a functional receptor for Cry2A toxins and that BmABCA2 deficiency-dependent Cry2A resistance does not confer cross-resistance to Cry1A, Cry1F, Cry1Ca, Cry1Da, or Cry9Aa toxins.

Ryanodine receptor mutations (G4946E and I4790K) differentially responsible for diamide insecticide resistance in diamondback moth, Plutella xylostella L.

This study examined diamondback moth (Plutella xylostella) strains showing high-level resistance to cyantraniliprole (KA17 strain) and to flubendiamide and chlorantraniliprole (KU13 strain). The LC50 value of the KA17 strain against cyantraniliprole was ca. 100-fold higher than that of the KU13 strain. The KA17 strain also exhibited high-level resistance to chlorantraniliprole and flubendiamide equivalent to those in the KU13 strain. The KU13 strain showed a higher LC50 value against cyantraniliprole than the susceptible strains. However, the LC50 value of the KU13 strain against cyantraniliprole was below the agriculturally recommended concentration. Subsequent QTL analysis using ddRAD-seq identified the resistance responsible regions of the KA17 and KU13 strains with different diamide resistance profiles. Ryanodine receptor (RyR) gene was included in the identified regions. Single nucleotide polymorphism calling in the RyR gene using RNA-seq found previously reported G4946E (amino acid mutation from glycine to glutamic acid at amino acid position 4946) and novel I4790K (amino acid mutation from isoleucine to lysine at amino acid position 4790) mutations, respectively, in the RyR of the KU13 and KA17 strains. Functional significance of I4790K in the resistance was confirmed in calcium imaging of the human embryonic kidney 293T cell line expressing Bombyx mori RyR with the mutation. This reporting is the first describing I4790K as a fundamental mechanism responsible for the resistance to the diamides including cyantraniliprole. From this study, we also report up-regulated expression of some degradation enzymes and that of the RyR gene in the KA17 and KU13 strains based on results of RNA-seq data analysis.

Spatial distribution of orally administered viral fusolin protein in the insect midgut and possible synergism between fusolin and digestive proteases to disrupt the midgut peritrophic matrix.

Oral inoculation of entomopoxvirus spindles, microstructures composed of fusolin protein, causes disruption of the peritrophic matrix (PM), a physical barrier against microbe infection, in the insect midgut. Although the atomic structure of fusolin has been determined, little has been directly elucidated of the mechanism of disruption of the PM. In the present study, we first performed an immunohistochemical examination to determine whether fusolin acts on the PM directly or indirectly in the midgut of Bombyx mori larvae that were inoculated with spindles of Anomala cuprea entomopoxvirus. This revealed that the PM, rather than the midgut cells, was the attachment site for fusolin. Fusolin broadly attached to the PM from the anterior to the posterior region, both to its ectoperitrophic and endoperitrophic surfaces and within the PM. These results likely explain why the whole of the PM is rapidly disintegrated. Second, we administered protease inhibitors mixed with spindles and observed decreased midgut protease activity and reduced disruption of the PM. This suggests that midgut protease(s) is also positively involved in PM disruption. Based on the present results, we propose an overall mechanism for the disruption of the PM by administration of fusolin.

Factors affecting eye drop instillation in glaucoma patients with visual field defect.

