Investigating the effects of percutaneous endovascular aneurysm repair for abdominal aortic aneurysm on the lumen size of the common femoral artery.

BACKGROUND: Percutaneous endovascular aneurysm repair (PEVAR) is the definitive therapy of choice for abdominal aortic aneurysms worldwide. However, current literature regarding the anatomic changes in the common femoral artery (CFA) post-PEVAR is sparse and contradictory, and a significant proportion of these studies did not control for the potential confounding effects of ethnicity. Thus, this study aims to investigate the anatomical effects of PEVAR on the CFA using an Asian study cohort. METHODS: Between January 2019 and September 2023, the records of 113 patients who received PEVAR were reviewed. Groins with previous surgical interventions were excluded. The most proximate pre- and postoperative CT angiography of patients receiving PEVAR via the Perclose ProGlide™ Suture-Mediated Closure System were retrospectively analysed for changes in both the CFA inner luminal diameter (ID) and outer diameter (OD), the latter also encompassing the arterial walls. Access site complications within 3 months post-PEVAR were also recorded per patient. RESULTS: One hundred seventeen groins from 60 patients were included in this study, with 1 report of pseudoaneurysm. The CFA ID exhibited a 0.167 mm decrease (p-value = 0.0403), while the OD decreased by 0.247 mm (p-value = 0.0107). This trend persisted when the data was separately analysed with the common cardiovascular risk factors of diabetes mellitus, hypertension and hyperlipidaemia. CONCLUSION: Our analysis demonstrated a statistically significant decrease in the CFA diameters post-PEVAR. However, the percentage changes were below established flow-limiting values, as reflected by the single access site complication reported. Hence, our findings give confidence in the safety profile of this procedure, even with the reported smaller baseline CFA lumen size in Asians. Moving forward, similar longer-term studies should be considered to characterise any late postoperative effects.

Novel recombinant vaccinia virus-vectored vaccine affords complete protection against homologous Borrelia burgdorferi infection in mice.

The rising prevalence of Lyme disease (LD) in North America and Europe has emerged as a pressing public health concern. Despite the availability of veterinary LD vaccines, no vaccine is currently available for human use. Outer surface protein C (OspC) found on the outer membrane of the causative agent, Borrelia burgdorferi, has been identified as a promising target for LD vaccine development due to its sustained expression during mammalian infection. However, the efficacy and immunological mechanisms of LD vaccines solely targeting OspC are not well characterized. In this study, we developed an attenuated Vaccinia virus (VV) vectored vaccine encoding type A OspC (VV-OspC-A). Two doses of the VV-OspC-A vaccine conferred complete protection against homologous B. burgdorferi challenge in mice. Furthermore, the candidate vaccine also prevented the development of carditis and lymph node hyperplasia associated with LD. When investigating the humoral immune response to vaccination, VV-OspC-A was found to induce a robust antibody response predominated by the IgG2a subtype, indicating a Th1-bias. Using a novel quantitative flow cytometry assay, we also determined that elicited antibodies were capable of inducing antibody-dependent cellular phagocytosis in vitro. Finally, we demonstrated that VV-OspC-A vaccination generated a strong antigen-specific CD4+ T-cell response characterized by the secretion of numerous cytokines upon stimulation of splenocytes with OspC peptides. This study suggests a promising avenue for LD vaccine development utilizing viral vectors targeting OspC and provides insights into the immunological mechanisms that confer protection against B. burgdorferi infection.

Fitness factors impacting survival of a subsurface bacterium in contaminated groundwater.

Many factors contribute to the ability of a microbial species to persist when encountering complexly contaminated environments including time of exposure, the nature and concentration of contaminants, availability of nutritional resources, and possession of a combination of appropriate molecular mechanisms needed for survival. Herein we sought to identify genes that are most important for survival of Gram-negative Enterobacteriaceae in contaminated groundwater environments containing high concentrations of nitrate and metals using the metal-tolerant Oak Ridge Reservation (ORR) isolate, Pantoea sp. MT58 (MT58). Survival fitness experiments in which a randomly barcoded transposon insertion (RB-TnSeq) library of MT58 was exposed directly to contaminated ORR groundwater samples from across a nitrate and mixed metal contamination plume were used to identify genes important for survival with increasing exposure times and concentrations of contaminants, and availability of a carbon source. Genes involved in controlling and using carbon, encoding transcriptional regulators, and related to Gram-negative outer membrane processes were among those found to be important for survival in contaminated ORR groundwater. A comparative genomics analysis of 75 Pantoea genus strains allowed us to further separate the survival determinants into core and non-core genes in the Pantoea pangenome, revealing insights into the survival of subsurface microorganisms during contaminant plume intrusion.

