Dextrin conjugation to colistin inhibits its toxicity, cellular uptake and acute kidney injury in vivo.

The acute kidney injury (AKI) and dose-limiting nephrotoxicity, which occurs in 20-60% of patients following systemic administration of colistin, represents a challenge in the effective treatment of multi-drug resistant Gram-negative infections. To reduce clinical toxicity of colistin and improve targeting to infected/inflamed tissues, we previously developed dextrin-colistin conjugates, whereby colistin is designed to be released by amylase-triggered degradation of dextrin in infected and inflamed tissues, after passive targeting by the enhanced permeability and retention effect. Whilst it was evident in vitro that polymer conjugation can reduce toxicity and prolong plasma half-life, without significant reduction in antimicrobial activity of colistin, it was unclear how dextrin conjugation would alter cellular uptake and localisation of colistin in renal tubular cells in vivo. We discovered that dextrin conjugation effectively reduced colistin's toxicity towards human kidney proximal tubular epithelial cells (HK-2) in vitro, which was mirrored by significantly less cellular uptake of Oregon Green (OG)-labelled dextrin-colistin conjugate, when compared to colistin. Using live-cell confocal imaging, we revealed localisation of both, free and dextrin-bound colistin in endolysosome compartments of HK-2 and NRK-52E cells. Using a murine AKI model, we demonstrated dextrin-colistin conjugation dramatically diminishes both proximal tubular injury and renal accumulation of colistin. These findings reveal new insight into the mechanism by which dextrin conjugation can overcome colistin's renal toxicity and show the potential of polymer conjugation to improve the side effect profile of nephrotoxic drugs.

Structure-Activity Relationships of Low Molecular Weight Alginate Oligosaccharide Therapy against Pseudomonas aeruginosa.

Low molecular weight alginate oligosaccharides have been shown to exhibit anti-microbial activity against a range of multi-drug resistant bacteria, including Pseudomonas aeruginosa. Previous studies suggested that the disruption of calcium (Ca2+)-DNA binding within bacterial biofilms and dysregulation of quorum sensing (QS) were key factors in these observed effects. To further investigate the contribution of Ca2+ binding, G-block (OligoG) and M-block alginate oligosaccharides (OligoM) with comparable average size DPn 19 but contrasting Ca2+ binding properties were prepared. Fourier-transform infrared spectroscopy demonstrated prolonged binding of alginate oligosaccharides to the pseudomonal cell membrane even after hydrodynamic shear treatment. Molecular dynamics simulations and isothermal titration calorimetry revealed that OligoG exhibited stronger interactions with bacterial LPS than OligoM, although this difference was not mirrored by differential reductions in bacterial growth. While confocal laser scanning microscopy showed that both agents demonstrated similar dose-dependent reductions in biofilm formation, OligoG exhibited a stronger QS inhibitory effect and increased potentiation of the antibiotic azithromycin in minimum inhibitory concentration and biofilm assays. This study demonstrates that the anti-microbial effects of alginate oligosaccharides are not purely influenced by Ca2+-dependent processes but also by electrostatic interactions that are common to both G-block and M-block structures.

Defining in vitro topical antimicrobial and antibiofilm activity of epoxy-tigliane structures against oral pathogens.

Background: Peri-implantitis has become an inexorable clinical challenge in implantology. Topical immunomodulatory epoxy-tiglianes (EBCs), derived from the Queensland blushwood tree, which induce remodeling and resolve dermal infection via induction of the inflammasome and biofilm disruption, may offer a novel therapeutic approach. Design: In vitro antimicrobial activity of EBC structures (EBC-46, EBC-1013 and EBC-147) against Streptococcus mutans, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis in minimum inhibitory concentration, growth curve and permeabilization assays were determined. Antibiofilm activity was assessed using minimum biofilm eradication concentration (MBEC) experiments. Biofilm formation and disruption assays were analyzed using confocal laser scanning microscopy, scanning electron microscopy and direct plate counting. Results: The observed antimicrobial efficacy of the tested compounds (EBC-1013 > EBC-46 > EBC-147) was directly related to significant membrane permeabilization and growth inhibition (p < 0.05) against planktonic S. mutans and P. gingivalis. Antibiofilm activity was evident in MBEC assays, with S. mutans biofilm formation assays revealing significantly lower biomass volume and increased DEAD:LIVE cell ratio observed for EBC-1013 (p < 0.05). Furthermore, biofilm disruption assays on titanium discs induced significant biofilm disruption in S. mutans and P. gingivalis (p < 0.05). Conclusions: EBC-1013 is a safe, semi-synthetic, compound, demonstrating clear antimicrobial biofilm disruption potential in peri-implantitis.

