Polymer Nanoantidotes.

Intoxication is one of the most common causes of accidental death globally. Although some antidotes capable of neutralizing the toxicity of certain xenobiotics have become well established, the current reality is that clinicians primarily rely on nonspecific extracorporeal techniques to remove toxins. Nano-intervention strategies in which nanoantidotes neutralize toxicity in situ via physical interaction, chemical bonding, or biomimetic clearance have begun to show clinical potential. However, most nanoantidotes remain in the proof-of-concept stage, and the difficulty of constructing clinical relevance models and the unclear pharmacokinetics of nanoantidotes hinder their translation to clinic. This Concept reviews the detoxification mechanisms of polymer nanoantidotes and predicts the opportunities and challenges associated with their clinical application.

Site-Specific Fluorescent Labeling of the Cysteine-Rich Toxin, DkTx, for TRPV1 Ion Channel Imaging and Membrane Binding Studies.

Peptide toxins secreted by venomous animals bind to mammalian ion channel proteins and modulate their function. The high specificity of these toxins for their target ion channels enables them to serve as powerful tools for ion channel biology. Toxins labeled with fluorescent dyes are employed for the cellular imaging of channels and also for studying toxin-channel and toxin-membrane interactions. Several of these toxins are cysteine-rich, rendering the production of properly folded fluorescently labeled toxins technically challenging. Herein, we evaluate a variety of site-specific protein bioconjugation approaches for producing fluorescently labeled double-knot toxin (DkTx), a potent TRPV1 ion channel agonist that contains an uncommonly large number of cysteines (12 out of a total of 75 amino acids present in the protein). We find that popular cysteine-mediated bioconjugation approaches are unsuccessful as the introduction of a non-native cysteine residue for thiol modification leads to the formation of misfolded toxin species. Moreover, N-terminal aldehyde-mediated bioconjugation approaches are also not suitable as the resultant labeled toxin lacks activity. In contrast to these approaches, C-terminal bioconjugation of DkTx via the sortase bioconjugation technology yields functionally active fluorescently labeled DkTx. We employ this labeled toxin for imaging rat TRPV1 heterologously expressed in Xenopus laevis oocytes, as well as for performing membrane binding studies on giant unilamellar vesicles composed of different lipid compositions. Our studies set the stage for using fluorescent DkTx as a tool for TRPV1 biology and provide an informative blueprint for labeling cysteine-rich proteins.

Multifunctional carbonized nanogels to treat lethal acute hepatopancreatic necrosis disease.

BACKGROUND: Shrimp aquaculture has suffered huge economic losses over the past decade due to the outbreak of acute hepatopancreatic necrosis disease (AHPND), which is mainly caused by the bacteria Vibrio parahaemolyticus (V. parahaemolyticus) with the virulence pVA1 plasmid, which encodes a secretory photorhabdus insect-related (Pir) toxin composed of PirA and PirB proteins. The Pir toxin mainly attacks the hepatopancreas, a major metabolic organ in shrimp, thereby causing necrosis and loss of function. The pandemic of antibiotic-resistant strains makes the impact worse. METHODS: Mild pyrolysis of a mixture of polysaccharide dextran 70 and the crosslinker 1,8-diaminooctane at 180 â„ƒ for 3 h to form carbonized nanogels (DAO/DEX-CNGs) through controlled cross-linking and carbonization. The multifunctional therapeutic CNGs inherit nanogel-like structures and functional groups from their precursor molecules. RESULTS: DAO/DEX-CNGs manifest broad-spectrum antibacterial activity against Vibrio parahaemolyticus responsible for AHPND and even multiple drug-resistant strains. The polymer-like structures and functional groups on graphitic-carbon within the CNGs exhibit multiple treatment effects, including disruption of bacterial membranes, elevating bacterial oxidative stress, and neutralization of PirAB toxins. The inhibition of Vibrio in the midgut of infected shrimp, protection of hepatopancreas tissue from Pir toxin, and suppressing overstimulation of the immune system in severe V. parahaemolyticus infection, revealing that CNGs can effectively guard shrimp from Vibrio invasion. Moreover, shrimps fed with DAO/DEX-CNGs were carefully examined, such as the expression of the immune-related genes, hepatopancreas biopsy, and intestinal microbiota. Few adverse effects on shrimps were observed. CONCLUSION: Our work proposes brand-new applications of multifunctional carbon-based nanomaterials as efficient anti-Vibrio agents in the aquatic industry that hold great potential as feed additives to reduce antibiotic overuse in aquaculture.

