Confocal microscopy analysis reveals that only a small proportion of extracellular vesicles are successfully labelled with commonly utilised staining methods

Assessing genuine extracellular vesicle (EV) uptake is crucial for understanding the functional roles of EVs. This study measured the bona fide labelling of EVs utilising two commonly used fluorescent dyes, PKH26 and C5-maleimide-Alexa633. MCF7 EVs tagged with mEmerald-CD81 were isolated from conditioned media by size exclusion chromatography (SEC) and characterised using Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), MACsPlex immunocapture assay and immunoblots. These fluorescently tagged EVs were subsequently stained with C5-maleimide-Alexa633 or PKH26, according to published protocols. Colocalisation of dual-labelled EVs was assessed by confocal microscopy and quantified using the Rank-Weighted Colocalisation (RWC) algorithm. We observed strikingly poor colocalisation between mEmerald-CD81-tagged EVs and C5-Maleimide-Alexa633 (5.4% ± 1.8) or PKH26 (4.6% ± 1.6), that remained low even when serum was removed from preparations. Our data confirms previous work showing that some dyes form contaminating aggregates. Furthermore, uptake studies showed that maleimide and mEmerald-CD81-tagged EVs can be often located into non-overlapping subcellular locations. By using common methods to isolate and stain EVs we observed that most EVs remained unstained and most dye signal does not appear to be EV associated. Our work shows that there is an urgent need for optimisation and standardisation in how EV researchers use these tools to assess genuine EV signals.

Simultaneous Exposure of Different Nanoparticles Influences Cell Uptake

Drug delivery using nano-sized carriers holds tremendous potential for curing a range of diseases. The internalisation of nanoparticles by cells, however, remains poorly understood, restricting the possibility for optimising entrance into target cells, avoiding off-target cells and evading clearance. The majority of nanoparticle cell uptake studies have been performed in the presence of only the particle of interest; here, we instead report measurements of uptake when the cells are exposed to two different types of nanoparticles at the same time. We used carboxylated polystyrene nanoparticles of two different sizes as a model system and exposed them to HeLa cells in the presence of a biomolecular corona. Using flow cytometry, we quantify the uptake at both average and individual cell level. Consistent with previous literature, we show that uptake of the larger particles is impeded in the presence of competing smaller particles and, conversely, that uptake of the smaller particles is promoted by competing larger particles. While the mechanism(s) underlying these observations remain(s) undetermined, we are partly able to restrain the likely possibilities. In the future, these effects could conceivably be used to enhance uptake of nano-sized particles used for drug delivery, by administering two different types of particles at the same time.

Interrogation of Carboxy-Terminus Localized GJA1 Variants Associated with Erythrokeratodermia Variabilis et Progressiva

Although inherited GJA1 (encoding Cx43) gene mutations most often lead to oculodentodigital dysplasia and related disorders, four variants have been linked to erythrokeratodermia variabilis et progressiva (EKVP), a skin disorder characterized by erythematous and hyperkeratotic lesions. While two autosomal-dominant EKVP-linked GJA1 mutations have been shown to lead to augmented hemichannels, the consequence(s) of keratinocytes harboring a de novo P283L variant alone or in combination with a de novo T290N variant remain unknown. Interestingly, these variants reside within or adjacent to a carboxy terminus polypeptide motif that has been shown to be important in regulating the internalization and degradation of Cx43. Cx43-rich rat epidermal keratinocytes (REKs) or Cx43-ablated REKs engineered to express fluorescent protein-tagged P283L and/or T290N variants formed prototypical gap junctions at cell–cell interfaces similar to wildtype Cx43. Dye coupling and dye uptake studies further revealed that each variant or a combination of both variants formed functional gap junction channels, with no evidence of augmented hemichannel function or induction of cell death. Tracking the fate of EKVP-associated variants in the presence of the protein secretion blocker brefeldin A, or an inhibitor of protein synthesis cycloheximide, revealed that P283L or the combination of P283L and T290N variants either significantly extended Cx43 residency on the cell surface of keratinocytes or delayed its degradation. However, caution is needed in concluding that this modest change in the Cx43 life cycle is sufficient to cause EKVP, or whether an additional underlying mechanism or another unidentified gene mutation is contributing to the pathogenesis found in patients. This question will be resolved if further patients are identified where whole exome sequencing reveals a Cx43 P283L variant alone or, in combination with a T290N variant, co-segregates with EKVP across several family generations.