Brain-tumor-seeking and serpin-inhibiting outer membrane vesicles restore plasmin-mediated attacks against brain metastases.

Many chemotherapeutic and molecular targeted drugs have been used to treat brain metastases, e.g., anti-angiogenic vandetanib. However, the blood-brain barrier and brain-specific resistance mechanisms make these systemic therapeutic approaches inefficacious. Brain metastatic cancer cells could mimic neurons to upregulate multiple serpins and secrete them into the extracellular environment to reduce local plasmin production to promote L1CAM-mediated vessel co-option and resist anti-angiogenesis therapy. Here, we developed brain-tumor-seeking and serpin-inhibiting outer membrane vesicles (DE@OMVs) to traverse across the blood-brain barrier, bypass neurons, and specially enter metastatic cancer cells via targeting GRP94 and vimentin. Through specific delivery of dexamethasone and embelin, reduced serpin secretion, restored plasmin production, significant L1CAM inactivation and tumor cell apoptosis were specially found in intracranial metastatic regions, leading to delayed tumor growth and prolonged survival in mice with brain metastases. By combining the brain-tumor-seeking properties with the regulation of the serpin/plasminogen activator/plasmin/L1CAM axis, this study provides a potent and highly-selective systemic therapeutic option for brain metastases.

Two new mouse alleles of Ocm and Slc26a5.

The genes Ocm (encoding oncomodulin) and Slc26a5 (encoding prestin) are expressed strongly in outer hair cells and both are involved in deafness in mice. However, it is not clear if they influence the expression of each other. In this study, we characterise the auditory phenotype resulting from two new mouse alleles, Ocmtm1e and Slc26a5tm1Cre. Each mutation leads to absence of detectable mRNA transcribed from the mutant allele, but there was no evidence that oncomodulin regulates expression of prestin or vice versa. The two mutants show distinctive patterns of auditory dysfunction. Ocmtm1e homozygotes have normal auditory brainstem response thresholds at 4 weeks old followed by progressive hearing loss starting at high frequencies, while heterozygotes show largely normal thresholds until 6 months of age, when signs of worse thresholds are detected. In contrast, Slc26a5tm1Cre homozygotes have stable but raised thresholds across all frequencies tested, 3 to 42 kHz, at least from 4 to 8 weeks old, while heterozygotes have raised thresholds at high frequencies. Distortion product otoacoustic emissions and cochlear microphonics show deficits similar to auditory brainstem responses in both mutants, suggesting that the origin of hearing impairment is in the outer hair cells. Endocochlear potentials are normal in the two mutants. Scanning electron microscopy revealed normal development of hair cells in Ocmtm1e homozygotes but scattered outer hair cell loss even at 4 weeks old when thresholds appeared normal, indicating that there is not a direct relationship between numbers of outer hair cells present and auditory thresholds.

In-host intra- and inter-species transfer of blaKPC-2 and blaNDM-1 in Serratia marcescens and its local and global epidemiology.

OBJECTIVES: This study aimed to investigate interspecies transfer of resistance gene blaNDM-1 and intraspecies transfer of blaKPC-2 in Serratia marcescens, and explore the epidemical and evolutionary characteristics of carbapenemase-producing S. marcescens (CPSM) regionally and globally. METHODS: Interspecies and intraspecies transfer of blaKPC-2- or blaNDM-1 were identified by antimicrobial susceptibility testing, plasmid conjugation and curing, discovery of transposable units (TUs), outer membrane vesicles (OMVs), qPCR, whole-genome sequencing and bioinformatic analysis. The genomic evolution of CPSM strains was explored by cgSNP and maximum-likelihood phylogenetic tree. RESULTS: CPSM S50079 strain, co-carrying blaKPC-2 and blaNDM-1 on one plasmid, was isolated from the blood of a patient with acute pancreatitis and could generate TUs carrying either blaKPC-2 or blaNDM-1. We identified the interspecies transfer of blaNDM-1-carrying plasmid from Providencia rettgeri P50213, producing the identical blaNDM-1-carrying TUs, to S. marcescens S50079K, an S50079 variant via plasmid curing, through blaNDM-1-harboring plasmid conjugation and OMVs transfer. Furthermore, the intraspecies transfer of blaKPC-2, mediated by IS26 from plasmid to chromosome in S50079, was identified. Likely, in another lung transplant patient, interspecies transfer of blaNDM-1 carried by IncX3 plasmid was also identified among S. marcescens and Citrobacter freundii as well as Enterobacter hormaechei via plasmid transfer. Furthermore, 11 CPSM from 349 non-repetitive S. marcescens strains were identified in the same hospital and clonal dissemination, with carbapenemase evolution from blaKPC-2 to both blaKPC-2 and blaNDM-1 was found in the 8 CPSM across four years. Finally, the analysis of 236 global CPSM from 835 non-repetitive S. marcescens genomes, retrieved from NCBI database, revealed long-term spread and evolution worldwide, and would cause the convergence of more carbapenemase genes. CONCLUSIONS: Interspecies transfer of resistance gene blaNDM-1 and intraspecies transfer of resistance gene blaKPC-2 in CPSM were identified. Nosocomial and global dissemination of CPSM were revealed and more urgent surveillance was acquired.

