The Efficiency of CBD Production Using Grafted Cannabis sativa L. Plants Is Highly Dependent on the Type of Rootstock: A Study.

The global cannabis market is continuously expanding and as a result, the cannabis industry demands new and improved agronomic cultivation practices to increase production efficiency of cannabidiol (CBD), which is valued for its therapeutic benefits. This study investigates the influence of three rootstock types on the survival rate, morphological parameters, and biochemical composition of cannabis: potentially dwarfing rootstocks (PDR), potentially vigorous rootstocks (PVR), and seedlings-as-rootstocks (SAR). Rootstocks were used for grafting two scion genotypes: 'ScionII' = chemotype II of industrial hemp, and 'ScionIII' = chemotype III of high CBD accumulating variety. Contrary to expectations, PVR and SAR did not outperform PDR on most of the measured variables. SAR showed the highest survival rate of the grafted cannabis plants (40-70%). The rootstock type had a statistically significant influence only on the bud compactness index in 'ScionII', with PDR being particularly noticeable. A comparative analysis of the 'rootstock/scion' combinations with their controls (non-grafted scions) revealed grafting's substantial improvement in most traits. Specifically, PDR increased CBD content by 27% in 'ScionIII', inflorescence yield and CBD yield per plant increased by 71% and 84%, respectively, when SAR was used in 'ScionII'. SAR showed to be the most effective rootstock type for CBD production. Our findings suggest grafting as a promising technique for optimizing cannabis's agronomic and medicinal potential, highlighting the necessity for further research on its underlying mechanisms to refine production efficiency and quality.

Antifungal Effect of Polymethyl Methacrylate Resin Base with Embedded Au Nanoparticles.

Full and partial restorations in dentistry must replicate the characteristics of the patient's natural teeth. Materials must have good mechanical properties and be non-toxic and biocompatible. Microbes, which can form biofilms, are constantly in contact with restorations. In this study, we investigate how well Candida albicans adheres to a polymethyl methacrylate (PMMA) resin base with gold (Au) nanoparticles. We synthesized Au nanoparticles and characterized them. The average size of Au nanoparticles embedded in PMMA was 11 nm. The color difference ΔE between PMMA and PMMA/Au composites was 2.7 and was still esthetically acceptable to patients. PMMA/Au surfaces are smoother and more hydrophilic than pure PMMA surfaces, and the isoelectric point of both types of surfaces was 4.3. Above the isoelectric point, PMMA/Au surfaces are more negatively charged than PMMA surfaces. The added Au nanoparticles decreased the tensile strength, while the hardness did not change significantly. Adhesion measurements showed that PMMA surfaces modified with Au nanoparticles reduced the extent of microbial adhesion of Candida albicans.

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.

Biophysical Properties of Foamed and Solid Polymers Used in Orthotics and Prosthetics.

Orthotic and prosthetic materials should have good mechanical and antibacterial properties. Therefore, in our study, we consider four common foamed closed-cells and two solid polymeric materials regarding their mechanical behaviour and tendency for bacterial adhesion. For all materials, the surface roughness, hydrophobicity, zeta potential, tensile properties, hardness and CIE color parameters were measured. We found that foamed polymeric materials have higher roughness, higher hydrophobicity, lower Young's modulus, lower maximum tensile strength and lower hardness than solid materials. Bacterial adhesion test measurements based on observation by scanning electron microscopy show much a lower adhesion extent of S. aureus on solid materials than on foamed materials. The measured biophysical properties could be the key data for users to select the optimal materials.

Decrease in Cellular Nanovesicles Concentration in Blood of Athletes More Than 15 Hours After Marathon.