Novel pyrazoles as potent growth inhibitors of staphylococci, enterococci and Acinetobacter baumannii bacteria

Background: Methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci and Acinetobacter baumannii cause serious antibiotic-resistant infections. Finding new antibiotics to treat these infections is imperative for combating this worldwide menace. Methods & Results: In this study, the authors designed and synthesized potent antimicrobial agents using 4-trifluoromethylphenyl-substituted pyrazole derivatives. In addition to their potency against planktonic bacteria, potent compounds effectively eradicated S. aureus and Enterococcus faecalis biofilms. Human cells tolerated these compounds with good selectivity factors. Furthermore, the authors provide evidence for the mode of action of compounds based on time-kill kinetics, flow cytometry analysis of propidium iodide-treated bacteria and oxygen uptake studies. Conclusion: This study demonstrated 20 novel compounds with potent antibacterial activity that are tolerated by human cell lines.

Ultrasound and Microbubbles Enhance Uptake of Doxorubicin in Murine Kidneys

The use of ultrasound and microbubble-enhanced drug delivery, commonly referred to as sonoporation, has reached numerous clinical trials and has shown favourable results. Nevertheless, the microbubbles and acoustic path also pass through healthy tissues. To date, the majority of studies have focused on the impact to diseased tissues and rarely evaluated the impact on healthy and collateral tissue. The aim of this study was to test the effect and feasibility of low-intensity sonoporation on healthy kidneys in a mouse model. In our work here, we used a clinical diagnostic ultrasound system (GE Vivid E9) with a C1-5 ultrasound transducer combined with a software modification for 20-µs-long pulses to induce the ultrasound-guided drug delivery of doxorubicin (DOX) in mice kidneys in combination with SonoVue® and Sonazoid™ microbubbles. The acoustic output settings were within the commonly used diagnostic ranges. Sonoporation with SonoVue® resulted in a significant decrease in weight vs. DOX alone (p = 0.0004) in the first nine days, whilst all other comparisons were not significant. Ultrasound alone resulted in a 381% increase in DOX uptake vs. DOX alone (p = 0.0004), whilst SonoVue® (p = 0.0001) and Sonazoid™ (p < 0.0001) further increased the uptake nine days after treatment (419% and 493%, respectively). No long-standing damage was observed in the kidneys via histology. In future sonoporation and drug uptake studies, we therefore suggest including an “ultrasound alone” group to verify the actual contribution of the individual components of the procedure on the drug uptake and to perform collateral damage studies to ensure there is no negative impact of low-intensity sonoporation on healthy tissues.

Some Uptake Studies of Metal Ions and the Formation of Cu₂0 Particles in Wool

<p>This research programme is concerned with the uptake studies of Cu2+, Zn2+ and Mn2+ at different conditions, by merino wool fibres and also uptake studies of Cu2+ ions by chemically modified wool fibres. Cu2O particles and Cu complexes are formed within merino wool by an in situ reaction with sodium borohydride and thioglycoloic acid respectively. The d-block elements have the ability to bind chemically to certain functional groups present within the keratin protein of wool. The absorption of the Cu2+, Mn2+ and Zn2+ from solution by wool fibres under different conditions notably, time, temperature and initial concentration have been studied. The optimum temperature and reaction time to give highest absorption of the Cu2+ by the wool fibre was found to be 90 oC and one hour without modifying the nature of the wool, from a solution of Cu2+ concentration of 450 mg L-1. Cu2+ was found to give the greatest absorption by the wool fibres, whereas Zn2+ and Mn2+ were found to be absorbed the least. The absorption of Cu2+ ions increases with increasing temperature. At the higher temperature of 90 oC, the -S-S- bonds in the cystine amino acids break more readily, generating thiol and cysteic acid groups to bind with copper ions. The uptake of Cu2+ by ethylenediaminetetraacetic dianhydride (14 mg g-1 of wool) or thioglycolic acid (42.5 mg g-1 of wool) or sodium borohydride (41.8 mg g-1 of wool) treated merino wool fibres increases with respect to unmodified wool (8 mg g-1 of wool). NaBH4 treated merino wool reduces Cu2+ ions to Cu2O particles which form within the wool fibres by an in situ reaction. TGA treated merino wool provides additional functional groups to bind with copper ions and Cu2O particles also likely to be formed within TGA treated wool composites. The metal ions were absorbed into the fibres under various conditions and the extent of absorption was quantified. The form and binding of the Cu2O particles or Cu2+ ions onto the wool fibres are studied using UV-Visible, FTIR, XRD, SEM, EDS and TEM methods.</p>