Genome-wide association across Saccharomyces cerevisiae strains reveals substantial variation in underlying gene requirements for toxin tolerance.

Cellulosic plant biomass is a promising sustainable resource for generating alternative biofuels and biochemicals with microbial factories. But a remaining bottleneck is engineering microbes that are tolerant of toxins generated during biomass processing, because mechanisms of toxin defense are only beginning to emerge. Here, we exploited natural diversity in 165 Saccharomyces cerevisiae strains isolated from diverse geographical and ecological niches, to identify mechanisms of hydrolysate-toxin tolerance. We performed genome-wide association (GWA) analysis to identify genetic variants underlying toxin tolerance, and gene knockouts and allele-swap experiments to validate the involvement of implicated genes. In the process of this work, we uncovered a surprising difference in genetic architecture depending on strain background: in all but one case, knockout of implicated genes had a significant effect on toxin tolerance in one strain, but no significant effect in another strain. In fact, whether or not the gene was involved in tolerance in each strain background had a bigger contribution to strain-specific variation than allelic differences. Our results suggest a major difference in the underlying network of causal genes in different strains, suggesting that mechanisms of hydrolysate tolerance are very dependent on the genetic background. These results could have significant implications for interpreting GWA results and raise important considerations for engineering strategies for industrial strain improvement.

Comparison of Circulating Levels of Uremic Toxins in Hemodialysis Patients Treated with Medium Cut-Off Membranes and High-Flux Membranes: Theranova in Sisli Hamidiye Etfal (THE SHE) Randomized Control Study.

INTRODUCTION: Removal of uremic toxins is a main objective of hemodialysis; however, whether high-flux and medium cut-off (MCO) membranes differ as regards removal of middle and large uremic toxins is not clear. OBJECTIVE: To compare medium cut-off and high-flux dialyzers as regards their intra- and interdialysis effect on circulating levels of middle and large uremic toxins and serum albumin. METHODS: Fifty-two patients were randomized to have hemodialysis with either 3 months of high-flux dialyzer followed by 3 months of MCO or vice versa. Blood samples were taken before and after dialysis at the first and last sessions of each dialyzer for analyses of middle and large uremic toxins including inflammatory mediators and vascular endothelial growth factor (VEGF), and serum albumin. RESULTS: Reduction rates were higher, and postdialysis levels of ß-2 microglobulin, free kappa and lambda light chains, and myoglobulin were lower at the first and last sessions with MCO dialyzers compared to high-flux dialyzers (p < 0.05 for all). Last session predialysis levels of ß-2 microglobulin, free kappa light chain, and free lambda light chain were lower than first session predialysis levels in MCO dialyzers as compared to high-flux dialyzers (p < 0.05 for all). Last session levels of interleukin-6, interleukin-10, interleukin-17, and interferon-gamma did not differ between dialyzers (p > 0.05 for all). VEGF level was lower in the MCO group compared to the high-flux group (p = 0.043). Last session level of serum albumin with MCO dialyzers was lower than that with high-flux dialyzers (3.62 [3.45-3.88] vs. 3.78 [3.58-4.02] g/L) (p = 0.04) and 6.7% lower (p < 0.001) than at the first session of MCO dialyzers. CONCLUSION: The decline in circulating levels of several middle and large uremic toxins including VEGF following hemodialysis was more pronounced when using MCO membranes as compared to high-flux membranes while their effect on inflammatory molecules was similar.