BACKGROUND: To investigate the success rate of eye drop instillation in glaucoma patients with visual field defect as well as non-glaucoma volunteers. Factors that may affect the success rate of eye drop instillation were also evaluated. DESIGN: A prospective, observational study. PARTICIPANTS: Seventy-eight glaucoma patients and 85 non-glaucoma volunteers were recruited in this study. METHODS: Open angle glaucoma patients with visual field defect as well as non-glaucoma volunteers were asked to video record their procedures of eye drop instillation using a 5-mL plastic bottle of artificial tear solution. Success of eye drop instillation was judged on video based on the first one drop of solution successfully applied on the cornea, by two investigators. MAIN OUTCOME MEASURES: Success rate of eye drop instillation in glaucoma patients and non-glaucoma volunteers. Factors related to success rate of eye drop instillation, such as visual field defect and clinical characteristics, were also analyzed using multivariable logistic regression. RESULTS: No significant deference in mean age was observed between two groups (glaucoma: 64.5 ± 14.4 years, non-glaucoma: 60.9 ± 14.1 years, P = 0.1156). Success rate of eye drop instillation was significantly lower (P = 0.0215) in glaucoma patients (30/78; 38.5%) than in non-glaucoma volunteers (48/85; 56.5%). The most frequent reason of instillation failure in glaucoma patients was touching the bulbar conjunctiva, cornea, eyelid or eyelashes with the tip of the bottle (29.5%). Multivariable logistic regression analysis identified lower corrected visual acuity (VA) (≤ 1.0; odds ratio [OR] = 0.20, 95% confidence interval [CI] 0.04-0.93, P = 0.0411), lower mean deviation (MD) (< -12 dB; OR = 0.20, 95% CI 0.05-0.86, P = 0.0307) and visual field defect (VFD) in the inferior hemifield (OR = 0.11, 95% CI 0.02-0.34, P < 0.001) to be significantly related to instillation failure in glaucoma patients. CONCLUSIONS: Success rate of eye drop instillation was significantly lower in glaucoma patients than in non-glaucoma volunteers. Corrected VA ≤ 1.0, MD < -12 dB and/or VFD in the inferior hemifield may be related to failure of eye drop instillation.

Single amino acid insertions in extracellular loop 2 of Bombyx mori ABCC2 disrupt its receptor function for Bacillus thuringiensis Cry1Ab and Cry1Ac but not Cry1Aa toxins.

In a previous report, seven Cry1Ab-resistant strains were identified in the silkworm, Bombyx mori; these strains were shown to have a tyrosine insertion at position 234 in extracellular loop 2 of the ABC transporter C2 (BmABCC2). This insertion was confirmed to destroy the receptor function of BmABCC2 and confer the strains resistance against Cry1Ab and Cry1Ac. However, these strains were susceptible to Cry1Aa. In this report, we examined the mechanisms of the loss of receptor function of the transporter by expressing mutations in Sf9 cells. After replacement of one or two of the five amino acid residues in loop 2 of the susceptible BmABCC2 gene [BmABCC2_S] with alanine, cells still showed susceptibility, retaining the receptor function. Five mutants with single amino acid insertions at position 234 in BmABCC2 were also generated, resulting in loop 2 having six amino acids, which corresponds to replacing the tyrosine insertion in the resistant BmABCC2 gene [BmABCC2_R(+(234)Y)] with another amino acid. All five mutants exhibited loss of function against Cry1Ab and Cry1Ac. These results suggest that the amino acid sequence in loop 2 is less important than the loop size (five vs. six amino acids) or loop structure for Cry1Ab and Cry1Ac activity. Several domain-swapped mutant toxins were then generated among Cry1Aa, Cry1Ab, and Cry1Ac, which are composed of three domains. Swapped mutants containing domain II of Cry1Ab or Cry1Ac did not kill Sf9 cells expressing BmABCC2_R(+(234)Y), suggesting that domain II of the Cry toxin is related to the interaction with the receptor function of BmABCC2. This also suggests that different reactions against Bt-toxins in some B. mori strains, that is, Cry1Ab resistance or Cry1Aa susceptibility, are attributable to structural differences in domain II of Cry1A toxins.

Sustainability of Intraocular Pressure Reduction of Travoprost Ophthalmic Solution in Subjects with Normal Tension Glaucoma.

