Comparative electrokinetic properties of extracellular vesicles produced by yeast and bacteria.

Extracellular vesicles (EVs) are nano-sized, biocolloidal proteoliposomes that have been shown to be produced by all cell types studied to date and are ubiquitous in the environment. Extensive literature on colloidal particles has demonstrated the implications of surface chemistry on transport behavior. Hence, one may anticipate that physicochemical properties of EVs, particularly surface charge-associated properties, may influence EV transport and specificity of interactions with surfaces. Here we compare the surface chemistry of EVs as expressed by zeta potential (calculated from electrophoretic mobility measurements). The zeta potentials of EVs produced by Pseudomonas fluorescens, Staphylococcus aureus, and Saccharomyces cerevisiae were largely unaffected by changes in ionic strength and electrolyte type, but were affected by changes in pH. The addition of humic acid altered the calculated zeta potential of the EVs, especially for those from S. cerevisiae. Differences in zeta potential were compared between EVs and their respective parent cell with no consistent trend emerging; however, significant differences were discovered between the different cell types and their EVs. These findings imply that, while EV surface charge (as estimated from zeta potential) is relatively insensitive to the evaluated environmental conditions, EVs from different organisms can differ regarding which conditions will cause colloidal instability.

Proteomic Profiling Reveals Distinct Bacterial Extracellular Vesicle Subpopulations with Possibly Unique Functionality.

Bacterial outer membrane vesicles (OMVs) are 20- to 200-nm secreted packages of lipids, small molecules, and proteins that contribute to diverse bacterial processes. In plant systems, OMVs from pathogenic and beneficial strains elicit plant immune responses that inhibit seedling growth and protect against future pathogen challenge. Previous studies of OMV-plant interactions suggest functionally important differences in the protein composition of Pseudomonas syringae and Pseudomonas fluorescens OMVs, and that their composition and activity differ as a result of medium culture conditions. Here, we show that plant apoplast-mimicking minimal medium conditions impact OMV protein content dramatically in P. syringae but not in P. fluorescens relative to complete medium conditions. Comparative, 2-way analysis of the four conditions reveals subsets of proteins that may contribute to OMV-mediated bacterial virulence and plant immune activation as well as those involved in bacterial stress tolerance or adaptation to a beneficial relationship with plants. Additional localization enrichment analysis of these subsets suggests the presence of outer-inner membrane vesicles (OIMVs). Collectively, these results reveal distinct differences in bacterial extracellular vesicle cargo and biogenesis routes from pathogenic and beneficial plant bacteria in different medium conditions and point to distinct populations of vesicles with diverse functional roles. IMPORTANCE Recent publications have shown that bacterial vesicles play important roles in interkingdom communication between bacteria and plants. Indeed, our recently published data reveal that bacterial vesicles from pathogenic and beneficial strains elicit immune responses in plants that protect against future pathogen challenge. However, the molecules underlying these striking phenomena remain unknown. Our recent work indicated that proteins packaged in vesicles are critically important for vesicle-mediated seedling growth inhibition, often considered an indirect measure of plant immune activation. In this study, we characterize the protein cargo of vesicles from Pseudomonas syringae pathovar tomato DC3000 and Pseudomonas fluorescens from two different medium conditions and show that distinct subpopulations of vesicles contribute to bacterial virulence and stress tolerance. Furthermore, we reveal differences in how beneficial and pathogenic bacterial species respond to harsh environmental conditions through vesicle packaging. Importantly, we find that protein cargo implicates outer-inner membrane vesicles in bacterial stress responses, while outer membrane vesicles are packaged for virulence.

The extracellular vesicle generation paradox: a bacterial point of view.

All bacteria produce secreted vesicles that carry out a variety of important biological functions. These extracellular vesicles can improve adaptation and survival by relieving bacterial stress and eliminating toxic compounds, as well as by facilitating membrane remodeling and ameliorating inhospitable environments. However, vesicle production comes with a price. It is energetically costly and, in the case of colonizing pathogens, it elicits host immune responses, which reduce bacterial viability. This raises an interesting paradox regarding why bacteria produce vesicles and begs the question as to whether the benefits of producing vesicles outweigh their costs. In this review, we discuss the various advantages and disadvantages associated with Gram-negative and Gram-positive bacterial vesicle production and offer perspective on the ultimate score. We also highlight questions needed to advance the field in determining the role for vesicles in bacterial survival, interkingdom communication, and virulence.

