Small Cellular Particles from European Spruce Needle Homogenate.

Small cellular particles (SCPs) are being considered for their role in cell-to-cell communication. We harvested and characterized SCPs from spruce needle homogenate. SCPs were isolated by differential ultracentrifugation. They were imaged by scanning electron microscope (SEM) and cryogenic transmission electron microscope (cryo TEM), assessed for their number density and hydrodynamic diameter by interferometric light microscopy (ILM) and flow cytometry (FCM), total phenolic content (TPC) by UV-vis spectroscopy, and terpene content by gas chromatography-mass spectrometry (GC-MS). The supernatant after ultracentrifugation at 50,000× g contained bilayer-enclosed vesicles whereas in the isolate we observed small particles of other types and only a few vesicles. The number density of cell-sized particles (CSPs) (larger than 2 µm) and meso-sized particles (MSPs) (cca 400 nm-2 µm) was about four orders of magnitude lower than the number density of SCPs (sized below 500 nm). The average hydrodynamic diameter of SCPs measured in 10,029 SCPs was 161 ± 133 nm. TCP decreased considerably due to 5-day aging. Volatile terpenoid content was found in the pellet after 300× g. The above results indicate that spruce needle homogenate is a source of vesicles to be explored for potential delivery use.

Trideset let delovanja Sole eksperimentalne kemije na Institutu "Jozef Stefan": trideset let motiviranja mladih generacij in utrjevanja poti naravoslovnega izobrazevanja.

Pred 30. leti, natancneje spomladi leta 1992, je bila, v okviru Odseka za anorgansko kemijo in tehnologijo Instituta "Jozef Stefan", ustanovljena Sola eksperimentalne kemije. Zaradi razvoja znanosti in interdisciplinarnih pristopov, je njen glavni namen priblizevanje kemije mladim generacijam in prikazovanje njene sirse uporabe v vsakdanjem zivljenju. Sola eksperimentalne kemije tako ustvarja pomemben most med raziskovanjem in izobrazevanjem ter aktivno prispeva k popularizaciji predmetnega podrocja v solah.

Interaction between Microalgae P. tricornutum and Bacteria Thalassospira sp. for Removal of Bisphenols from Conditioned Media.

We studied the efficiency of three culture series of the microalgae Phaeodactylum tricornutum (P. tricornutum) and bacteria Thalassospira sp. (axenic microalgae, bacterial culture and co-culture of the two) in removing bisphenols (BPs) from their growth medium. Bacteria were identified by 16S ribosomal RNA polymerase chain reaction (16S rRNA PCR). The microorganism growth rate was determined by flow cytometry. Cultures and isolates of their small cellular particles (SCPs) were imaged by scanning electron microscopy (SEM) and cryogenic transmission electron microscopy (Cryo-TEM). BPs were analyzed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). Our results indicate that some organisms may have the ability to remove a specific pollutant with high efficiency. P. tricornutum in axenic culture and in mixed culture removed almost all (more than 99%) of BPC2. Notable differences in the removal of 8 out of 18 BPs between the axenic, mixed and bacterial cultures were found. The overall removals of BPs in axenic P. tricornutum, mixed and bacterial cultures were 11%, 18% and 10%, respectively. Finding the respective organisms and creating microbe societies seems to be key for the improvement of wastewater treatment. As a possible mediating factor, numerous small cellular particles from all three cultures were detected by electron microscopy. Further research on the mechanisms of interspecies communication is needed to advance the understanding of microbial communities at the nano-level.

Enrichment of plasma in platelets and extracellular vesicles by the counterflow to erythrocyte settling.

In order to prepare optimal platelet and extracellular vesicle (EV)-rich plasma for the treatment of chronic temporal bone inflammation, we studied effects of centrifugation parameters on redistribution of blood constituents in blood samples of 23 patients and 20 volunteers with no record of disease. Concentrations of blood cells and EVs were measured by flow cytometry. Sample content was inspected by scanning electron microscopy. A mathematical model was constructed to interpret the experimental results. The observed enrichment of plasma in platelets and EVs after a single spin of blood depended on the erythrocyte sedimentation rate, thereby indicating the presence of a flow of plasma that carried platelets and EVs in the direction opposite to settling of erythrocytes. Prolonged handling time correlated with the decrease of concentration of platelets and larger EVs in platelet and EV-rich plasma (PVRP), R = -0.538, p = 0.003, indicating cell fragmentation during the processing of samples. In further centrifugation of the obtained plasma, platelet and EV enrichment depended on the average distance of the sample from the centrifuge rotor axis. Based on the agreement of the model predictions with observations, we propose the centrifugation protocol optimal for platelet and EV enrichment and recovery in an individual sample, adjusted to the dimensions of the centrifuge rotor, volume of blood and erythrocyte sedimentation rate.[Figure: see text].

Stability of Erythrocyte-Derived Nanovesicles Assessed by Light Scattering and Electron Microscopy.

