Oxygen matters: Unraveling the role of oxygen in the neuronal response to cisplatin.

BACKGROUND AND AIMS: Cell culture is a fundamental experimental tool for understanding cell physiology. However, translating these findings to in vivo settings has proven challenging. Replicating donor tissue conditions, including oxygen levels, is crucial for achieving meaningful results. Nevertheless, oxygen culture conditions are often overlooked, particularly in the context of chemotherapy-induced neurotoxicity. METHODS: In this study, we investigated the role of oxygen levels in primary neuronal cultures by comparing neuronal performance under cisplatin exposure (1 µg/mL) in supraphysiological normoxia (representing atmospheric conditions in a standard incubator; 18.5% O2) and physioxia (representing physiologic oxygen conditions in nervous tissue; 5% O2). Experiments were also conducted to assess survival, neurite development, senescence marker expression, and proinflammatory cytokine secretion. RESULTS: Under control conditions, both oxygen concentration conditions exhibited similar behaviors. However, after cisplatin administration, sensory neurons cultured under supraphysiological normoxic conditions show higher mortality, exhibit an evolutionarily proinflammatory cytokine profile over time, and activate apoptotic-regulated neuron death markers. In contrast, under physiological conditions, neurons treated with cisplatin exhibited senescence marker expression and an attenuated inflammatory secretome. INTERPRETATION: These results underscore the critical role of oxygen in neuronal culture, particularly in studying compounds where neuronal damage is mechanistically linked to oxidative stress. Even at identical doses of evaluated neurotoxic drugs, distinct cellular phenotypic fates can emerge, impacting translatability to the in vivo setting.

The RIN4-like/NOI proteins NOI10 and NOI11 modulate the response to biotic stresses mediated by RIN4 in Arabidopsis.

KEY MESSAGE: NOI10 and NOI11 are two RIN4-like/NOI proteins that participate in the immune response of the Arabidopsis plant and affect the RIN4-regulated mechanisms involving the R-proteins RPM1 and RPS2. The immune response in plants depends on the regulation of signaling pathways triggered by pathogens and herbivores. RIN4, a protein of the RIN4-like/NOI family, is considered to be a central immune signal in the interactions of plants and pathogens. In Arabidopsis thaliana, four of the 15 members of the RIN4-like/NOI family (NOI3, NOI5, NOI10, and NOI11) were induced in response to the plant herbivore Tetranychus urticae. While overexpressing NOI10 and NOI11 plants did not affect mite performance, opposite callose accumulation patterns were observed when compared to RIN4 overexpressing plants. In vitro and in vivo analyses demonstrated the interaction of NOI10 and NOI11 with the RIN4 interactors RPM1, RPS2, and RIPK, suggesting a role in the context of the RIN4-regulated immune response. Transient expression experiments in Nicotiana benthamiana evidenced that NOI10 and NOI11 differed from RIN4 in their functionality. Furthermore, overexpressing NOI10 and NOI11 plants had significant differences in susceptibility with WT and overexpressing RIN4 plants when challenged with Pseudomonas syringae bacteria expressing the AvrRpt2 or the AvrRpm1 effectors. These results demonstrate the participation of NOI10 and NOI11 in the RIN4-mediated pathway. Whereas RIN4 is considered a guardee protein, NOI10 and NOI11 could act as decoys to modulate the concerted activity of effectors and R-proteins.

From In Vitro Promise to In Vivo Reality: An Instructive Account of Infection Model Evaluation of Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are regarded as a promising alternative to traditional antibiotics in the face of ever-increasing resistance. However, many AMPs fail to progress into clinics due to unexpected difficulties found in preclinical in vivo phases. Our research has focused on crotalicidin (Ctn), an AMP from snake venom, and a fragment thereof, Ctn[15-34], with improved in vitro antimicrobial and anticancer activities and remarkable serum stability. As the retroenantio versions of both AMPs maintained favorable profiles, in this work, we evaluate the in vivo efficacy of both the native-sequence AMPs and their retroenantio counterparts in a murine infection model with Acinetobacter baumannii. A significant reduction in bacterial levels is found in the mice treated with Ctn[15-34]. However, contrary to expectations, the retroenantio analogs either exhibit toxicity or lack efficacy when administered to mice. Our findings underscore the critical importance of in vivo infection model evaluation to fully calibrate the therapeutic potential of AMPs.

Identification of Reference Genes for Precise Expression Analysis during Germination in Chenopodium quinoa Seeds under Salt Stress.