Role of Enzymic Antioxidants in Mediating Oxidative Stress and Contrasting Wound Healing Capabilities in Oral Mucosal/Skin Fibroblasts and Tissues.

Unlike skin, oral mucosal wounds are characterized by rapid healing and minimal scarring, attributable to the "enhanced" healing properties of oral mucosal fibroblasts (OMFs). As oxidative stress is increasingly implicated in regulating wound healing outcomes, this study compared oxidative stress biomarker and enzymic antioxidant profiles between patient-matched oral mucosal/skin tissues and OMFs/skin fibroblasts (SFs) to determine whether superior oral mucosal antioxidant capabilities and reduced oxidative stress contributed to these preferential healing properties. Oral mucosa and skin exhibited similar patterns of oxidative protein damage and lipid peroxidation, localized within the lamina propria/dermis and oral/skin epithelia, respectively. SOD1, SOD2, SOD3 and catalase were primarily localized within epithelial tissues overall. However, SOD3 was also widespread within the lamina propria localized to OMFs, vasculature and the extracellular matrix. OMFs were further identified as being more resistant to reactive oxygen species (ROS) generation and oxidative DNA/protein damage than SFs. Despite histological evaluation suggesting that oral mucosa possessed higher SOD3 expression, this was not fully substantiated for all OMFs examined due to inter-patient donor variability. Such findings suggest that enzymic antioxidants have limited roles in mediating privileged wound healing responses in OMFs, implying that other non-enzymic antioxidants could be involved in protecting OMFs from oxidative stress overall.

An investigation into the robustness of a double-ended wideband impedance-based fault location technique.

The double-ended impedance-based fault location technique (DEFLT) uses the wideband frequency content of the transient generated by the fault to determine the impedance from the point of measurement to the fault. This paper evaluates and develops the DEFLT experimentally for a Shipboard Power System (SPS) to determine its robustness to source impedance, the presence of interconnected loads ("tapped" loads) and tapped lines. Results demonstrate that the estimated impedance (and therefore distance to the fault) is influenced by the presence of tapped loads when the source impedance is large, or when the tapped load is comparable to the rated load of the system. Therefore, a scheme is proposed that compensates for any tapped load without requiring any additional measurements. Using the proposed scheme, the maximum error is significantly reduced from 92 to 13%. Simulation and experimental results show that a high accuracy for the estimated fault location can be achieved.

Alginate oligosaccharides enhance the antifungal activity of nystatin against candidal biofilms.