INTRODUCTION: Cellular nanovesicles (CNVs), that are shed from cells, have been recognized as promising indicators of health status. We analyzed the effect of long-distance running on concentration of CNVs, along with some standard blood parameters, in 27 athletes two days before and >15 hours after physical effort. METHODS: CNVs were isolated by repetitive centrifugation and washing of samples, and assessed by flow cytometry. Cholinesterase (ChE) and glutathione S-transferase (GST) activity were measured spectrophotometrically. Interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) concentrations were measured using enzyme-linked immunosorbent assay (ELISA). C-reactive protein (CRP) was measured with immunoturbidimetric determination and lipidogram parameters were measured by enzymatic colorimetric assay. Flow cytometry was used for blood cell count and mean platelet volume (MPV) measurement. RESULTS: More than 15 hours after physical effort a decrease was found in CNVs' concentration in isolates from blood (46%; p<0.05), in ChE activity in whole blood (47%; p<0.001), in plasma (34%; p<0.01), and in erythrocyte suspension (54%; p<0.001), as well as in GST activity in erythrocyte suspension (16%; p<0.01) and in IL-6 concentration in plasma (63%; p<0.05). We found no change in GST activity in plasma and in TNF-α concentration in plasma. Correlations (>0.8; p<0.001) between CNVs' concentration and ChE activity, and GST activity, respectively, in erythrocyte suspension were found. CONCLUSION: We found that >15 hours post-physical effort, CNVs' concentration was below the initial value, concomitant with other measured parameters: ChE and GST activity as well as IL-6 concentration, indicating a favorable effect of physical effort on health status. CNVs' concentration and ChE activity in isolates from peripheral blood proved to have potential as indicators of the response of the human body to inflammation after physical effort. Physical activity should be considered as an important factor in preparation of subjects for blood sampling in procedures focusing on CNV-containing diagnostic and therapeutic compounds.

Extracellular Vesicle Isolation Yields Increased by Low-Temperature Gaseous Plasma Treatment of Polypropylene Tubes.

Novel Extracellular Vesicles (EVs) based diagnostic techniques are promising non-invasive procedures for early stage disease detection which are gaining importance in the medical field. EVs are cell derived particles found in body liquids, especially blood, from which they are isolated for further analysis. However, techniques for their isolation are not fully standardized and require further improvement. Herein modification of polypropylene (PP) tubes by cold Atmospheric Pressure Plasma Jet (APPJ) is suggested to minimize the EVs to surface binding and thus increase EVs isolation yields. The influence of gaseous plasma treatment on surface morphology was studied by Atomic Force Microscopy (AFM), changes in surface wettability by measuring the Water Contact Angle (WCA), while surface chemical changes were analyzed by X-Ray Photoelectron Spectroscopy (XPS). Moreover, PP tubes from different manufacturers were compared. The final isolation yields of EVs were evaluated by flow cytometry. The results of this study suggest that gaseous plasma treatment is an intriguing technique to uniformly alter surface properties of PP tubes and improve EVs isolation yields up to 42%.

Viscosity of Plasma as a Key Factor in Assessment of Extracellular Vesicles by Light Scattering.

Extracellular vesicles (EVs) isolated from biological samples are a promising material for use in medicine and technology. However, the assessment methods that would yield repeatable concentrations, sizes and compositions of the harvested material are missing. A plausible model for the description of EV isolates has not been developed. Furthermore, the identity and genesis of EVs are still obscure and the relevant parameters have not yet been identified. The purpose of this work is to better understand the mechanisms taking place during harvesting of EVs, in particular the role of viscosity of EV suspension. The EVs were harvested from blood plasma by repeated centrifugation and washing of samples. Their size and shape were assessed by using a combination of static and dynamic light scattering. The average shape parameter of the assessed particles was found to be ρ ~ 1 (0.94-1.1 in exosome standards and 0.7-1.2 in blood plasma and EV isolates), pertaining to spherical shells (spherical vesicles). This study has estimated the value of the viscosity coefficient of the medium in blood plasma to be 1.2 mPa/s. It can be concluded that light scattering could be a plausible method for the assessment of EVs upon considering that EVs are a dynamic material with a transient identity.