Chenopodium quinoa Willd. (quinoa), a member of the Amaranthaceae family, is an allotetraploid annual plant, endemic to South America. The plant of C. quinoa presents significant ecological plasticity with exceptional adaptability to several environmental stresses, including salinity. The resilience of quinoa to several abiotic stresses, as well as its nutritional attributes, have led to significant shifts in quinoa cultivation worldwide over the past century. This work first defines germination sensu stricto in quinoa where the breakage of the pericarp and the testa is followed by endosperm rupture (ER). Transcriptomic changes in early seed germination stages lead to unstable expression levels in commonly used reference genes that are typically stable in vegetative tissues. Noteworthy, no suitable reference genes have been previously identified specifically for quinoa seed germination under salt stress conditions. This work aims to identify these genes as a prerequisite step for normalizing qPCR data. To this end, germinating seeds from UDEC2 and UDEC4 accessions, with different tolerance to salt, have been analyzed under conditions of absence (0 mM NaCl) and in the presence (250 mM NaCl) of sodium chloride. Based on the relevant literature, six candidate reference genes, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Monensin sensitivity1 (MON1), Polypyrimidine tract-binding protein (PTB), Actin-7 (ACT7), Ubiquitin-conjugating enzyme (UBC), and 18S ribosomal RNA (18S), were selected and assessed for stability using the RefFinder Tool encompassing the statistical algorithms geNorm, NormFinder, BestKeeper, and ΔCt in the evaluation. The data presented support the suitability of CqACT7 and CqUBC as reference genes for normalizing gene expression during seed germination under salinity stress. These recommended reference genes can be valuable tools for consistent qPCR studies on quinoa seeds.

Repair of Long Peripheral Nerve Defects in Sheep: A Translational Model for Nerve Regeneration.

Despite advances in microsurgery, full functional recovery of severe peripheral nerve injuries is not commonly attained. The sheep appears as a good preclinical model since it presents nerves with similar characteristics to humans. In this study, we induced 5 or 7 cm resection in the peroneal nerve and repaired with an autograft. Functional evaluation was performed monthly. Electromyographic and ultrasound tests were performed at 6.5 and 9 months postoperation (mpo). No significant differences were found between groups with respect to functional tests, although slow improvements were seen from 5 mpo. Electrophysiological tests showed compound muscle action potentials (CMAP) of small amplitude at 6.5 mpo that increased at 9 mpo, although they were significantly lower than the contralateral side. Ultrasound tests showed significantly reduced size of tibialis anterior (TA) muscle at 6.5 mpo and partially recovered size at 9 mpo. Histological evaluation of the grafts showed good axonal regeneration in all except one sheep from autograft 7 cm (AG7) group, while distal to the graft there was a higher number of axons than in control nerves. The results indicate that sheep nerve repair is a useful model for investigating long-gap peripheral nerve injuries.

A novel decellularized nerve graft for repairing peripheral nerve long gap injury in the rat.

Decellularized nerve allografts are an alternative to autograft for repairing severe nerve injuries, since they have higher availability and do not induce rejection. In this study, we have assessed the regenerative potential of a novel decellularization protocol for human and rat nerves for repairing nerve resections, compared to the gold standard autograft. A 15-mm gap in the sciatic nerve was repaired with decellularized rat allograft (DC-RA), decellularized human xenograft (DC-HX), or fresh autograft (AG). Electrophysiology tests were performed monthly to evaluate muscle reinnervation, whereas histological and immunohistochemical analyses of the grafts were evaluated at 4 months. A short-term study was also performed to compare the differences between the two decellularized grafts (DC-RA and DC-HX) in early phases of regeneration. The decellularization process eliminated cellularity while preserving the ECM and endoneurial tubules of both rat and human nerves. Higher amount of reinnervation was observed in the AG group compared to the DC-RA group, while only half of the animals of the DC-HX showed distal muscle reinnervation. The number of regenerating myelinated axons in the mid-graft was similar between AG and DC-RA and lower in DC-HX graft, but significantly lower in both DC grafts distally. At short term, fibroblasts repopulated the DC-RA graft, supporting regenerated axons, whereas an important fibrotic reaction was observed around DC-HX grafts. In conclusion, the decellularized allograft sustained regeneration through a long gap in the rat although at a slower rate compared to the ideal autograft, whereas regeneration was limited or even failed when using a decellularized xenograft.

Sodium solute symporter and cadherin proteins act as Bacillus thuringiensis Cry3Ba toxin functional receptors in Tribolium castaneum.