INTRODUCTION: We examined the sustainability of the intraocular pressure (IOP)-lowering efficacy of travoprost (0.004%) ophthalmic solution in subjects with normal tension glaucoma (NTG). METHODS: Travoprost ophthalmic solution was given once daily at 9 PM to subjects with newly diagnosed NTG or with NTG who had not received any ocular hypotensives within the previous 30 days. IOP was measured at three time points (9 AM, 1 PM, and 5 PM) at baseline and week 12 visits, and at one time point (9 AM) at week 4 and week 8 visits. Conjunctival hyperemia, superficial punctate keratopathy, and other adverse events were evaluated during the observation period. RESULTS: Thirty subjects (12 males and 18 females; mean age 65.6 years) from 32 subjects enrolled were included in the efficacy analysis. The mean IOPs (±standard deviation) of 16.6 ± 1.4, 15.7 ± 1.8, and 15.7 ± 2.2 mmHg at 9 AM, 1 PM, and 5 PM, respectively, at baseline reduced significantly to the mean IOPs of 13.0 ± 1.8, 12.7 ± 1.8, and 12.8 ± 1.6 mmHg, respectively, at week 12 (P < 0.0001 for every time point). Together with the mean IOPs of 13.4 ± 1.9 mmHg at week 4 and 13.2 ± 1.9 mmHg at week 8, the pooled IOP during the observation period for up to 12 weeks showed a statistically and clinically significant reduction of IOP at 9 AM. (3.4 mmHg or 20.3% reduction from baseline, P < 0.0001). There were no adverse events leading to treatment discontinuation. CONCLUSION: This multi-center collaborative study suggests that IOP-lowering efficacy of travoprost ophthalmic solution persists during the day at the clinically relevant level in subjects with NTG. FUNDING: Alcon Japan Ltd. TRIAL REGISTRATION: University Hospital Medical Information Network, UMIN ID: 000011621.

Successful displacement of a traumatic submacular hemorrhage in a 13-year-old boy treated by vitrectomy, subretinal injection of tissue plasminogen activator and intravitreal air tamponade: a case report.

BACKGROUND: The natural course of submacular hemorrhage resulting from traumatic choroidal rupture generally has a poor outcome unless treated. The intravitreal injection of gas only or gas with recombinant tissue plasminogen activator (rt-PA) has been reported to be effective, but has also been reported to induce severe complications such as retinal detachment and vitreous hemorrhage. Recently, we reported a safe and effective procedure for treating submacular hemorrhage due to polypoidal choroidal vasculopathy (PCV) with a low dose of rt-PA. Here we report the application of this procedure to a case of traumatic submacular hemorrhage in a 13-year-old boy, which achieved a good visual outcome. CASE PRESENTATION: A 13-year-old Japanese boy presented with a thick submacular hemorrhage in his left eye as a result of blunt trauma from being hit by a sinker. Best-corrected visual acuity (BCVA) was assessed as only able to perceive hand motions. We carried out a vitrectomy, subretinal injection of 4,000 IU rt-PA (6.9 µg) and air tamponade. The day after surgery, most of the submacular hemorrhage had moved to the inferior periphery. One month after the surgery, we observed cataract formation, thin remnants of the submacular hemorrhage and juxtafoveal choroidal rupture. We carried out cataract surgery and injected bevacizumab intravitreally to prevent the development of choroidal neovascularization. Two months after the second surgery, the submacular hemorrhage had totally disappeared and the patient had a BCVA of 20/40. CONCLUSION: Vitrectomy, subretinal injection of rt-PA, and intravitreal air tamponade may be a promising strategy for treating traumatic submacular hemorrhage in young patients.

The complete genome sequence of the Alphaentomopoxvirus Anomala cuprea entomopoxvirus, including its terminal hairpin loop sequences, suggests a potentially unique mode of apoptosis inhibition and mode of DNA replication.

Complete genome sequence of Anomala cuprea entomopoxvirus, which belongs to the genus Alphaentomopoxvirus, including its terminal hairpin loop sequences, is reported. This is the first genome sequence of Alphaentomopoxvirus reported, and hairpin loops in entomopoxviruses have not previously been sequenced. The genome is 245,717 bp, which is smaller than had previously been estimated for Alphaentomopoxvirus. The inverted terminal repeats are quite long, and experimental results suggest that one genome molecule has one type of hairpin at one end and another type at the other end. The genome contains unexpected ORFs, e.g., that for the ubiquitin-conjugating enzyme E2 of eukaryotes. The BIR and RING domains found in a single ORF for an inhibitor of apoptosis in baculoviruses and entomopoxviruses occurred in two different, widely separated ORFs. Furthermore, an ORF in the genome contains a serpin domain that was previously found in vertebrate poxviruses for apoptosis inhibition but not in insect viruses.