Nanobioconjugates for Signal Amplification in Electrochemical Biosensing.

Nanobioconjugates are hybrid materials that result from the coalescence of biomolecules and nanomaterials. They have emerged as a strategy to amplify the signal response in the biosensor field with the potential to enhance the sensitivity and detection limits of analytical assays. This critical review collects a myriad of strategies for the development of nanobioconjugates based on the conjugation of proteins, antibodies, carbohydrates, and DNA/RNA with noble metals, quantum dots, carbon- and magnetic-based nanomaterials, polymers, and complexes. It first discusses nanobioconjugates assembly and characterization to focus on the strategies to amplify a biorecognition event in biosensing, including molecular-, enzymatic-, and electroactive complex-based approaches. It provides some examples, current challenges, and future perspectives of nanobioconjugates for the amplification of signals in electrochemical biosensing.

Worker Defensive Behavior Associated with Toxins in the Neotropical Termite Neocapritermes braziliensis (Blattaria, Isoptera, Termitidae, Termitinae).

Termite societies are abundant in the tropics, and are therefore exposed to multiple enemies and predators, especially during foraging activity. Soldiers constitute a specialized defensive caste, although workers also participate in this process, and even display suicidal behavior, which is the case with the species Neocapritermes braziliensis. Here we describe the morphology, mechanisms of action, and proteomics of the salivary weapon in workers of this species, which due to the autothysis of the salivary glands causes their body rupture, in turn releasing a defensive secretion, observed during aggressiveness bioassays. Salivary glands are paired, composed of two translucent reservoirs, ducts and a set of multicellular acini. Histological and ultrastructural techniques showed that acini are composed of two types of central cells, and small parietal cells located in the acinar periphery. Type I central cells were abundant and filled with a large amount of secretion, while type II central cells were scarce and presented smaller secretion. Parietal cells were often paired and devoid of secretion. The gel-free proteomic approach (shotgun) followed by mass spectrometry revealed 235 proteins in the defensive secretion, which were classified into functional groups: (i) toxins and defensins, (ii) folding/conformation and post-translational modifications, (iii) salivary gland detoxification, (iv) housekeeping proteins and (v) uncharacterized and hypothetical proteins. We highlight the occurrence of neurotoxins previously identified in arachnid venoms, which are novelties for termite biology, and contribute to the knowledge regarding the defense strategies developed by termite species from the Neotropical region.

Increasing the removal of protein-bound uremic toxins by liposome-supported hemodialysis.

Protein-bound uremic toxins (PBUTs) accumulate at high plasma levels and cause various deleterious effects in end-stage renal disease patients because their removal by conventional hemodialysis is severely limited by their low free-fraction levels in plasma. Here, we assessed the extent to which solute removal can be increased by adding liposomes to the dialysate. The uptake of liposomes by direct incubation in vitro showed an obvious dose-response relationship for p-cresyl sulfate (PCS) and indoxyl sulfate (IS) but not for hippuric acid (HA). The percent removal of both PCS and IS but not of HA was gradually increased with the increased concentration of liposomes in a rapid equilibrium dialysis setup. In vitro closed circulation showed that adding liposomes to the dialysate markedly increased the dialysances of PBUTs without greatly altering that of urea and creatinine. In vivo experiments in uremic rats demonstrated that adding liposomes to the dialysate resulted in higher reduction ratios (RRs) and more total solute removal (TSR) for several PBUTs compared to the conventional dialysate, which was approximately similar to the addition of bovine serum albumin to the dialysate. These findings highlight that as an adjunct to conventional hemodialysis, addition of liposomes to the dialysate could significantly improve the removal of protein-bound uremic solutes without greatly altering the removal of small, water-soluble solutes.

Membrane innovation: closer to native kidneys.