Understanding how Bacillus thuringiensis (Bt) toxins interact with proteins in the midgut of susceptible coleopteran insects is crucial to fully explain the molecular bases of Bt specificity and insecticidal action. In this work, aminopeptidase N (TcAPN-I), E-cadherin (TcCad1), and sodium solute symporter (TcSSS) have been identified by ligand blot as putative Cry3Ba toxin-binding proteins in Tribolium castaneum (Tc) larvae. RNA interference knockdown of TcCad1 or TcSSS proteins resulted in decreased susceptibility to Cry3Ba toxin, demonstrating the Cry toxin receptor functionality for these proteins. In contrast, TcAPN-I silencing had no effect on Cry3Ba larval toxicity, suggesting that this protein is not relevant in the Cry3Ba toxin mode of action in Tc. Remarkable features of TcSSS protein were the presence of cadherin repeats in its amino acid sequence and that a TcSSS peptide fragment containing a sequence homologous to a binding epitope found in Manduca sexta and Tenebrio molitor Bt cadherin functional receptors enhanced Cry3Ba toxicity. This is the first time that the involvement of a sodium solute symporter protein as a Bt functional receptor has been demonstrated. The role of this novel receptor in Bt toxicity against coleopteran insects together with the lack of receptor functionality of aminopeptidase N proteins might account for some of the differences in toxin specificity between Lepidoptera and Coleoptera insect orders.

Beyond the limiting gap length: peripheral nerve regeneration through implantable nerve guidance conduits.

Peripheral nerve damage results in the loss of sensorimotor and autonomic functions, which is a significant burden to patients. Furthermore, nerve injuries greater than the limiting gap length require surgical repair. Although autografts are the preferred clinical choice, their usage is impeded by their limited availability, dimensional mismatch, and the sacrifice of another functional donor nerve. Accordingly, nerve guidance conduits, which are tubular scaffolds engineered to provide a biomimetic environment for nerve regeneration, have emerged as alternatives to autografts. Consequently, a few nerve guidance conduits have received clinical approval for the repair of short-mid nerve gaps but failed to regenerate limiting gap damage, which represents the bottleneck of this technology. Thus, it is still necessary to optimize the morphology and constituent materials of conduits. This review summarizes the recent advances in nerve conduit technology. Several manufacturing techniques and conduit designs are discussed, with emphasis on the structural improvement of simple hollow tubes, additive manufacturing techniques, and decellularized grafts. The main objective of this review is to provide a critical overview of nerve guidance conduit technology to support regeneration in long nerve defects, promote future developments, and speed up its clinical translation as a reliable alternative to autografts.

Tribolium castaneum Apolipophorin-III acts as an immune response protein against Bacillus thuringiensis Cry3Ba toxic activity.

In this study, a 2.1-fold Apolipophorin-III mRNA up-regulation was found in Tribolium castaneum larvae challenged with Bacillus thuringiensis Cry3Ba spore-crystal mixture. Knockdown of Apolipophorin-III by RNAi resulted in increased T. castaneum larvae susceptibility following Cry3Ba spore-crystal treatment, demonstrating Apolipophorin-III involvement in insect defense against B. thuringiensis. We showed that Apolipophorin-III participates in T. castaneum immune response to B. thuringiensis activating the prophenoloxidase cascade since: (i) phenoloxidase activity significantly increased after Cry3Ba spore-crystal treatment compared to untreated or Cry1Ac spore-crystal treated larvae and (ii) phenoloxidase activity in Cry3Ba spore-crystal treated Apolipophorin-III silenced larvae was 71±14% lower than that of non-silenced intoxicated larvae.

Prohibitin, an essential protein for Colorado potato beetle larval viability, is relevant to Bacillus thuringiensis Cry3Aa toxicity.