Microbial vesicle-mediated communication: convergence to understand interactions within and between domains of life.

All cells produce extracellular vesicles (EVs). These biological packages contain complex mixtures of molecular cargo and have a variety of functions, including interkingdom communication. Recent discoveries highlight the roles microbial EVs may play in the environment with respect to interactions with plants as well as nutrient cycling. These studies have also identified molecules present within EVs and associated with EV surfaces that contribute to these functions. In parallel, studies of engineered nanomaterials have developed methods to track and model small particle behavior in complex systems and measure the relative importance of various surface features on transport and function. While studies of EV behavior in complex environmental conditions have not yet employed transdisciplinary approaches, it is increasingly clear that expertise from disparate fields will be critical to understand the role of EVs in these systems. Here, we outline how the convergence of biology, soil geochemistry, and colloid science can both develop and address questions surrounding the basic principles governing EV-mediated interkingdom interactions.

Protective plant immune responses are elicited by bacterial outer membrane vesicles.

Bacterial outer membrane vesicles (OMVs) perform a variety of functions in bacterial survival and virulence. In mammalian systems, OMVs activate immune responses and are exploited as vaccines. However, little work has focused on the interactions of OMVs with plant hosts. Here, we report that OMVs from Pseudomonas syringae and P. fluorescens activate plant immune responses that protect against bacterial and oomycete pathogens. OMV-mediated immunomodulatory activity from these species displayed different sensitivity to biochemical stressors, reflecting differences in OMV content. Importantly, OMV-mediated plant responses are distinct from those triggered by conserved bacterial epitopes or effector molecules alone. Our study shows that OMV-induced protective immune responses are independent of the T3SS and protein, but that OMV-mediated seedling growth inhibition largely depends on proteinaceous components. OMVs provide a unique opportunity to understand the interplay between virulence and host response strategies and add a new dimension to consider in host-microbe interactions.

Delay or Avoidance of Medical Care Because of COVID-19-Related Concerns - United States, June 2020.

Temporary disruptions in routine and nonemergency medical care access and delivery have been observed during periods of considerable community transmission of SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19) (1). However, medical care delay or avoidance might increase morbidity and mortality risk associated with treatable and preventable health conditions and might contribute to reported excess deaths directly or indirectly related to COVID-19 (2). To assess delay or avoidance of urgent or emergency and routine medical care because of concerns about COVID-19, a web-based survey was administered by Qualtrics, LLC, during June 24-30, 2020, to a nationwide representative sample of U.S. adults aged ≥18 years. Overall, an estimated 40.9% of U.S. adults have avoided medical care during the pandemic because of concerns about COVID-19, including 12.0% who avoided urgent or emergency care and 31.5% who avoided routine care. The estimated prevalence of urgent or emergency care avoidance was significantly higher among the following groups: unpaid caregivers for adults* versus noncaregivers (adjusted prevalence ratio [aPR] = 2.9); persons with two or more selected underlying medical conditions† versus those without those conditions (aPR = 1.9); persons with health insurance versus those without health insurance (aPR = 1.8); non-Hispanic Black (Black) adults (aPR = 1.6) and Hispanic or Latino (Hispanic) adults (aPR = 1.5) versus non-Hispanic White (White) adults; young adults aged 18-24 years versus adults aged 25-44 years (aPR = 1.5); and persons with disabilities§ versus those without disabilities (aPR = 1.3). Given this widespread reporting of medical care avoidance because of COVID-19 concerns, especially among persons at increased risk for severe COVID-19, urgent efforts are warranted to ensure delivery of services that, if deferred, could result in patient harm. Even during the COVID-19 pandemic, persons experiencing a medical emergency should seek and be provided care without delay (3).

Minimally invasive microneedles for ocular drug delivery.