Background: The increasing prevalence of invasive fungal infections in immuno-compromised patients is a considerable cause of morbidity and mortality. With the rapid emergence of antifungal resistance and an inadequate pipeline of new therapies, novel treatment strategies are now urgently required. Methods: The antifungal activity of the alginate oligosaccharide OligoG in conjunction with nystatin was tested against a range of Candida spp. (C. albicans, C. glabrata, C. parapsilosis, C. auris, C. tropicalis and C. dubliniensis), in both planktonic and biofilm assays, to determine its potential clinical utility to enhance the treatment of candidal infections. The effect of OligoG (0-6%) ± nystatin on Candida spp. was examined in minimum inhibitory concentration (MIC) and growth curve assays. Antifungal effects of OligoG and nystatin treatment on biofilm formation and disruption were characterized using confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and ATP cellular viability assays. Effects on the cell membrane were determined using permeability assays and transmission electron microscopy (TEM). Results: MIC and growth curve assays demonstrated the synergistic effects of OligoG (0-6%) with nystatin, resulting in an up to 32-fold reduction in MIC, and a significant reduction in the growth of C. parapsilosis and C. auris (minimum significant difference = 0.2 and 0.12 respectively). CLSM and SEM imaging demonstrated that the combination treatment of OligoG (4%) with nystatin (1 µg/ml) resulted in significant inhibition of candidal biofilm formation on glass and clinical grade silicone surfaces (p < 0.001), with increased cell death (p < 0.0001). The ATP biofilm disruption assay demonstrated a significant reduction in cell viability with OligoG (4%) alone and the combined OligoG/nystatin (MIC value) treatment (p < 0.04) for all Candida strains tested. TEM studies revealed the combined OligoG/nystatin treatment induced structural reorganization of the Candida cell membrane, with increased permeability when compared to the untreated control (p < 0.001). Conclusions: Antimicrobial synergy between OligoG and nystatin against Candida spp. highlights the potential utility of this combination therapy in the prevention and topical treatment of candidal biofilm infections, to overcome the inherent tolerance of biofilm structures to antifungal agents.

Topical, immunomodulatory epoxy-tiglianes induce biofilm disruption and healing in acute and chronic skin wounds.

The management of antibiotic-resistant, bacterial biofilm infections in chronic skin wounds is an increasing clinical challenge. Despite advances in diagnosis, many patients do not derive benefit from current anti-infective/antibiotic therapies. Here, we report a novel class of naturally occurring and semisynthetic epoxy-tiglianes, derived from the Queensland blushwood tree (Fontainea picrosperma), and demonstrate their antimicrobial activity (modifying bacterial growth and inducing biofilm disruption), with structure/activity relationships established against important human pathogens. In vitro, the lead candidate EBC-1013 stimulated protein kinase C (PKC)-dependent neutrophil reactive oxygen species (ROS) induction and NETosis and increased expression of wound healing-associated cytokines, chemokines, and antimicrobial peptides in keratinocytes and fibroblasts. In vivo, topical EBC-1013 induced rapid resolution of infection with increased matrix remodeling in acute thermal injuries in calves. In chronically infected diabetic mouse wounds, treatment induced cytokine/chemokine production, inflammatory cell recruitment, and complete healing (in six of seven wounds) with ordered keratinocyte differentiation. These results highlight a nonantibiotic approach involving contrasting, orthogonal mechanisms of action combining targeted biofilm disruption and innate immune induction in the treatment of chronic wounds.

The SARS-CoV2 envelope differs from host cells, exposes procoagulant lipids, and is disrupted in vivo by oral rinses.

The lipid envelope of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an essential component of the virus; however, its molecular composition is undetermined. Addressing this knowledge gap could support the design of antiviral agents as well as further our understanding of viral-host protein interactions, infectivity, pathogenicity, and innate immune system clearance. Lipidomics revealed that the virus envelope comprised mainly phospholipids (PLs), with some cholesterol and sphingolipids, and with cholesterol/phospholipid ratio similar to lysosomes. Unlike cellular membranes, procoagulant amino-PLs were present on the external side of the viral envelope at levels exceeding those on activated platelets. Accordingly, virions directly promoted blood coagulation. To investigate whether these differences could enable selective targeting of the viral envelope in vivo, we tested whether oral rinses containing lipid-disrupting chemicals could reduce infectivity. Products containing PL-disrupting surfactants (such as cetylpyridinium chloride) met European virucidal standards in vitro; however, components that altered the critical micelle concentration reduced efficacy, and products containing essential oils, povidone-iodine, or chlorhexidine were ineffective. This result was recapitulated in vivo, where a 30-s oral rinse with cetylpyridinium chloride mouthwash eliminated live virus in the oral cavity of patients with coronavirus disease 19 for at least 1 h, whereas povidone-iodine and saline mouthwashes were ineffective. We conclude that the SARS-CoV-2 lipid envelope i) is distinct from the host plasma membrane, which may enable design of selective antiviral approaches; ii) contains exposed phosphatidylethanolamine and phosphatidylserine, which may influence thrombosis, pathogenicity, and inflammation; and iii) can be selectively targeted in vivo by specific oral rinses.