Bacillus thuringienesis (Bt) Cry toxins constitute the most extensively used environmentally safe biopesticide and their mode of action relies on the interaction of the toxins with membrane proteins in the midgut of susceptible insects that mediate toxicity and insect specificity. Therefore, identification of Bt Cry toxin interacting proteins in the midgut of target insects and understanding their role in toxicity is of great interest to exploit their insecticidal action. Using ligand blot, we demonstrated that Bt Cry3Aa toxin bound to a 30kDa protein in Colorado potato beetle (CPB) larval midgut membrane, identified by sequence homology as prohibitin-1 protein. Prohibitins comprise a highly conserved family of proteins implicated in important cellular processes. We obtained the complete CPB prohibitin-1 DNA coding sequence of 828pb, in silico translated into a 276-amino acid protein. The analysis at the amino acid level showed that the protein contains a prohibitin-homology domain (Band7_prohibitin, cd03401) conserved among prohibitin proteins. A striking feature of the CPB identified prohibitin-1 is the predicted presence of cadherin elements, potential binding sites for Cry toxins described in other Bt susceptible insects. We also showed that CPB prohibitin-1 protein partitioned into both, detergent soluble and insoluble membrane fractions, as well as a prohibitin-2 homologous protein, previously reported to form functional complexes with prohibitin-1 in other organisms. Prohibitin complexes act as membrane scaffolds ensuring the recruitment of membrane proteases to facilitate substrate processing. Accordingly, sequestration of prohibitin-1 by an anti-prohibitin-1 antibody impaired the Cry3Aa toxin inhibition of the proteolytic cleavage of a fluorogenic synthetic substrate of an ADAM-like metalloprotease previously reported to proteolize this toxin. In this work, we also demonstrated that prohibitin-1 RNAi silencing in CPB larvae produced deleterious effects and together with a LD50 Cry3Aa toxin treatment resulted in a highly efficient short term response since 100% larval mortality was achieved just 5days after toxin challenge. Therefore, the combination of prohibitin RNAi and Cry toxin reveals as an effective strategy to improve crop protection.

Decellularized Graft for Repairing Severe Peripheral Nerve Injuries in Sheep.

BACKGROUND AND OBJECTIVES: Peripheral nerve injuries resulting in a nerve defect require surgical repair. The gold standard of autograft (AG) has several limitations, and therefore, new alternatives must be developed. The main objective of this study was to assess nerve regeneration through a long gap nerve injury (50 mm) in the peroneal nerve of sheep with a decellularized nerve allograft (DCA). METHODS: A 5-cm long nerve gap was made in the peroneal nerve of sheep and repaired using an AG or using a DCA. Functional tests were performed once a month and electrophysiology and echography evaluations at 6.5 and 9 months postsurgery. Nerve grafts were harvested at 9 months for immunohistochemical and morphological analyses. RESULTS: The decellularization protocol completely eliminated the cells while preserving the extracellular matrix of the nerve. No significant differences were observed in functional tests of locomotion and pain response. Reinnervation of the tibialis anterior muscles occurred in all animals, with some delay in the DCA group compared with the AG group. Histology showed a preserved fascicular structure in both AG and DCA; however, the number of axons distal to the nerve graft was higher in AG than in DCA. CONCLUSION: The decellularized graft assayed supported effective axonal regeneration when used to repair a 5-cm long gap in the sheep. As expected, a delay in functional recovery was observed compared with the AG because of the lack of Schwann cells.

Comparative and evolutionary analysis of Arabidopsis RIN4-like/NOI proteins induced by herbivory.

The spider mite Tetranychus urticae is an economically important agricultural pest, which feeds on a broad spectrum of plant species. In an RNAseq experiment performed in our laboratory, 4 of the 15 members of the RIN4-like/NOI family of Arabidopsis thaliana were significantly overexpressed after T. urticae infestation. Two of them (NOI3 and NOI5) are shorter and harbour one NOI domain, which characterises this family, and the other two (NOI10 and NOI11) have two-NOI domains. The only member of this family characterized is RIN4, a two-NOI intrinsically disordered protein anchored to the plasma membrane and involved in plant defence against bacterial pathogens. The function of all other members of the RIN4-like/NOI Arabidopsis family and their putative role in herbivore defence remains unknown. We perform a comparative genomic analysis of RIN4-like/NOI sequences to study the evolutionary features of this protein family and the distribution of its members among species. We show that short one-NOI proteins were more numerous and exhibited lower disorder propensity compared to two-NOI members. NOI10 and NOI11, from the two-NOI group, are included in a clade-specific expansion of Brassicaceae with unique predicted posttranslational modification sites and clear predicted structural differences from RIN4. Our analysis suggests that the members of the RIN4-like/NOI family upregulated after mite feeding have novel functions different from those assigned to RIN4, likely involving adaptation to stress specialisation.

New insights into peripheral nerve regeneration: The role of secretomes.