Engineering Planar Gram-Negative Outer Membrane Mimics Using Bacterial Outer Membrane Vesicles.

Antibiotic resistance is a major challenge in modern medicine. The unique double membrane structure of gram-negative bacteria limits the efficacy of many existing antibiotics and adds complexity to antibiotic development by limiting transport of antibiotics to the bacterial cytosol. New methods to mimic this barrier would enable high-throughput studies for antibiotic development. In this study, we introduce an innovative approach to modify outer membrane vesicles (OMVs) from Aggregatibacter actinomycetemcomitans, to generate planar supported lipid bilayer membranes. Our method first involves the incorporation of synthetic lipids into OMVs using a rapid freeze-thaw technique to form outer membrane hybrid vesicles (OM-Hybrids). Subsequently, these OM-Hybrids can spontaneously rupture when in contact with SiO2 surfaces to form a planar outer membrane supported bilayer (OM-SB). We assessed the formation of OM-Hybrids using dynamic light scattering and a fluorescence quenching assay. To analyze the formation of OM-SBs from OM-Hybrids we used quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence recovery after photobleaching (FRAP). Additionally, we conducted assays to detect surface-associated DNA and proteins on OM-SBs. The interaction of an antimicrobial peptide, polymyxin B, with the OM-SBs was also assessed. These findings emphasize the capability of our platform to produce planar surfaces of bacterial outer membranes, which in turn, could function as a valuable tool for streamlining the development of antibiotics.

Immunodominant extracellular loops of Treponema pallidum FadL outer membrane proteins elicit antibodies with opsonic and growth-inhibitory activities.

The global resurgence of syphilis has created a potent stimulus for vaccine development. To identify potentially protective antibodies (Abs) against Treponema pallidum (TPA), we used Pyrococcus furiosus thioredoxin (PfTrx) to display extracellular loops (ECLs) from three TPA outer membrane protein families (outer membrane factors for efflux pumps, eight-stranded ß-barrels, and FadLs) to assess their reactivity with immune rabbit serum (IRS). Five ECLs from the FadL orthologs TP0856, TP0858 and TP0865 were immunodominant. Rabbits and mice immunized with these five PfTrx constructs produced ECL-specific Abs that promoted opsonophagocytosis of TPA by rabbit peritoneal and murine bone marrow-derived macrophages at levels comparable to IRS and mouse syphilitic serum. ECL-specific rabbit and mouse Abs also impaired viability, motility, and cellular attachment of spirochetes during in vitro cultivation. The results support the use of ECL-based vaccines and suggest that ECL-specific Abs promote spirochete clearance via Fc receptor-independent as well as Fc receptor-dependent mechanisms.

Incidence and Clinical Characteristics of COVID-19 Ophthalmopathy Following the Termination of Dynamic Zero COVID-19 Strategy in China.