Alginate oligosaccharides enhance diffusion and activity of colistin in a mucin-rich environment.

In a number of chronic respiratory diseases e.g. cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD), the production of viscous mucin reduces pulmonary function and represents an effective barrier to diffusion of inhaled therapies e.g. antibiotics. Here, a 2-compartment Transwell model was developed to study impaired diffusion of the antibiotic colistin across an artificial sputum (AS) matrix/medium and to quantify its antimicrobial activity against Pseudomonas aeruginosa NH57388A biofilms (alone and in combination with mucolytic therapy). High-performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) revealed that the presence of AS medium significantly reduced the rate of colistin diffusion (> 85% at 48 h; p < 0.05). Addition of alginate oligosaccharide (OligoG CF-5/20) significantly improved colistin diffusion by 3.7 times through mucin-rich AS medium (at 48 h; p < 0.05). Increased diffusion of colistin with OligoG CF-5/20 was shown (using confocal laser scanning microscopy and COMSTAT image analysis) to be associated with significantly increased bacterial killing (p < 0.05). These data support the use of this model to study drug and small molecule delivery across clinically-relevant diffusion barriers. The findings indicate the significant loss of colistin and reduced effectiveness that occurs with mucin binding, and support the use of mucolytics to improve antimicrobial efficacy and lower antibiotic exposure.

A physicochemical assessment of the thermal stability of dextrin-colistin conjugates.

Attachment of polysaccharide carriers is increasingly being used to achieve precision delivery and improved effectiveness of protein and peptide drugs. Although it is clear that their clinical effectiveness relies on the purity and integrity of the conjugate in storage, as well as following administration, instability of polysaccharide-based conjugates can reduce the protective efficacy of the polymer, which may adversely affect the bioactive's potency. As a model, these studies used dextrin-colistin conjugates, with varying degrees of polymer modification (1, 2.5 and 7.5 mol% succinoylation) to assess the effect of storage temperature (- 20, 4, 21 and 37 °C) and duration (up to 12 months) on saccharide and colistin release and antimicrobial activity. Estimation of the proportion of saccharide release (by comparison of area under the curve from size exclusion chromatograms) was more pronounced at higher temperatures (up to 3 and 35% at - 20 °C and 37 °C, respectively after 12 months), however, repeated freeze-thaw did not produce any measurable release of saccharides, while addition of amylase (20, 100, 500 IU/L) caused rapid release of saccharides (> 70% total within 24 h). At all temperatures, conjugates containing the lowest degree of succinoylation released the highest proportion of free colistin, which increased with storage temperature, however no trend in saccharide release was observed. Despite the clear physical effects of prolonged storage, antimicrobial activity of all samples was only altered after storage at 37 °C for 12 months (> threefold decreased activity). These results demonstrate significant release of saccharides from dextrin-colistin conjugates during prolonged storage in buffered solution, especially at elevated temperature, which, in most cases, did not affect antimicrobial activity. These findings provide vital information about the structure-activity relationship of dextrin-colistin conjugates, prior to full-scale commercial development, which can subsequently be applied to other polysaccharide-protein and -peptide conjugates.

Quantifying the effects of antibiotic treatment on the extracellular polymer network of antimicrobial resistant and sensitive biofilms using multiple particle tracking.

Novel therapeutics designed to target the polymeric matrix of biofilms requires innovative techniques to accurately assess their efficacy. Here, multiple particle tracking (MPT) was developed to characterize the physical and mechanical properties of antimicrobial resistant (AMR) bacterial biofilms and to quantify the effects of antibiotic treatment. Studies employed nanoparticles (NPs) of varying charge and size (40-500 nm) in Pseudomonas aeruginosa PAO1 and methicillin-resistant Staphylococcus aureus (MRSA) biofilms and also in polymyxin B (PMB) treated Escherichia coli biofilms of PMB-sensitive (PMBSens) IR57 and PMB-resistant (PMBR) PN47 strains. NP size-dependent and strain-related differences in the diffusion coefficient values of biofilms were evident between PAO1 and MRSA. Dose-dependent treatment effects induced by PMB in PMBSens E. coli biofilms included increases in diffusion and creep compliance (P < 0.05), not evident in PMB treatment of PMBR E. coli biofilms. Our results highlight the ability of MPT to quantify the diffusion and mechanical effects of antibiotic therapies within the AMR biofilm matrix, offering a valuable tool for the pre-clinical screening of anti-biofilm therapies.