Neurons of the peripheral nervous system retain the intrinsic capability of regenerate their axons after injury, by triggering a complex activation response. This genetic switch is dependent of signals from the injured axon. Schwann cells (SCs) in the distal stump of an injured nerve also play an active role in the local regulation of axonal programs, by using cell-to-cell contacts but also secreted signals, the so-called secretome. Secretome contains all the proteins (cytokines, growth factors and others) secreted by the cell and includes extracellular vesicles. The released vesicles can transport signaling proteins and both coding and regulatory RNAs, thus facilitating multilevel communication. It is nowadays clear that secretome of SCs is fundamental to both orchestrate Wallerian degeneration and to sustain axonal regeneration. Therefore, the use of secretome has emerged as an alternative to cell therapy in the field of tissue regeneration. In fact, separate components of SC secretome have been extensively used in experimental models to enhance peripheral nerve regeneration after injury. However, the most used secretome in neural therapies has been the one derived from mesenchymal (MSC) or other derived stem cells. In fact, the effects of cell therapy with MSCs have been mainly associated with the secretion of bioactive molecules and extracellular vesicles, which constitute their secretome. In this review, we first describe the role of SC and macrophage secretomes on Wallerian degeneration and axonal regeneration after peripheral nerve injury. Then, we review the different works reported in the literature that have used secretomes of SCs or MSCs in the treatment of peripheral nerve injuries in experimental models, to highlight the use of secretomes as a promising cell-free therapeutic approach, that reduces some of the risks associated with the use of cells, such as tumor formation or rejection.

Repair of Long Nerve Defects with a New Decellularized Nerve Graft in Rats and in Sheep.

Decellularized nerve allografts (DC) are an alternative to autografts (AG) for repairing severe peripheral nerve injuries. We have assessed a new DC provided by VERIGRAFT. The decellularization procedure completely removed cellularity while preserving the extracellular matrix. We first assessed the DC in a 15 mm gap in the sciatic nerve of rats, showing slightly delayed but effective regeneration. Then, we assayed the DC in a 70 mm gap in the peroneal nerve of sheep compared with AG. Evaluation of nerve regeneration and functional recovery was performed by clinical, electrophysiology and ultrasound tests. No significant differences were found in functional recovery between groups of sheep. Histology showed a preserved fascicular structure in the AG while in the DC grafts regenerated axons were grouped in small units. In conclusion, the DC was permissive for axonal regeneration and allowed to repair a 70 mm long gap in the sheep nerve.

Plant Defenses Against Tetranychus urticae: Mind the Gaps.

The molecular interactions between a pest and its host plant are the consequence of an evolutionary arms race based on the perception of the phytophagous arthropod by the plant and the different strategies adopted by the pest to overcome plant triggered defenses. The complexity and the different levels of these interactions make it difficult to get a wide knowledge of the whole process. Extensive research in model species is an accurate way to progressively move forward in this direction. The two-spotted spider mite, Tetranychus urticae Koch has become a model species for phytophagous mites due to the development of a great number of genetic tools and a high-quality genome sequence. This review is an update of the current state of the art in the molecular interactions between the generalist pest T. urticae and its host plants. The knowledge of the physical and chemical constitutive defenses of the plant and the mechanisms involved in the induction of plant defenses are summarized. The molecular events produced from plant perception to the synthesis of defense compounds are detailed, with a special focus on the key steps that are little or totally uncovered by previous research.

A neurotoxin that specifically targets Anopheles mosquitoes.

Clostridial neurotoxins, including tetanus and botulinum neurotoxins, generally target vertebrates. We show here that this family of toxins has a much broader host spectrum, by identifying PMP1, a clostridial-like neurotoxin that selectively targets anopheline mosquitoes. Isolation of PMP1 from Paraclostridium bifermentans strains collected in anopheline endemic areas on two continents indicates it is widely distributed. The toxin likely evolved from an ancestral form that targets the nervous system of similar organisms, using a common mechanism that disrupts SNARE-mediated exocytosis. It cleaves the mosquito syntaxin and employs a unique receptor recognition strategy. Our research has an important impact on the study of the evolution of clostridial neurotoxins and provides the basis for the use of P. bifermentans strains and PMP1 as innovative, environmentally friendly approaches to reduce malaria through anopheline control.

Tribolium castaneum immune defense genes are differentially expressed in response to Bacillus thuringiensis toxins sharing common receptor molecules and exhibiting disparate toxicity.