Further research on the biological function of inclusion bodies of Anomala cuprea entomopoxvirus, with special reference to the effect on the insecticidal activity of a Bacillus thuringiensis formulation.

BACKGROUND: Entomopoxviruses (EVs) form two types of inclusion body: spheroids, which contain virions, and spindles, which do not. The authors tested whether the spindles from a coleopteran EV, Anomala cuprea EV (ACEV), enhanced the insecticidal activity of a commercial Bacillus thuringiensis (Bt) formulation and the susceptibility of scarabaeid pest species in Japan to the virus's spheroids, to assess whether ACEV inclusion bodies are potential biological control agents for pest insects. RESULTS: Peroral inoculation with both ACEV spindles and the Bt toxin only or the complete Bt formulation shortened the survival and increased the mortality of treated insects compared with those of insects inoculated with Bt without the spindles (8-38 h of decrease in LT50 values among assays). ACEV showed high infectivity to a major scarabaeid pest species in Japanese sugar cane fields. CONCLUSION: The results suggest that spindles or the constituent protein fusolin can be used as a coagent with Bt formulations, and that fusolin coexpression with a Bt toxin in crops might improve the insecticidal efficacy. In addition, the spheroids are potential biocontrol agents for some scarabaeid pests that are not easy to control because of their underground habitation.

KONAGAbase: a genomic and transcriptomic database for the diamondback moth, Plutella xylostella.

BACKGROUND: The diamondback moth (DBM), Plutella xylostella, is one of the most harmful insect pests for crucifer crops worldwide. DBM has rapidly evolved high resistance to most conventional insecticides such as pyrethroids, organophosphates, fipronil, spinosad, Bacillus thuringiensis, and diamides. Therefore, it is important to develop genomic and transcriptomic DBM resources for analysis of genes related to insecticide resistance, both to clarify the mechanism of resistance of DBM and to facilitate the development of insecticides with a novel mode of action for more effective and environmentally less harmful insecticide rotation. To contribute to this goal, we developed KONAGAbase, a genomic and transcriptomic database for DBM (KONAGA is the Japanese word for DBM). DESCRIPTION: KONAGAbase provides (1) transcriptomic sequences of 37,340 ESTs/mRNAs and 147,370 RNA-seq contigs which were clustered and assembled into 84,570 unigenes (30,695 contigs, 50,548 pseudo singletons, and 3,327 singletons); and (2) genomic sequences of 88,530 WGS contigs with 246,244 degenerate contigs and 106,455 singletons from which 6,310 de novo identified repeat sequences and 34,890 predicted gene-coding sequences were extracted. The unigenes and predicted gene-coding sequences were clustered and 32,800 representative sequences were extracted as a comprehensive putative gene set. These sequences were annotated with BLAST descriptions, Gene Ontology (GO) terms, and Pfam descriptions, respectively. KONAGAbase contains rich graphical user interface (GUI)-based web interfaces for easy and efficient searching, browsing, and downloading sequences and annotation data. Five useful search interfaces consisting of BLAST search, keyword search, BLAST result-based search, GO tree-based search, and genome browser are provided. KONAGAbase is publicly available from our website (http://dbm.dna.affrc.go.jp/px/) through standard web browsers. CONCLUSIONS: KONAGAbase provides DBM comprehensive transcriptomic and draft genomic sequences with useful annotation information with easy-to-use web interfaces, which helps researchers to efficiently search for target sequences such as insect resistance-related genes. KONAGAbase will be continuously updated and additional genomic/transcriptomic resources and analysis tools will be provided for further efficient analysis of the mechanism of insecticide resistance and the development of effective insecticides with a novel mode of action for DBM.

The ATP-binding cassette transporter subfamily C member 2 in Bombyx mori larvae is a functional receptor for Cry toxins from Bacillus thuringiensis.