Modern methods in analytical biochemistry have established that uraemia is associated with the retention of proteins, both in their native state and post-translationally modified, over a wide range of molecular weights up to 60 kDa. Evidence is accumulating that these higher molecular weight retention solutes are important uraemic toxins, and therapies such as online haemodiafiltration (HDF), which enhance their removal, are associated with improved outcomes. However, HDF has limitations regarding cost, clinical implementation and the need for an external source of sterile substitution solution to maintain fluid balance. New membranes that have a solute removal profile more closely approaching that of the glomerular filtration barrier when used for conventional haemodialysis, while at the same time not allowing the passage of clinically significant amounts of beneficial proteins, are needed to address these limitations. Tighter control of the molecular characteristics of the polymers used for membrane fabrication, along with the introduction of additives and improvements in the manufacturing process, has led to membranes with a tighter pore size distribution that allows the use of an increased absolute pore size without leaking substantial amounts of albumin. At the same time, the wall thickness and internal diameter of membrane fibres have been decreased, enhancing convective transport within the dialyser without the need for an external source of substitution solution. These new expanded range membranes provide a solute removal profile more like that of the native kidney than currently available membranes when used in conventional haemodialysis.

Current approaches to middle molecule removal: room for innovation.

Aggressive removal of middle molecules or larger low-molecular-weight proteins (LMWPs) has been a growing concern following studies on their harmful effects on the mortality and morbidity of chronic dialysis patients. To remove larger LMWPs and some protein-bound uremic toxins (PBUTs), high- and medium-cutoff (HCOs and MCOs, respectively) membranes, convective therapy and protein adsorptive membranes are available. When we use HCO or MCO membranes for convective therapy, we have to take care to avoid massive albumin leakage during a dialysis session. Convection volume is an important element to increase middle molecule removal; however, a larger convection volume has a risk of larger leakage of albumin. Predilution hemodiafiltration is a useful measurement to increase larger LMWPs without massive albumin leakage. ß2-microglobulin (B2M), α1-microglobulin (A1M) and albumin leakage during a dialysis session are useful parameters for assessing middle-molecule removal. Reduction ratios of B2M >80% and of A1M >35% are favorable to improve severe dialysis-related symptoms. The efficacy of middle molecule removal should be evaluated in comparison with clinical outcomes, mortality, morbidity and the improvement of dialysis-related symptoms. Recently some dialysis-related symptoms such as sleep disturbance, skin itchiness and dialysis hypotension have been recognized as good surrogate makers for mortality. Further studies to evaluate the relationship between middle molecule or PBUTs removal and the improvement of patient symptoms should be performed in well-designed randomized controlled trials.

Large uremic toxins: an unsolved problem in end-stage kidney disease.

Patients with end-stage kidney disease (ESKD) on maintenance hemodialysis are subject to a high burden of inflammation and cardiovascular disease, driven at least in part by retention of uremic solutes. Existing dialysis technologies using high-flux membranes offer limited clearance of solutes >15 kDa. New approaches to improve the removal of large uremic toxins include the novel medium cut-off dialysis membranes with pores larger than those in high-flux membranes. These new membranes provide the potential to improve the clearance of large middle molecules up to 50 kDa. In this review, we discuss 18 uremic toxins with molecular weights between 15 and 60 kDa that are retained in ESKD, for which there is evidence of a link to inflammation and/or cardiovascular disease. These include inflammatory proteins, cytokines, adipokines and other signaling proteins. Improved clearance of this group of difficult to remove molecules has the potential to lead to improved outcomes in dialysis patients by reducing the burden of cardiovascular disease, which now needs to be assessed in robust clinical trials.

The utility of saliva testing in the estimation of uremic toxin levels in serum.