Phenotypic and Genotypic Adaptations in Pseudomonas aeruginosa Biofilms following Long-Term Exposure to an Alginate Oligomer Therapy.

Chronic Pseudomonas aeruginosa lung infections in cystic fibrosis (CF) evolve to generate environmentally adapted biofilm communities, leading to increased patient morbidity and mortality. OligoG CF-5/20, a low-molecular-weight inhaled alginate oligomer therapy, is currently in phase IIb/III clinical trials in CF patients. Experimental evolution of P. aeruginosa in response to OligoG CF-5/20 was assessed using a bead biofilm model allowing continuous passage (45 days; ∼245 generations). Mutants isolated after OligoG CF-5/20 treatment typically had a reduced biofilm-forming ability and altered motility profile. Genotypically, OligoG CF-5/20 provided no selective pressure on genomic mutations within morphotypes. Chronic exposure to azithromycin, a commonly prescribed antibiotic in CF patients, with or without OligoG CF-5/20 in the biofilm evolution model also had no effect on rates of resistance acquisition. Interestingly, however, cross-resistance to other antibiotics (e.g., aztreonam) was reduced in the presence of OligoG CF-5/20. Collectively, these findings show no apparent adverse effects from long-term exposure to OligoG CF-5/20, instead resulting in both fewer colonies with multidrug resistance (MDR)-associated phenotypes and improved antibiotic susceptibility of P. aeruginosaIMPORTANCE The emergence of multidrug-resistant (MDR) pathogens within biofilms in the cystic fibrosis lung results in increased morbidity. An inhalation therapy derived from alginate, OligoG CF-5/20, is currently in clinical trials for cystic fibrosis patients. OligoG CF-5/20 has been shown to alter sputum viscoelasticity, disrupt mucin polymer networks, and disrupt MDR pseudomonal biofilms. Long-term exposure to inhaled therapeutics may induce selective evolutionary pressures on bacteria within the lung biofilm. Here, a bead biofilm model with repeated exposure of P. aeruginosa to OligoG CF-5/20 (alone and in combination with azithromycin) was conducted to study these long-term effects and characterize the phenotypic and genotypic adaptations which result. These findings, over 6 weeks, show that long-term use of OligoG CF-5/20 does not lead to extensive mutational changes and may potentially decrease the pathogenicity of the bacterial biofilm and improve the susceptibility of P. aeruginosa to other classes of antibiotics.

A New Look at the Purported Health Benefits of Commercial and Natural Clays.

Clays attributed to have medicinal properties have been used since prehistoric times and are still used today as complementary medicines, which has given rise to unregulated "bioceutical" clays to treat skin conditions. Recently, clays with antibacterial characteristics have been proposed as alternatives to antibiotics, potentially overcoming modern day antibiotic resistance. Clays with suggested antibacterial properties were examined to establish their effects on common wound-infecting bacteria. Geochemical, microscopical, and toxicological characterization of clay particulates, their suspensions and filtered leachates was performed on THP-1 and HaCaT cell lines. Cytoskeletal toxicity, cell proliferation/viability (MTT assays), and migration (scratch wounds) were further evaluated. Clays were assayed for antibacterial efficacy using minimum inhibitory concentration assays. All clays possessed a mineral content with antibacterial potential; however, clay leachates contained insufficient ions to have any antibacterial effects. All clay leachates displayed toxicity towards THP-1 monocytes, while clay suspensions showed less toxicity, suggesting immunogenicity. Reduced clay cytotoxicity on HaCaTs was shown, as many leachates stimulated wound-healing responses. The "Green" clay exhibited antibacterial effects and only in suspension, which was lost upon neutralization. pH and its interaction with clay particle surface charge is more significant than previously understood to emphasize dangers of unregulated marketing and unsubstantiated bioceutical claims.