In Tribolium castaneum larvae we have demonstrated by RNA interference knockdown that the Bacillus thuringiensis Cry3Ba toxin receptors Cadherin-like and Sodium solute symporter proteins are also functional receptors of the less active Cry3Aa toxin. Differences in susceptibility to B. thuringiensis infection might not only rely on toxin-receptor interaction but also on host defense mechanisms. We compared the expression of the immune related genes encoding Apolipophorin-III and two antimicrobial peptides, Defensin3 and Defensin2 after B. thuringiensis challenge. All three genes were up-regulated following Cry3Ba spore-crystal intoxication whereas only Defensins gene expression was induced upon Cry3Aa spore-crystal treatment, evidencing a possible association between host immune response and larval susceptibility to B. thuringiensis. We assessed the antimicrobial activity spectra of T. castaneum defensins peptide fragments and found that a peptide fragment of Defensin3 was effective against the human microbial pathogens, Escherichia coli, Staphylococcus aureus and Candida albicans, being S. aureus the most susceptible one.

Proteome response of Tribolium castaneum larvae to Bacillus thuringiensis toxin producing strains.

Susceptibility of Tribolium castaneum (Tc) larvae was determined against spore-crystal mixtures of five coleopteran specific and one lepidopteran specific Bacillus thuringiensis Cry toxin producing strains and those containing the structurally unrelated Cry3Ba and Cry23Aa/Cry37Aa proteins were found toxic (LC(50) values 13.53 and 6.30 µg spore-crystal mixture/µL flour disc, respectively). Using iTRAQ combined with LC-MS/MS allowed the discovery of seven novel differentially expressed proteins in early response of Tc larvae to the two active spore-crystal mixtures. Proteins showing a statistically significant change in treated larvae compared to non-intoxicated larvae fell into two major categories; up-regulated proteins were involved in host defense (odorant binding protein C12, apolipophorin-III and chemosensory protein 18) and down-regulated proteins were linked to metabolic pathways affecting larval metabolism and development (pyruvate dehydrogenase Eα subunit, cuticular protein, ribosomal protein L13a and apolipoprotein LI-II). Among increased proteins, Odorant binding protein C12 showed the highest change, 4-fold increase in both toxin treatments. The protein displayed amino acid sequence and structural homology to Tenebrio molitor 12 kDa hemolymph protein b precursor, a non-olfactory odorant binding protein. Analysis of mRNA expression and mortality assays in Odorant binding protein C12 silenced larvae were consistent with a general immune defense function of non-olfactory odorant binding proteins. Regarding down-regulated proteins, at the transcriptional level, pyruvate dehydrogenase and cuticular genes were decreased in Tc larvae exposed to the Cry3Ba producing strain compared to the Cry23Aa/Cry37Aa producing strain, which may contribute to the developmental arrest that we observed with larvae fed the Cry3Ba producing strain. Results demonstrated a distinct host transcriptional regulation depending upon the Cry toxin treatment. Knowledge on how insects respond to Bt intoxication will allow designing more effective management strategies for pest control.

Functional significance of membrane associated proteolysis in the toxicity of Bacillus thuringiensis Cry3Aa toxin against Colorado potato beetle.

Bacillus thuringiensis Cry toxins are widely used as biocontrol agents in bioinsecticides and transgenic plants. In the three domain-Cry toxins, domain II has been identified as an important determinant of their highly specific activity against insects. In this work, we assessed the role in membrane associated proteolysis and toxicity in Colorado potato beetle (CPB) of a previously reported ADAM recognition motif present in Cry3Aa toxin domain II. We used site-directed mutagenesis to modify the Bacillus thuringiensis cry3A gene in amino acid residues 344, 346, 347, 351 and 353 of the ADAM recognition motif in Cry3Aa toxin. Cry3Aa toxin mutants displayed decreased toxicity when compared to the wild type toxin and impaired ability to compete CPB brush border membrane associated cleavage of an ADAM fluorogenic substrate. Although the proteolytic profile of Cry3Aa toxin mutants generated by brush border membrane associated proteases was similar to that of Cry3Aa toxin, the metalloprotease inhibitor 1,10-phenanthroline was less efficient on the proteolysis of mutants than on that of the wild type toxin. The relevance of the Cry3Aa-ADAM interaction through the predicted recognition sequence was further confirmed by analyzing the effect of membrane integrity disturbance on Cry3Aa toxin membrane associated proteolysis and CPB larvae toxicity. Data support that Cry3Aa proteolysis, as a result of the interaction with ADAM through the Cry3Aa recognition motif, is essential for Cry3Aa toxic action in CPB. Detailed knowledge of Cry3Aa interaction with CPB midgut membrane should facilitate the development of more effective Bt based products against this devastating pest and other Coleoptera.