Background p-Cresol sulfate (pCS) and indoxyl sulfate (IS) are uremic toxins, high concentrations of which are related to renal failure progression. Saliva could become the first-line diagnostic sample of choice, especially for monitoring purposes. Recently, a method for determination of pCS and IS in saliva was developed. Since no data exist on correlations between the levels of toxins in saliva and serum, the applicability of saliva as a diagnostic material is yet to be established. Here, we present a study on the assessment of the utility of saliva testing in the estimation of uremic toxin levels in serum. Methods The study material included serum and unstimulated, fasting saliva obtained from healthy volunteers (n=26) and patients at all stages of chronic kidney diseases (CKD, n=93). The concentration of pCS and IS in saliva and serum (total and unbound fractions) was determined. The daytime variation of the toxins was studied. Results A correlation was found between pCS and IS in saliva and biological active fractions in serum (0.74; 0.81). The variation of the serum/saliva ratio during the day was negligible, with a median of 10% for pCS and 6% for IS, making saliva a reliable material for the estimation of the uremic toxins in circulation at any time of the day. Significant correlations were observed between salivary toxin levels and estimated glomerular filtration rate (pCS: -0.61; IS: -0.70) as well as significant differences in toxin levels between the stages of CKD. Conclusions Saliva could be a valuable diagnostic material for the estimation of toxin levels in circulation.

Adsorption of Protein-Bound Uremic Toxins Through Direct Hemoperfusion With Hexadecyl-Immobilized Cellulose Beads in Patients Undergoing Hemodialysis.

An accumulation of protein-bound uremic toxins (PBUTs) is one of major reasons for development of uremia-related complications. We examined the PBUT removal ability of a hexadecyl-immobilized cellulose bead (HICB)-containing column for patients undergoing hemodialysis. Adsorption of indoxyl sulfate (IS), a representative PBUT, to HICBs was examined in vitro. The HICB column was used in patients undergoing hemodialysis for direct hemoperfusion with a regular hemodialyzer. The serum IS, indole acetic acid (IAA), phenyl sulfate (PhS), and p-cresyl sulfate (PCS) levels were measured before and after passing the column. HICBs adsorbed protein-free (free) IS in a dose- and time-dependent manner in vitro (55.4 ± 1.4% adsorption of 1 millimolar, 251 µg/mL, IS for 1 h). In clinical studies, passing the HICB-containing column decreased the serum level of free IS, IAA, PhS, and PCS levels significantly (by 34.4 ± 30.0%, 34.8 ± 25.4%, 28.4 ± 18.0%, and 34.9 ± 22.1%, respectively), but not protein-bound toxins in maintenance hemodialysis patients. HICBs absorbed some amount of free PBUTs, but the clinical trial to use HICB column did not show effect to reduce serum PBUTs level in hemodialysis patients. Adsorption treatment by means of direct hemoperfusion with regular hemodialysis may become an attractive blood purification treatment to increase PBUT removal when more effective materials to adsorb PBUTs selectively will be developed.

Profiling of Hura crepitans L. latex by ultra-high-performance liquid chromatography/atmospheric pressure chemical ionisation linear ion trap Orbitrap mass spectrometry.

INTRODUCTION: The phytochemistry of the latex of Hura crepitans L. (Euphorbiaceae), a widespread tree in the Amazonian forest having many uses, is little known. Only huratoxin, a daphnane diterpene orthoester, has been described despite the high pharmacological potential of this kind of compounds. Glucosphingolipids (cerebrosides) are also known to be distributed in Euphorbiaceae latexes. OBJECTIVE: To tentatively identify daphnanes diterpenes and cerebrosides in the latex of H. crepitans. METHODS: An ethanolic extract of the lyophilised latex of H. crepitans was analysed by ultra-high-performance liquid chromatography (UHPLC) coupled with positive and negative atmospheric pressure chemical ionisation high-resolution mass spectrometry (APCI-HRMS) method using a quadrupole/linear ion trap/Orbitrap (LTQ-Orbitrap). Tandem mass spectrometry (MS/MS) spectra were recorded by two different fragmentation modes: collision induced dissociation (CID) and higher-energy collisional dissociation (HCD). RESULTS: The analysis of CID- and HCD-MS/MS spectra allowed to propose fragmentation patterns for daphnane esters and cerebrosides and highlight diagnostic ions in positive and negative ion modes. A total of 34 compounds including 24 daphnane esters and 10 cerebrosides have been tentatively annotated. Among them, 17 daphnane diterpenes bearing one or two acyl chains are new compounds and the cerebrosides are described in the genus Hura for the first time. CONCLUSION: This study revealed the chemical constituents of the latex of H. crepitans and particularly its richness and chemical diversity in daphnane diterpenes, more frequently encountered in the species of Thymelaeaceae.