Bi-Functional Alginate Oligosaccharide-Polymyxin Conjugates for Improved Treatment of Multidrug-Resistant Gram-Negative Bacterial Infections.

The recent emergence of resistance to colistin, an antibiotic of last resort with dose-limiting toxicity, has highlighted the need for alternative approaches to combat infection. This study aimed to generate and characterise alginate oligosaccharide ("OligoG")-polymyxin (polymyxin B and E (colistin)) conjugates to improve the effectiveness of these antibiotics. OligoG-polymyxin conjugates (amide- or ester-linked), with molecular weights of 5200-12,800 g/mol and antibiotic loading of 6.1-12.9% w/w, were reproducibly synthesised. In vitro inflammatory cytokine production (tumour necrosis factor alpha (TNFα) ELISA) and cytotoxicity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) of colistin (2.2-9.3-fold) and polymyxin B (2.9-27.2-fold) were significantly decreased by OligoG conjugation. Antimicrobial susceptibility tests (minimum inhibitory concentration (MIC), growth curves) demonstrated similar antimicrobial efficacy of ester- and amide-linked conjugates to that of the parent antibiotic but with more sustained inhibition of bacterial growth. OligoG-polymyxin conjugates exhibited improved selectivity for Gram-negative bacteria in comparison to mammalian cells (approximately 2-4-fold). Both OligoG-colistin conjugates caused significant disruption of Pseudomonas aeruginosa biofilm formation and induced bacterial death (confocal laser scanning microscopy). When conjugates were tested in an in vitro "time-to-kill" (TTK) model using Acinetobacter baumannii, only ester-linked conjugates reduced viable bacterial counts (~2-fold) after 4 h. Bi-functional OligoG-polymyxin conjugates have potential therapeutic benefits in the treatment of multidrug-resistant (MDR) Gram-negative bacterial infections, directly reducing toxicity whilst retaining antimicrobial and antibiofilm activities.

Operationally Relevant Behavior Assessment Using the Robotic On-Board Trainer for Research (ROBoT-r).

INTRODUCTION: Spaceflight can strain astronaut physical, physiological, and mental well-being, whereas maintaining astronaut operational performance remains an essential goal. Although various cognitive tests have been used for spaceflight assessment, these have been challenged on their lack of operational relevance.METHODS: To address this gap, we developed and characterized the Robotic On-Board Trainer for Research (ROBoT-r) system, based on the Robotic On-Board Trainer (ROBoT) currently used for astronaut training on Canadarm2 track-and-capture activities. The task requires use of dual hand-controllers (6 degrees of freedom) to grapple an incoming vehicle in free-drift in a time-limited setting. After developing a platform for conducting research studies, characterization testing of ROBoT-r was completed by 14 astronaut-like volunteers (35 ± 11 yr; N = 5 women) over 16 sessions each.RESULTS: We describe the design and capabilities of the ROBoT-r system for conducting operationally relevant research on human performance. Version 6.2 of the system supports H-II Transfer Vehicle track-and-capture operations within a multimillion component, physics-enabled 3D model using NASA's DOUG graphics platform. It has configurable task initialization and auto-run capabilities, saves 38 variables continuously at 20 Hz throughout each run, provides the user quantitative feedback after each run, and provides summaries after each session. Detailed performance characterization data is reported for future experimental planning purposes.DISCUSSION: ROBoT-r's range of performance variables enables detailed and quantitative performance assessment. Its use in spaceflight will help provide insight into operational performance, as well as allowing investigators to compare these results with more traditional cognitive tests to help better understand the interaction between individual cognitive abilities and operational performance.Ivkovic V, Sommers B, Cefaratti DA, Newman G, Thomas DW, Alexander DG, Strangman GE. Operationally relevant behavior assessment using the Robotic On-Board Trainer for Research (ROBoT-r). Aerosp Med Hum Perform. 2019; 90(9):819-825.