Ocular involvement is not uncommon in patients with COVID-19. However, the incidence of COVID-19 ophthalmopathy in COVID-19 patients is still not clear. In this prospective case series study, we recruited 2445 consecutive cases presenting at Neuro-ophthalmology clinic of our Eye Center during the last resurgence of SARS-CoV-2 infection from 8 December 2022 to 15 March 2023 in China, 149 cases were diagnosed as COVID-19 ophthalmopathy, 87 cases were female, with a mean age of 43.2 years, and the mean follow-up time was 15.4 weeks. One hundred and twenty of 149 cases suffered from systemic symptoms mostly manifesting as fever, cough and muscle pain prior to or soon after ocular involvement. The most common COVID-19 ophthalmopathy was optic neuritis (51/149), followed by acute zonal occult outer retinopathy complex disease (31/149), uveitis (17/149), ocular mobility disorder-related (third, fourth, or sixth) cranial nerve neuritis (15/149), anterior ischaemic optic neuropathy (9/149), retinal artery occlusion (8/149), retinal microangiopathy including retinal haemorrhage and cotton wool spot (8/149), viral conjunctivitis (7/149), retinal vein occlusion (3/149), viral keratitis (2/149), ptosis (2/149), and other rare ocular diseases. Except 5 cases with central retinal artery occlusion, other 144 COVID-19 ophthalmopathy cases showed good response to steroid therapy. Our study revealed an incidence of 6.09% for COVID-19 ophthalmopathy in outpatients at our Neuro-ophthalmology clinic during last resurgence of COVID-19 in China, and demonstrated that SARS-CoV-2 infection could induce an initial onset or a relapse of ophthalmic diseases, and that ocular involvement might manifest as the initial or even the only presentation of COVID-19.

Analysis of Bacterial Extracellular Vesicles by Immunogold Transmission Electron Microscopy.

Bacterial extracellular vesicles (BEVs) have emerged as mediators of transkingdom communication with numerous potential biotechnological applications. As such, investigation of BEV's protein composition holds promise to uncover new biological mechanisms, such as in microbiome-host communication or pathogen infection. Additionally, bioengineering of BEV protein composition can enhance their therapeutic potential. However, accurate assessment of BEV protein cargo is limited by their nanometer size, which precludes light microscopy imaging, as well as by co-isolation of protein impurities during separation processes. A solution to these challenges is found in immunogold transmission electron microscopy (TEM), which combines antibody-based labeling with direct visualization of BEVs. Several challenges are commonly encountered during immunogold TEM analysis of BEVs, most notably inefficient antibody labeling and poor contrast. Here, we present an optimized protocol for immunogold TEM analysis of BEVs that overcomes such challenges.

Methods for the Characterization of the Colloidal Properties of Bacterial Membrane Vesicles.

Bacterial membrane vesicles (BMVs) are extracellular vesicles secreted by either Gram-positive or Gram-negative bacteria. These BMVs typically possess a diameter between 20 and 250 nm. Due to their size, when these BMVs are suspended in another medium, they could be constituents of a colloidal system. It has been hypothesized that investigating BMVs as colloidal particles could help characterize BMV interactions with other environmentally relevant surfaces. Developing a more thorough understanding of BMV interactions with other surfaces would be critical for developing predictive models of their environmental fate. However, this bio-colloidal perspective has been largely overlooked for BMVs, despite the wealth of methods and expertise available to characterize colloidal particles. A particular strength of taking a more colloid-centric approach to BMV characterization is the potential to quantify a particle's attachment efficiency (α). These values describe the likelihood of attachment during particle-particle or particle-surface interactions, especially those interactions which are governed by physicochemical interactions (such as those described by DLVO and xDLVO theory). Elucidating the influence of physical and electrochemical properties on these attachment efficiency values could give insights into the primary factors driving interactions between BMVs and other surfaces. This chapter details methods for the characterization of BMVs as colloids, beginning with size and surface charge (i.e., electrophoretic mobility/zeta potential) measurements. Afterward, this chapter will address experimental design, especially column experiments, targeted for BMV investigation and the determination of α values.

Use of Synchrotron Radiation Circular Dichroism to Analyze the Interaction and Insertion of Proteins into Bacterial Outer Membrane Vesicles.

Circular dichroism (CD) is a spectroscopic technique commonly used for the analysis of proteins. Particularly, it allows the determination of protein secondary structure content in various media, including the membrane environment. In this chapter, we present how CD applications can be used to analyze the interaction of proteins with bacterial outer membrane vesicles (OMVs). Most CD studies characterizing the structure of proteins inserted into membranes rely on artificial lipid bilayers, mimicking natural membranes. Nevertheless, these artificial models lack the important features of the true membrane, especially for the outer membrane of Gram-negative bacteria. These features include lipid diversity, glycosylation, and asymmetry. Here, we show how to analyze the interactions of proteins, either integral or peripheral, with OMVs in solution and with supported membranes of OMVs, using conventional CD and orientated circular dichroism (OCD). We explain how to decipher the spectroscopic signals to obtain information on the molecular structure of the protein upon its interaction with an OMV and through its potential insertion into an OMV membrane.