Bacillus thuringiensis is the most widely used biopesticide, and its Cry toxin genes are essential transgenes for the generation of insect-resistant transgenic crops. Recent reports have suggested that ATP-binding cassette transporter subfamily C2 (ABCC2) proteins are implicated in Cry intoxication, and that a single amino acid insertion results in high levels of resistance to Cry1 toxins. However, there is currently no available direct evidence of functional interactions between ABCC2 and Cry toxins. To address this important knowledge gap, we investigated the role of Bombyx mori ABCC2 (BmABCC2) or its mutant from a Cry1Ab-resistant B. mori strain on Cry1A toxin action. When we expressed BmABCC2 ectopically on Sf9 cells, it served as a functional receptor, and the single amino acid insertion found in BmABCC2 from Cry1Ab-resistant larvae resulted in lack of susceptibility to Cry1Ab and Cry1Ac. Using the same expression system, we found that Bo. mori cadherin-like receptor (BtR175) conferred susceptibility to Cry1A toxins, albeit to a lower degree than BmABCC2. Coexpression of BtR175 and BmABCC2 resulted in the highest cell susceptibility to Cry1A, Cry1F, and even the phylogenetically distant Cry8Ca toxin, when compared with expression of either receptor alone. The susceptibility observed in the coexpressing cells and that in Bo. mori larvae are likely to be correlated, suggesting that BtR175 and BmABCC2 are important factors determining larval susceptibility. Our study demonstrates, for the first time, Cry toxin receptor functionality for ABCC2, and highlights the crucial role of this protein and cadherin in the mechanism of action of Cry toxin.

Involvement of stem cell factor and c-kit in corneal wound healing in mice.

PURPOSE: To study the roles played by stem cell factor (SCF) and SCF receptor c-kit in wound healing of corneal epithelial cells. METHODS: A 2 mm corneal epithelial wound was made in control (WBB6F1(+/+)), SCF (Sl/Sl(d))-, and c-kit (W/W(v)) mutant mice, and the speed of wound healing, 5-bromo-2'-deoxyuridine (BrdU) incorporation, and scanning electron microscopic (SEM) morphology of the corneas were examined. The incorporation of BrdU and the degree of cell attachment in cultured mouse corneal epithelial cells (MCECs) isolated from WBB6F1(+/+), Sl/Sl(d), and W/W(v) mice were examined. Cultured immortalized human corneal epithelial cells (HCECs) were examined by a cell attachment assay after their exposure to anti-SCF antibodies, tyrosine kinase inhibitor (genistein), and competitive Arg-Gly-Asp (RGD) peptide, as well as on cultures treated with extracellular matrix. RESULTS: The speed of corneal wound healing was slower in Sl/Sl(d) and W/W(v) mice than in controls (p<0.01) and the speed of healing in Sl/Sl(d) mice recovered after topical application of SCF (8 ng/ml). No significant difference was found in the BrdU incorporation assay either in vivo or in vitro. Loosened epithelial cells were detected at wound margins in W/W(v) mice by SEM. The cell attachment rate was increased by 157% in cells from WBB6F1(+/+) and 252% in Sl/Sl(d) MCECs by recombinant mouse SCF; however, no significant difference was found in W/W(v) MCECs. Anti-SCF antibodies (Ab), genistein, and RGD peptide reduced the percentage of attached HCECs. Anti-SCF Ab inhibited the attachment of HCECs on fibronectin, laminin, or type IV collagen coated dishes. CONCLUSIONS: These findings indicate that the SCF/c-kit system may play a role in corneal wound healing through epithelial cell attachment.

Single amino acid mutation in an ATP-binding cassette transporter gene causes resistance to Bt toxin Cry1Ab in the silkworm, Bombyx mori.