Some Uptake Studies of Metal Ions and the Formation of Cu₂0 Particles in Wool

<p>This research programme is concerned with the uptake studies of Cu2+, Zn2+ and Mn2+ at different conditions, by merino wool fibres and also uptake studies of Cu2+ ions by chemically modified wool fibres. Cu2O particles and Cu complexes are formed within merino wool by an in situ reaction with sodium borohydride and thioglycoloic acid respectively. The d-block elements have the ability to bind chemically to certain functional groups present within the keratin protein of wool. The absorption of the Cu2+, Mn2+ and Zn2+ from solution by wool fibres under different conditions notably, time, temperature and initial concentration have been studied. The optimum temperature and reaction time to give highest absorption of the Cu2+ by the wool fibre was found to be 90 oC and one hour without modifying the nature of the wool, from a solution of Cu2+ concentration of 450 mg L-1. Cu2+ was found to give the greatest absorption by the wool fibres, whereas Zn2+ and Mn2+ were found to be absorbed the least. The absorption of Cu2+ ions increases with increasing temperature. At the higher temperature of 90 oC, the -S-S- bonds in the cystine amino acids break more readily, generating thiol and cysteic acid groups to bind with copper ions. The uptake of Cu2+ by ethylenediaminetetraacetic dianhydride (14 mg g-1 of wool) or thioglycolic acid (42.5 mg g-1 of wool) or sodium borohydride (41.8 mg g-1 of wool) treated merino wool fibres increases with respect to unmodified wool (8 mg g-1 of wool). NaBH4 treated merino wool reduces Cu2+ ions to Cu2O particles which form within the wool fibres by an in situ reaction. TGA treated merino wool provides additional functional groups to bind with copper ions and Cu2O particles also likely to be formed within TGA treated wool composites. The metal ions were absorbed into the fibres under various conditions and the extent of absorption was quantified. The form and binding of the Cu2O particles or Cu2+ ions onto the wool fibres are studied using UV-Visible, FTIR, XRD, SEM, EDS and TEM methods.</p>

Calcium Phosphate-Coated Lipid Nanoparticles as a Potential Tool in Bone Diseases Therapy

The treatment of bone diseases (including osteoporosis, osteoarthritis, and bone cancer) often results in reduced efficiency and/or adverse reactions due to the fact that it is not specifically targeted to the site of action. The employment of a suitable carrier should increase drug location to the site of bone disease. The purpose of this study is to prepare and characterize lipid nanoparticles (NPs) coated with calcium phosphate (CaP-NPs). A coating method, to date used only to obtain liposomes covered with CaP, is herein partially-modified to prepare CaP-coated lipid NPs. An extensive physico-chemical characterization was achieved by employing several techniques (DLS, SEM and TEM, and both combined with EDS, XRD, and FTIR) that confirmed the feasibility of the developed coating method. Preliminary uptake studies on human osteosarcoma cells (U-2OS) were performed by entrapping, as a lipid probe, Sudan Red III in NPs. The obtained data provided evidence that CaP-NPs showed higher cell accumulation than uncoated NPs. This result may have important implications for the development of drug loaded CaP-NPs to be tested in vitro with a view of planning future treatment of bone diseases, and indicate that CaP-NPs are potential vehicles for selective drug delivery to bone tissue.

Structural insights into a novel family of integral membrane siderophore reductases

Gram-negative bacteria take up the essential ion Fe3+ as ferric-siderophore complexes through their outer membrane using TonB-dependent transporters. However, the subsequent route through the inner membrane differs across many bacterial species and siderophore chemistries and is not understood in detail. Here, we report the crystal structure of the inner membrane protein FoxB (from Pseudomonas aeruginosa) that is involved in Fe-siderophore uptake. The structure revealed a fold with two tightly bound heme molecules. In combination with in vitro reduction assays and in vivo iron uptake studies, these results establish FoxB as an inner membrane reductase involved in the release of iron from ferrioxamine during Fe-siderophore uptake.