Application of Peak Intensity Analysis to Measurements of Protein Binding to Lipid Vesicles and Erythrocytes Using Fluorescence Correlation Spectroscopy: Dependence on Particle Size.

Fluorescence correlation spectroscopy (FCS) is a sensitive analytical tool for investigation of processes accompanied by changes in the mobility of molecules and complexes. In the present work, peak intensity analysis (PIA) in combination with the solution stirring using FCS setup was applied to explore the interaction between fluorescently labeled protein ligands and corresponding receptors located on membranes. In the system composed of biotinylated liposomes and fluorescently labeled streptavidin as a ligand, PIA allowed us to determine the optimum receptor concentration and demonstrate the essential dependence of the binding efficacy on the length of the linker between the biotin group and the polar head group of the lipid. The binding was dependent on the size of liposomes which was varied by lipid extrusion through filters of different pore diameters. The sensitivity of the method was higher with the liposomes of larger sizes. The PIA approach can be applied not only to liposomes but also to relatively large objects, e.g., erythrocytes or Sepharose beads derivatized with lactose as a receptor for the binding of viscumin and ricin.

A Herbivore Tag-and-Trace System Reveals Contact- and Density-Dependent Repellence of a Root Toxin.

Foraging behavior of root feeding organisms strongly affects plant-environment-interactions and ecosystem processes. However, the impact of plant chemistry on root herbivore movement in the soil is poorly understood. Here, we apply a simple technique to trace the movement of soil-dwelling insects in their habitats without disturbing or restricting their interactions with host plants. We tagged the root feeding larvae of Melolontha melolontha with a copper ring and repeatedly located their position in relation to their preferred host plant, Taraxacum officinale, using a commercial metal detector. This method was validated and used to study the influence of the sesquiterpene lactone taraxinic acid ß-D-glucopyranosyl ester (TA-G) on the foraging of M. melolontha. TA-G is stored in the latex of T. officinale and protects the roots from herbivory. Using behavioral arenas with TA-G deficient and control plants, we tested the impact of physical root access and plant distance on the effect of TA-G on M. melolontha. The larvae preferred TA-G deficient plants to control plants, but only when physical root contact was possible and the plants were separated by 5 cm. Melolontha melolontha showed no preference for TA-G deficient plants when the plants were grown 15 cm apart, which may indicate a trade-off between the cost of movement and the benefit of consuming less toxic food. We demonstrate that M. melolontha integrates host plant quality and distance into its foraging patterns and suggest that plant chemistry affects root herbivore behavior in a plant-density dependent manner.

Functional Nanoparticles Activate a Decellularized Liver Scaffold for Blood Detoxification.

Extracorporeal devices have great promise for cleansing the body of virulence factors that are caused by venomous injuries, bacterial infections, and biological weaponry. The clinically used extracorporeal devices, such as artificial liver-support systems that are mainly based on dialysis or electrostatic interaction, are limited to remove a target toxin. Here, a liver-mimetic device is shown that consists of decellularized liver scaffold (DLS) populated with polydiacetylene (PDA) nanoparticles. DLS has the gross shape and 3D architecture of a liver, and the PDA nanoparticles selectively capture and neutralize the pore-forming toxins (PFTs). This device can efficiently and target-orientedly remove PFTs in human blood ex vivo without changing blood components or activating complement factors, showing potential application in antidotal therapy. This work provides a proof-of-principle for blood detoxification by a nanoparticle-activated DLS, and can lead to the development of future medical devices for antidotal therapy.