Methods to Analyze the Surface Composition of Bacterial Membrane Vesicles.

Bacterial membrane vesicles (BMVs) are small, spherical structures released by Gram-positive and Gram-negative bacteria that play essential roles in intercellular communication, nutrient acquisition, and antibiotic resistance. BMVs typically range from 40 to 400 nm in diameter and contain a single membrane derived from the bacterial membrane, comprising proteins, lipids, nucleic acids, and other biomolecules. Notably, the molecules located on the surface of BMVs facilitate interactions with neighboring cells, including the transfer of functional genes, coordination of bacterial growth through quorum sensing, and delivery of toxins during infections. In addition, BMVs exhibit heterogeneity in their surface composition, which influences their interactions with host and bacterial cells. It is therefore essential to understand not just the composition of BMVs, but the localization of the molecules of interest, particularly those on the surface. In this chapter, we describe several methods that can be used to quantify and characterize the protein and nucleic acid composition, particularly on the surface of BMVs. We describe quantitative immunoblot and ELISA protocols that enable quantification of the concentration of a particular protein of interest. We also describe an enzymatic digestion protocol to determine whether the protein of interest is located on the surface or within the lumen of the BMV, as well as a nucleic acid staining procedure that enables quantification of dsDNA specifically located on the surface of the BMVs. Together, these tools provide a detailed analysis of the protein and nucleic acid composition of BMVs that can be further combined with various separation techniques to study variations within different populations.

Methods for Assessing Plant Immune System Activation by Bacterial Extracellular Vesicles and Other Elicitors.

Bacterial extracellular vesicles (BEVs) are released from the surface of bacterial cells and contain a diverse molecular cargo. Studies conducted primarily with bacterial pathogens of mammals have shown that BEVs are involved in multiple processes such as cell-cell communication, the delivery of RNA, DNA, and proteins to target cells, protection from stresses, manipulation of host immunity, and other functions. Until a decade ago, the roles of BEVs in plant-bacteria interactions were barely investigated. However, recent studies have shown that BEVs of plant pathogens possess similar functions as their mammalian pathogen counterparts, and more research is now devoted to study their roles and interactions with plants. In the following methods chapter, we provide five well-validated assays to examine the interaction of BEVs with the plant immune system. These assays rely on different markers or immune outputs, which indicate the activation of plant immunity (defense marker gene expression, reactive oxygen species burst, seedling inhibition). Furthermore, we offer assays that directly evaluate the priming of the immune system following BEV challenge and the effectiveness of its response to subsequent local or systemic infection. Altogether, these assays provide a thorough examination to the interactions of BEVs and the plant immune system.

Methods for Bioinformatic Prediction of Genuine sRNAs from Outer Membrane Vesicles.

The molecular pathogenesis of Gram-negative bacteria remains a complex and incompletely understood phenomenon. Various factors are believed to contribute to the pathogenicity of these bacteria. One key mechanism utilized by Gram-negative bacteria is the production of outer membrane vesicles (OMVs), which are small spherical particles derived from the bacterial outer membrane. These OMVs are crucial in delivering virulence factors to the host, facilitating host-pathogen interactions. Within these OMVs, small regulatory RNAs (sRNAs) have been identified as important players in modulating the host immune response. One of the main challenges in studying OMVs and their cargo of sRNAs is the difficulty in isolating and purifying sufficient quantities of OMVs, as well as accurately predicting genuine sRNAs computationally. In this chapter, we present protocols aimed at overcoming these obstacles.

Multiple Particle Tracking: A Method for Probing Biologically Relevant Mobility of Bacterial Extracellular Vesicles.

Bacterial extracellular vesicles (bEVs) are produced by both Gram-negative and Gram-positive bacteria. These biological nanoparticles transport small molecules, nucleic acids, and proteins, enabling communication with both bacterial and mammalian cells. bEVs can evade and disrupt biological barriers, and their lipid membranes protect their cargo from degradation, facilitating long-distance communication in vivo. Furthermore, bacteria are easily manipulated and easily cultured. These combined factors make bEVs an ideal candidate for drug delivery applications. Thus, the study of how bEVs interact with biological barriers is interesting from both a signaling and drug delivery perspective. Here we describe methods for tracking bEV motion in biological matrices ex vivo. We outline methods for growth, isolation, quantification, and labeling, as well as techniques for tracking bEV motion ex vivo and quantifying these data. The methods described here are relevant to bEV communication with host cells as well as drug delivery applications using bEVs.