Bt toxins derived from the arthropod bacterial pathogen Bacillus thuringiensis are widely used for insect control as insecticides or in transgenic crops. Bt resistance has been found in field populations of several lepidopteran pests and in laboratory strains selected with Bt toxin. Widespread planting of crops expressing Bt toxins has raised concerns about the potential increase of resistance mutations in targeted insects. By using Bombyx mori as a model, we identified a candidate gene for a recessive form of resistance to Cry1Ab toxin on chromosome 15 by positional cloning. BGIBMGA007792-93, which encodes an ATP-binding cassette transporter similar to human multidrug resistance protein 4 and orthologous to genes associated with recessive resistance to Cry1Ac in Heliothis virescens and two other lepidopteran species, was expressed in the midgut. Sequences of 10 susceptible and seven resistant silkworm strains revealed a common tyrosine insertion in an outer loop of the predicted transmembrane structure of resistant alleles. We confirmed the role of this ATP-binding cassette transporter gene in Bt resistance by converting a resistant silkworm strain into a susceptible one by using germline transformation. This study represents a direct demonstration of Bt resistance gene function in insects with the use of transgenesis.

A 50-kilodalton Cry2A peptide is lethal to Bombyx mori and Lymantria dispar.

The Cry2Aa3 gene was introduced into asporogenic Bacillus thuringiensis, and the synthesized protoxin killed Bombyx mori and Lymantria dispar larvae. Chymotrypsin hydrolyzed the linkages between 49Tyr/Val50 and 145Lys/Ser146 in the protoxin, and 50- and 58-kDa fragments were generated, respectively. Both peptides killed the larvae of both insects.

Clinical study of low back pain and radicular pain pathways by using l2 spinal nerve root infiltration: a randomized, controlled, clinical trial.

STUDY DESIGN: Randomized control trial (RCT) for L2 spinal nerve infiltration (L2 block) in clinical cases. OBJECTIVES: To confirm or refute the effect of L2 block using RCT, and to study the pathway of low back pain (LBP) and radicular pain in clinical cases. SUMMARY OF BACKGROUND DATA: It has been reported in animal experiments that one of the main pathways of pain originating from the lumbar spine is the sympathetic trunk through the L2 spinal nerve rootvia sympathetic afferents. METHODS: To evaluate the effectiveness of L2 block, patients who had LBP and were treated with nonsteroidal anti-inflammatory drugs for at least 2 weeks were then randomized to the L2 block or control block groups. The intensities of LBP and radicular pain were measured using visual analog scale and face scale before and at 5 minutes and 7 days after the injection. These values were compared, and the effects of the injections on the pain pathway were studied. RESULTS: The average visual analog scale scores for LBP before and at 5 minutes and 7 days after the injection were 69, 14, and 44 mm in the L2 block group and 68, 62, and 59 mm in the control block group, respectively. After L2 block, 28 patients reported adequate therapeutic effect at 10 weeks, and the effect lasted for more than 24 weeks in 10 of these patients. After control block, 9 patients reported adequate therapeutic effect at 10 and 24 weeks. CONCLUSION: The LBP and radicular pain pathways were likely interrupted by L2 block. An L2 block is useful in reducing LBP due to the disorders of L2 spinal nerve-innervated structures, such as the disc, facet joint, and sacroiliac joint. However, the therapeutic value of an L2 block may be occasionally insufficient to alleviate pain completely because of the short duration of its' effect.

The N-terminal region of an entomopoxvirus fusolin is essential for the enhancement of peroral infection, whereas the C-terminal region is eliminated in digestive juice.

The spindles of Anomala cuprea entomopoxvirus (AncuEPV), which are composed of glycoprotein fusolin, are known to enhance the peroral infectivity of AncuEPV itself and of nucleopolyhedroviruses. This has been demonstrated to involve the disruption of intestinal peritrophic membrane (PM), composed of chitin matrix, glycosaminoglycans, and proteins. To identify essential and nonessential regions for this enhancement activity, AncuEPV fusolin and its deletion mutants were expressed in Sf21 cells using a baculovirus system, and their enhancement abilities were analyzed. The recombinant fusolin enhanced the peroral infectivity of Bombyx mori nucleopolyhedrovirus up to 320-fold and facilitated the infection of host insect with AncuEPV. Deletion mutagenesis revealed that the N-terminal region (amino acids 1 to 253), a possible chitin-binding domain, is essential for the enhancement of infection, whereas the C-terminal region is entirely dispensable. The glycosylation-defective mutants N191Q, whose Asn(191) is replaced with Gln, and DeltaSIG, whose signal peptide is deleted, showed considerably reduced and abolished enhancing activities, respectively, indicating that the carbohydrate chain is important in the enhancing activity. Interestingly, the C-terminal dispensable region was digested by a serine protease(s) in insect digestive juice. Moreover, both the N-terminal conserved region and the carbohydrate chain were necessary not only for chitin binding but also for stability in digestive juice. A triple amino acid replacement mutant, IHE (Ile-His-Glu(161) to Ala-Ala-Ala), was stable in digestive juice and had chitin-binding ability but did not retain its enhancing activity. These results suggest that the enhancement of infectivity involves more than the tolerance to digestive juice and chitin-binding ability.