INTRODUCTION: Anterior and posterior segment eye diseases are highly challenging to treat, due to the barrier properties and relative inaccessibility of the ocular tissues. Topical eye drops and systemically delivered treatments result in low bioavailability. Alternatively, direct injection of medication into the ocular tissues is clinically employed to overcome the barrier properties, but injections cause significant tissue damage and are associated with a number of untoward side effects and poor patient compliance. Microneedles (MNs) has been recently introduced as a minimally invasive means for localizing drug formulation within the target ocular tissues with greater precision and accuracy than the hypodermic needles. Areas covered: This review article seeks to provide an overview of a range of challenges that are often faced to achieve efficient ocular drug levels within targeted tissue(s) of the eye. It also describes the problems encountered using conventional hypodermic needle-based ocular injections for anterior and posterior segment drug delivery. It discusses research carried out in the field of MNs, to date. Expert opinion: MNs can aid in localization of drug delivery systems within the selected ocular tissue. And, hold the potential to revolutionize the way drug formulations are administered to the eye. However, the current limitations and challenges of MNs application warrant further research in this field to enable its widespread clinical application.

Punctal plug: a medical device to treat dry eye syndrome and for sustained drug delivery to the eye.

Punctal plugs (PPs) are miniature medical implants that were initially developed for the treatment of dry eyes. Since their introduction in 1975, many PPs made from different materials and designs have been developed. PPs, albeit generally successful, suffer from drawbacks such as epiphora and suppurative canaliculitis. To overcome these issues intelligent designs of PPs were proposed (e.g. SmartPLUG™ and Form Fit™). PPs are also gaining interest among pharmaceutical scientists for sustaining drug delivery to the eye. This review aims to provide an overview of PPs for dry eye treatment and drug delivery to treat a range of ocular diseases. It also discusses current challenges in using PPs for ocular diseases.

Microneedle-mediated intrascleral delivery of in situ forming thermoresponsive implants for sustained ocular drug delivery.

OBJECTIVES: This paper describes use of minimally invasive hollow microneedle (HMN) to deliver in situ forming thermoresponsive poloxamer-based implants into the scleral tissue to provide sustained drug delivery. METHODS: In situ forming poloxamer formulations were prepared and investigated for their rheological properties. HMN devices 400, 500 and 600 µm in height were fabricated from hypodermic needles (i.e. 27, 29 and 30 G) and tested for depth of penetration into rabbit sclera. Maximum force and work required to expel different volumes of poloxamer formulations was also investigated. Release of fluorescein sodium (FS) from intrasclerally injected implants was also investigated. Optical coherence tomography (OCT) was used to examine implant localisation and scleral pore-closure. KEY FINDINGS: Poloxamer formulations showed Newtonian behaviour at 20°C and pseudoplastic (shear-thinning) behaviour at 37°C. Maximum force and work required to expel different volumes of poloxamer formulations with different needles ranged from 0.158 to 2.021 N and 0.173 to 6.000 N, respectively. OCT showed intrascleral localisation of implants and scleral pore-closure occurred within 2-3 h. Sustain release of FS was noticed over 24 h and varied with depth of implant delivery. CONCLUSIONS: This study shows that the minimally invasive HMN device can localise in situ forming implants in the scleral tissue and provide sustained drug delivery.

Solvent induced phase inversion-based in situ forming controlled release drug delivery implants.

In situ forming (ISF) drug delivery implants have gained tremendous levels of interest over the last few decades. This is due to their wide range of biomedical applications such as in tissue engineering, cell encapsulation, microfluidics, bioengineering and drug delivery. Drug delivery implants forming upon injection has shown a range of advantages which include localized drug delivery, easy and less invasive application, sustained drug action, ability to tailor drug delivery, reduction in side effects associated with systemic delivery and also improved patient compliance and comfort. Different factors such as temperature, pH, ions, and exchange of solvents are involved in in situ implant formation. This review especially focuses on ISF implants that are formed through solvent induced phase inversion (SPI) technique. The article critically reviews and compares a wide range of polymers, solvents, and co-solvents that have been used in SPI implant preparation for control release of a range of drug molecules. Major drawback of SPI systems has been their high burst release. In this regard, the article exhaustively discusses factors that affect the burst release and different modification strategies that has been utilised to reduce the burst effect from these implants. Performance and controversial issues associated with the use of different biocompatible solvents in SPI systems is also discussed. Biodegradation, formulation stability, methods of characterisation and sterilisation techniques of SPI systems is comprehensively reviewed. Furthermore, the review also examines current SPI-based marketed products, their therapeutic application and associated clinical data. It also exemplifies the interest of multi-billion dollar pharma companies worldwide for further developments of SPI systems to a range of therapeutic applications. The authors believe that this will be the first review article that extensively investigate and discusses studies done to date on SPI systems. In so doing, this article will undoubtedly serve as an enlightening tool for the scientists working in the concerned area.