On-Demand Vaccine Production via Dock-and-Display of Biotinylated Antigens on Bacterial Extracellular Vesicles.

Engineered outer membrane vesicles (OMVs) derived from Gram-negative bacteria are a promising vaccine technology for developing immunity against diverse pathogens. However, antigen display on OMVs can be challenging to control and highly variable due to bottlenecks in protein expression and localization to the bacterial host cell's outer membrane, especially for bulky and complex antigens. Here, we describe methods related to a universal vaccine technology called AvidVax (avidin-based vaccine antigen crosslinking) for rapid and simplified assembly of antigens on the exterior of OMVs during vaccine development. The AvidVax platform involves remodeling the OMV surface with multiple copies of a synthetic antigen-binding protein (SNAP), which is an engineered fusion protein comprised of an outer membrane scaffold protein linked to a biotin-binding protein. The resulting SNAPs enable efficient decoration of OMVs with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, nucleic acids, and short peptides. We detail the key steps in the AvidVax vaccine production pipeline including preparation and isolation of SNAP-OMVs, biotinylation and enrichment of vaccine antigens, and formulation and characterization of antigen-loaded SNAP-OMVs.

Separation of Bacterial Extracellular Vesicles via High-Performance Anion Exchange Chromatography.

Bacterial extracellular vesicles (BEVs) have extraordinary biotechnological potential, but traditional purification methods lack desirable scalability and commonly co-isolate protein impurities, limiting clinical translation. Anion exchange chromatography (AEC) separates molecules based on differences in net charge and is widely used for industrial biomanufacturing of protein therapeutics. Recently, AEC has recently been applied for purification of EVs from both mammalian and bacterial sources. Since most bacteria produce BEVs with a negative surface membrane change, AEC can potentially be widely used for BEV purification. Here, we describe a method utilizing high-performance AEC (HPAEC) in tandem with size-based tangential flow filtration for improved BEV purification. We have previously found this method can reduce co-isolated protein impurities and potentiate anti-inflammatory bioactivity of probiotic BEVs. Thus, this method holds promise as a scalable alternative for improved BEV purification.

Assessing In Vivo Bacterial Extracellular Vesicle (BEV) Biodistribution Using Fluorescent Lipophilic Membrane Stains.

Bacterial extracellular vesicles (BEVs) are nano-size vesicles containing a cargo of bioactive molecules that can play key roles in microbe-microbe and microbe-host interactions. In tracking their biodistribution in vivo, BEVs can cross several physical host barriers including the intestinal epithelium, vascular endothelium, and blood-brain-barrier (BBB) to ultimately accumulate in tissues such as the liver, lungs, spleen, and the brain. This tissue-specific dissemination has been exploited for the delivery of biomolecules such as vaccines for mucosal delivery. Although numerous strategies for labeling and tracking BEVs have been described, most have constraints that impact on interpreting in vivo bioimaging patterns. Here, we describe a general method for labeling BEVs using lipophilic fluorescent membrane stains which can be adopted by non-expert users. We also describe how the procedure can be used to overcome potential limitations. Furthermore, we outline methods of quantitative ex vivo tissue imaging that can be used to evaluate BEV organ trafficking.

Engineering Outer Membrane Vesicles to Carry Enzymes: Encapsulation, Isolation, Characterization, and Modification.

Outer membrane vesicles (OMVs) are small, spherical, nanoscale proteoliposomes released from Gram-negative bacteria that play an important role in cellular defense, pathogenesis, and signaling, among other functions. The functionality of OMVs can be enhanced by engineering developed for biomedical and biochemical applications. Here, we describe methods for directed packaging of enzymes into bacterial OMVs of E. coli using engineered molecular systems, such as localizing proteins to the inner or outer surface of the vesicle. Additionally, we detail some modification strategies for OMVs such as lyophilization and surfactant conjugation that enable the protection of activity of the packaged enzyme when exposed to non-physiological conditions such as elevated temperature, organic solvents, and repeated freeze/thaw that otherwise lead to a substantial loss in the activity of the free enzyme.