Selective inhibition of binding of Bacillus thuringiensis Cry1Ab toxin to cadherin-like and aminopeptidase proteins in brush-border membranes and dissociated epithelial cells from Bombyx mori.

Binding analyses with denatured epithelial membrane proteins from Bt (Bacillus thuringiensis) demonstrated at least two kinds of proteins, APNs (aminopeptidases N) and cadherin-like proteins, as possible receptors for the Cry1A class of Bt toxins. Two alternative models have been proposed, both based on initial toxin binding to a cadherin-like protein, but one involving APN and the other not. We have used two Bombyx mori strains (J65 and Kin), which are highly susceptible to Cry1Ab, to study the role of these two types of receptors on Cry1Ab toxin binding and cytotoxicity by means of the inhibitory effect of antibodies. BBMVs (brush-border membrane vesicles) of strain J65 incubated with labelled 125I-Cry1Ab revealed a marked reduction in reversible and irreversible binding when anti-BtR175 (a cadherin-like protein) was used for BBMV pre-treatment. By contrast, the anti-APN1 antibody specifically affected the irreversible binding, while the reversible binding component was not affected. This is the first time that binding of Cry1Ab to APN1 and to a cadherin-like protein from BBMVs in solution has been shown. Dissociated epithelial cells from the Kin strain were used to test the inhibitory effect of the antibodies on the cytotoxicity of Cry1Ab. Pre-incubation of the cells with the anti-BtR175 antibody conferred protection against Cry1Ab, but not the anti-APN1 antibody. Therefore our results seem to support the two models of the mode of action of Cry1Ab in Lepidoptera, depending on whether BBMVs or intact dissociated cells are used, suggesting that both pathways may co-operate for the toxicity of Cry1A toxins in vivo.

Binding of Bacillus thuringiensis Cry1A toxins to brush border membrane vesicles of midgut from Cry1Ac susceptible and resistant Plutella xylostella.

Plutella xylostella strain resistant (PXR) to Bacillus thuringiensis Cry1Ac toxin was not killed at even more than 1000 microg Cry1Ac/g diet but killed by Cry1Ab at 0.5 microg/g diet. In contrast, susceptible strain (PXS) was killed by Cry1Ac at 1 microg/g diet. Cy3-labeld Cry1A(s) binding to brush border membrane vesicles (BBMV) prepared from both strains were analyzed with direct binding assay. The Kd value of Cry1Aa to both BBMV was almost identical: 213.2 and 205.8 nM, and 263.5 and 265.0 nM for Cry1Ac. The highest Kd values were in Cry1Ab which showed most effective insecticidal activity in PXS and PXR, 2126 and 2463 nM, respectively. These results clearly showed that the BBMV from PXR and PXS could equally bind to Cry1Ac. The binding between BBMV and Cy3-labeled Cry1Ac was inhibited only by anti-175 kDa cadherin-like protein (CadLP) and -252 kDa protein antisera, but not by anti-120 kDa aminopeptidase. This supports that resistance in PXR resulted from the abortion of pore formation after the binding of Cry1Ac to the BBMV. And furthermore, the importance of 175K CadLP and P252 proteins in those bindings was suggested. We briefly discuss possible mechanisms of the resistance.