Cellulose Nanofibril Formulations Incorporating a Low-Molecular-Weight Alginate Oligosaccharide Modify Bacterial Biofilm Development.

Cellulose nanofibrils (CNFs) from wood pulp are a renewable material possessing advantages for biomedical applications because of their customizable porosity, mechanical strength, translucency, and environmental biodegradability. Here, we investigated the growth of multispecies wound biofilms on CNF formulated as aerogels and films incorporating the low-molecular-weight alginate oligosaccharide OligoG CF-5/20 to evaluate their structural and antimicrobial properties. Overnight microbial cultures were adjusted to 2.8 × 109 colony-forming units (cfu) mL-1 in Mueller Hinton broth and growth rates of Pseudomonas aeruginosa PAO1 and Staphylococcus aureus 1061A monitored for 24 h in CNF dispersions sterilized by γ-irradiation. Two CNF formulations were prepared (20 g m-2) with CNF as air-dried films or freeze-dried aerogels, with or without incorporation of an antimicrobial alginate oligosaccharide (OligoG CF-5/20) as a surface coating or bionanocomposite, respectively. The materials were structurally characterized by scanning electron microscopy (SEM) and laser profilometry (LP). The antimicrobial properties of the formulations were assessed using single- and mixed-species biofilms grown on the materials and analyzed using LIVE/DEAD staining with confocal laser scanning microscopy (CLSM) and COMSTAT software. OligoG-CNF suspensions significantly decreased the growth of both bacterial strains at OligoG concentrations >2.58% (P < 0.05). SEM showed that aerogel-OligoG bionanocomposite formulations had a more open three-dimensional structure, whereas LP showed that film formulations coated with OligoG were significantly smoother than untreated films or films incorporating PEG400 as a plasticizer (P < 0.05). CLSM of biofilms grown on films incorporating OligoG demonstrated altered biofilm architecture, with reduced biomass and decreased cell viability. The OligoG-CNF formulations as aerogels or films both inhibited pyocyanin production (P < 0.05). These novel CNF formulations or bionanocomposites were able to modify bacterial growth, biofilm development, and virulence factor production in vitro. These data support the potential of OligoG and CNF bionanocomposites for use in biomedical applications where prevention of infection or biofilm growth is required.

Anti-pseudomonad Activity of Manuka Honey and Antibiotics in a Specialized ex vivo Model Simulating Cystic Fibrosis Lung Infection.

Pseudomonas aeruginosa causes problematic chronic lung infections in those suffering from cystic fibrosis. This is due to its antimicrobial resistance mechanisms and its ability to form robust biofilm communities with increased antimicrobial tolerances. Using novel antimicrobials or repurposing current ones is required in order to overcome these problems. Manuka honey is a natural antimicrobial agent that has been used for many decades in the treatment of chronic surface wounds with great success, particularly those infected with P. aeruginosa. Here we aim to determine whether the antimicrobial activity of manuka honey could potentially be repurposed to inhibit pulmonary P. aeruginosa infections using two ex vivo models. P. aeruginosa isolates (n = 28) from an international panel were tested for their susceptibility to manuka honey and clinically relevant antibiotics (ciprofloxacin, ceftazidime, and tobramycin), alone and in combination, using conventional antimicrobial susceptibility testing (AST). To increase clinical applicability, two ex vivo porcine lung (EVPL) models (using alveolar and bronchiolar tissue) were used to determine the anti-biofilm effects of manuka honey alone and in combination with antibiotics. All P. aeruginosa isolates were susceptible to manuka honey, however, varying incidences of resistance were seen against antibiotics. The combination of sub-inhibitory manuka honey and antibiotics using conventional AST had no effect on activity against the majority of isolates tested. Using the two ex vivo models, 64% (w/v) manuka honey inhibited many of the isolates where abnormally high concentrations of antibiotics could not. Typically, combinations of both manuka honey and antibiotics had increased antimicrobial activity. These results highlight the potential of manuka honey as a future antimicrobial for the treatment of pulmonary P. aeruginosa isolates, clearing potential infection reservoirs within the upper airway.