Extracellular vesicles (EVs) are gaining increasing amounts of attention due to their potential use in diagnostics and therapy, but the poor reproducibility of the studies that have been conducted on these structures hinders their breakthrough into routine practice. We believe that a better understanding of EVs stability and methods to control their integrity are the key to resolving this issue. In this work, erythrocyte EVs (hbEVs) were isolated by centrifugation from suspensions of human erythrocytes that had been aged in vitro. The isolate was characterised by scanning (SEM) and cryo-transmission electron microscopy (cryo-TEM), flow cytometry (FCM), dynamic/static light scattering (LS), protein electrophoresis, and UV-V spectrometry. The hbEVs were exposed to various conditions (pH (4-10), osmolarity (50-1000 mOsm/L), temperature (15-60 °C), and surfactant Triton X-100 (10-500 µM)). Their stability was evaluated by LS by considering the hydrodynamic radius (Rh), intensity of scattered light (I), and the shape parameter (ρ). The morphology of the hbEVs that had been stored in phosphate-buffered saline with citrate (PBS-citrate) at 4 °C remained consistent for more than 6 months. A change in the media properties (50-1000 mOsm/L, pH 4-10) had no significant effect on the Rh (=100-130 nm). At pH values below 6 and above 8, at temperatures above 45 °C, and in the presence of Triton X-100, hbEVs degradation was indicated by a decrease in I of more than 20%. Due to the simple preparation, homogeneous morphology, and stability of hbEVs under a wide range of conditions, they are considered to be a suitable option for EV reference material.

Autologous Platelet- and Extracellular Vesicle-Rich Plasma Is an Effective Treatment Modality for Chronic Postoperative Temporal Bone Cavity Inflammation: Randomized Controlled Clinical Trial.

PURPOSE: To determine the efficacy of autologous platelet- and extracellular vesicle-rich plasma (PVRP) to treat chronic postoperative temporal bone cavity inflammation (CPTBCI) after exhausting surgical and standard conservative therapies. MATERIALS AND METHODS: Patients were randomly allocated to treatment with PVRP (PVRP group) or standard conservative methods (control group) in a setting of four once-monthly checkups and subsequent follow-up. The treatment outcome was measured with the Chronic Otitis Media Questionnaire-12 (COMQ-12), CPTBCI focus surface area, and CPTBCI symptom-free time after the fourth checkup. RESULTS: Eleven patients from each group completed the trial; 95% of patients suffered from chronically discharging mastoid cavity (the type of CPTBCI). Within four checkups, the COMQ-12 score decreased statistically significantly in the PVRP group (p < 0.001) but not in the control group (p = 0.339). The CPTBCI foci surface area decreased statistically significantly between the first and second checkups (p < 0.0005) but not between other checkups (p > 0.05) in the PVRP group. No statistically significant differences in CPTBCI foci surface area were detected between checkups in the control group (p = 0.152). Nine patients from the PVRP group and three patients from the control group were CPTBCI symptom-free at the fourth checkup. The median symptom-free time was 9.2 months (95% CI [7.4, 11.9]) in the PVRP group. Cumulatively, 49% of patients in the PVRP group remained CPTBCI symptom-free for 12.7 months after the fourth checkup. CONCLUSION: Autologous PVRP represents a novel additional and successful treatment modality for a chronically discharging radical mastoid cavity when the surgical and standard conservative treatment methods have been exhausted. TRIAL NUMBER: https://clinicaltrials.gov (NCT04281901).

Magneto-mechanical actuation of barium-hexaferrite nanoplatelets for the disruption of phospholipid membranes.

HYPOTHESIS: The magneto-mechanical actuation (MMA) of magnetic nanoparticles with a low-frequency alternating magnetic field (AMF) can be used to destroy cancer cells. So far, MMA was tested on different cells using different nanoparticles and different field characteristics, which makes comparisons and any generalizations about the results of MMA difficult. In this paper we propose the use of giant unilamellar vesicles (GUVs) as a simple model system to study the effect of MMA on a closed lipid bilayer membrane, i.e., a basic building block of any cell. EXPERIMENTS: The GUVs were exposed to barium-hexaferrite nanoplatelets (NPLs, ~50 nm wide and 3 nm thick) with unique magnetic properties dominated by a permanent magnetic moment that is perpendicular to the platelet, at different concentrations (1-50 µg/mL) and pH values (4.2-7.4) of the aqueous suspension. The GUVs were observed with an optical microscope while being exposed to a uniaxial AMF (3-100 Hz, 2.2-10.6 mT). FINDINGS: When the NPLs were electrostatically attached to the GUV membranes, the MMA induced cyclic fluctuations of the GUVs' shape corresponding to the AMF frequency at the low NPL concentration (1 µm/mL), whereas the GUVs were bursting at the higher concentration (10 µg/mL). Theoretical considerations suggested that the bursting of the GUVs is a consequence of the local action of an assembly of several NPLs, rather than a collective effect of all the absorbed NPLs.

Stereopure Functionalized Benzosultams via Ruthenium(II)-Catalyzed Dynamic Kinetic Resolution-Asymmetric Transfer Hydrogenation.

A highly diastereo- and enantioselective Ru(II)-catalyzed dynamic kinetic resolution-asymmetric transfer hydrogenation (DKR-ATH) of α-(N-sulfonylimino) and α-(N-sulfonylamino) aryl ketones to 4-hydroxy-benzo-δ- and 3-(α-hydroxy-arylmethyl)-benzo-γ-sultams is presented. By employing enantiopure ansa-Ru[PipSO2DPEN(CH2)4Ph] cat. II with S/C = 10 000 in a HCO2H/Et3N binary mix, up to >99.9% ee and dr >99:1 are obtained with 100% conversion under mild conditions. Application to access the stereopure "structurally simplified TsDPEN" N,N-ligand syn-3-(α-aminobenzyl)-benzo-γ-sultam ("syn-ULTAM") and its structural isomer trans-4-amino-3-phenyl-benzo-δ-sultam (trans-4) is demonstrated.