Supramolecular complexations of natural products.

Complexations of natural products with synthetic receptors as well as the use of natural products as host compounds are reviewed, with an emphasis on possible practical uses or on biomedical significance. Applications such as separation, sensing, enzyme monitoring, and protection of natural drugs are first outlined. We then discuss examples of complexes with all important classes of natural compounds, such as amino acids, peptides, nucleosides/nucleotides, carbohydrates, catecholamines, flavonoids, terpenoids/steroids, alkaloids, antibiotics and toxins.

Association of Filifactor alocis and its RTX toxin gene ftxA with periodontal attachment loss, and in synergy with Aggregatibacter actinomycetemcomitans.

The Gram-positive bacterium, Filifactor alocis is an oral pathogen, and approximately 50% of known strains encode a recently identified repeat-in-toxin (RTX) protein, FtxA. By assessing a longitudinal Ghanaian study population of adolescents (10-19 years of age; mean age 13.2 years), we recently discovered a possible correlation between deep periodontal pockets measured at the two-year follow-up, presence of the ftxA gene, and a high quantity of F. alocis. To further understand the contribution of F. alocis and FtxA in periodontal disease, we used qPCR in the present study to assess the carriage loads of F. alocis and the prevalence of its ftxA gene in subgingival plaque specimens, sampled at baseline from the Ghanaian cohort (n=500). Comparing these results with the recorded clinical attachment loss (CAL) longitudinal progression data from the two-year follow up, we concluded that carriers of ftxA-positive F. alocis typically exhibited higher loads of the bacterium. Moreover, high carriage loads of F. alocis and concomitant presence of the ftxA gene were two factors that were both associated with an enhanced prevalence of CAL progression. Interestingly, CAL progression appeared to be further promoted upon the simultaneous presence of F. alocis and the non-JP2 genotype of Aggregatibacter actinomycetemcomitans. Taken together, our present findings are consistent with the notion that F. alocis and its ftxA gene promotes CAL during periodontal disease.

Structural Analysis of Membrane-associated Forms of Helicobacter pylori VacA Toxin.

Helicobacter pylori colonizes the stomach in about half of the human population, leading to an increased risk of peptic ulcer disease and gastric cancer. H. pylori secretes an 88 kDa VacA toxin that contributes to pathogenesis. VacA assembles into oligomeric complexes in solution and forms anion-selective channels in cell membranes. Cryo-electron microscopy (cryo-EM) analyses of VacA oligomers in solution provided insights into VacA oligomerization but failed to reveal the structure of the hydrophobic N-terminal region predicted to be a pore-forming domain. In this study, we incubated VacA with liposomes and used single particle cryo-EM to analyze detergent-extracted VacA oligomers. A 3D structure of detergent-solubilized VacA hexamers revealed the presence of six α-helices extending from the center of the oligomers, a feature not observed in previous studies of water-soluble VacA oligomers. Cryo-electron tomography analysis and 2D averages of VacA associated with liposomes confirmed that central regions of the membrane-associated VacA oligomers can insert into the lipid bilayer. However, insertion is heterogenous, with some membrane-associated oligomers appearing only partially inserted and others sitting on top of the bilayer. These studies indicate that VacA undergoes a conformational change when contacting the membrane and reveal an α-helical region positioned to extend into the membrane. Although the reported VacA 3D structure does not represent a selective anion channel, our combined single particle 3D analysis, cryo-electron tomography, and modeling allow us to propose a model for the structural organization of the VacA N-terminus in the context of a hexamer as it inserts into the membrane.