Polymer Masked-Unmasked Protein Therapy: Identification of the Active Species after Amylase Activation of Dextrin-Colistin Conjugates.

Polymer masked-unmasked protein therapy (PUMPT) uses conjugation of a biodegradable polymer, such as dextrin, hyaluronic acid, or poly(l-glutamic acid), to mask a protein or peptide's activity; subsequent locally triggered degradation of the polymer at the target site regenerates bioactivity in a controllable fashion. Although the concept of PUMPT is well established, the relationship between protein unmasking and reinstatement of bioactivity is unclear. Here, we used dextrin-colistin conjugates to study the relationship between the molecular structure (degree of unmasking) and biological activity. Size exclusion chromatography was employed to collect fractions of differentially degraded conjugates and ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) employed to characterize the corresponding structures. Antimicrobial activity was studied using a minimum inhibitory concentration (MIC) assay and confocal laser scanning microscopy of LIVE/DEAD-stained biofilms with COMSTAT analysis. In vitro toxicity of the degraded conjugate was assessed using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. UPLC-MS revealed that the fully "unmasked" dextrin-colistin conjugate composed of colistin bound to at least one linker, whereas larger species were composed of colistin with varying lengths of glucose units attached. Increasing the degree of dextrin modification by succinoylation typically led to a greater number of linkers bound to colistin. Greater antimicrobial and antibiofilm activity were observed for the fully "unmasked" conjugate compared to the partially degraded species (MIC = 0.25 and 2-8 µg/mL, respectively), whereas dextrin conjugation reduced colistin's in vitro toxicity toward kidney cells, even after complete unmasking. This study highlights the importance of defining the structure-antimicrobial activity relationship for novel antibiotic derivatives and demonstrates the suitability of LC-MS to aid the design of biodegradable polymer-antibiotic conjugates.

Controlled release of dextrin-conjugated growth factors to support growth and differentiation of neural stem cells.

An essential aspect of stem cell in vitro culture and in vivo therapy is achieving sustained levels of growth factors to support stem cell survival and expansion, while maintaining their multipotency and differentiation potential. This study investigated the ability of dextrin (~74,000 g/mol; 27.8 mol% succinoylation) conjugated to epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF; or FGF-2) (3.9 and 6.7% w/w protein loading, respectively) to support the expansion and differentiation of stem cells in vitro via sustained, controllable growth factor release. Supplementation of mouse neural stem cells (mNSCs) with dextrin-growth factor conjugates led to greater and prolonged proliferation compared to unbound EGF/bFGF controls, with no detectable apoptosis after 7 days of treatment. Immunocytochemical detection of neural precursor (nestin) and differentiation (Olig2, MAP2, GFAP) markers verified that controlled release of dextrin-conjugated growth factors preserves stem cell properties of mNSCs for up to 7 days. These results show the potential of dextrin-growth factor conjugates for localized delivery of bioactive therapeutic agents to support stem cell expansion and differentiation, and as an adjunct to direct neuronal repair.

Stochastic electromagnetic field propagation- measurement and modelling.

This paper reviews recent progress in the measurement and modelling of stochastic electromagnetic fields, focusing on propagation approaches based on Wigner functions and the method of moments technique. The respective propagation methods are exemplified by application to measurements of electromagnetic emissions from a stirred, cavity-backed aperture. We discuss early elements of statistical electromagnetics in Heaviside's papers, driven mainly by an analogy of electromagnetic wave propagation with heat transfer. These ideas include concepts of momentum and directionality in the realm of propagation through confined media with irregular boundaries. We then review and extend concepts using Wigner functions to propagate the statistical properties of electromagnetic fields. We discuss in particular how to include polarization in this formalism leading to a Wigner tensor formulation and a relation to an averaged Poynting vector.This article is part of the theme issue 'Celebrating 125 years of Oliver Heaviside's 'Electromagnetic Theory''.