Concentration of extracellular vesicles isolated from blood relative to the clinical pathological status of dogs with mast cell tumours.

Extracellular vesicles (EVs) are membrane-enclosed fragments shed from all cell types, including tumour cells. EVs contain a wide range of proteins, biolipids and genetic material derived from mother cells and therefore may be potential biomarkers for tumour diagnosis, disease progression and treatment success. We studied the effect of canine mast cell tumours (MCTs) on EV concentrations in blood isolates in association with MCT's histological grade, Ki-67 proliferative index, KIT-staining pattern and number of PLT. The average EV concentration in blood isolates from nine dogs with MCTs was considerably higher than that in blood from eight healthy dogs. But there were no statistically significant differences in EVs concentration in the population of dogs with MCT according to a different histological grade of malignancy (Patnaik, Kiupel), KIT-staining pattern and Ki-67 proliferation index. The results show that these variables statistically do not significantly predicted EV concentrations in blood isolates (P > .05), except the KIT-staining pattern I which added statistically significantly to the prediction (P < .05). The results confirmed the impact of neoplasms on the morphological changes to cell membranes, which result in greater vesiculability and higher EV concentrations.

Applicability of extracellular vesicles in clinical studies.

BACKGROUND: Extracellular vesicles (EVs) are submicron cellular fragments that mediate intercellular communication. EVs have in the last decade attracted major interest as biomarkers or platforms for biomarkers of health and disease. To better understand the reasons why despite great expectations and considerable effort, EV-based methods have not yet been introduced into clinical practice, we present a systematic analysis of published results of clinical studies. MATERIALS AND METHODS: Clinical studies on populations of body fluid samples, published from 2010 to including 2015, applying centrifugation of fluid human samples with centrifuge accelerations up to about 25 000 g and flow cytometry for detection of EVs were analysed with respect to statistical significance (p), statistical power (P), clinical significance (CS), defined as the difference between the means divided by the sum of standard deviations, and size of the populations (Nmin ), defined as the number of samples in the smaller group. RESULTS: Final analysis included 65 publications with 716 comparisons reporting 308 (43%) statistically significant differences (P < 0·05), 242 (34%) had statistical power P > 0·8 and 88 (12%) had clinical importance CS > 1·96. None of comparison with CS > 1·96 included populations in which the smaller group consisted of 50 or more samples. CONCLUSIONS: To fulfil claimed expectations for EV-based methods as promising diagnostic tools, more evidence on EV-based mechanisms of diseases should be gathered. Also, the methods of EV harvesting and assessment should be improved to yield better repeatability and thus allow clinical studies with larger number of samples.

Effect of shear stress in the flow through the sampling needle on concentration of nanovesicles isolated from blood.

During harvesting of nanovesicles (NVs) from blood, blood cells and other particles in blood are exposed to mechanical forces which may cause activation of platelets, changes of membrane properties, cell deformation and shedding of membrane fragments. We report on the effect of shear forces imposed upon blood samples during the harvesting process, on the concentration of membrane nanovesicles in isolates from blood. Mathematical models of blood flow through the needle during sampling with vacuumtubes and with free flow were constructed, starting from the Navier-Stokes formalism. Blood was modeled as a Newtonian fluid. Work of the shear stress was calculated. In experiments, nanovesicles were isolated by repeated centrifugation (up to 17,570×g) and washing, and counted by flow cytometry. It was found that the concentration of nanovesicles in the isolates positively corresponded with the work by the shear forces in the flow of the sample through the needle. We have enhanced the effect of the shear forces by shaking the samples prior to isolation with glass beads. Imaging of isolates by scanning electron microscopy revealed closed globular structures of a similar size and shape as those obtained from unshaken plasma by repetitive centrifugation and washing. Furthermore, the sizes and shapes of NVs obtained by shaking erythrocytes corresponded to those isolated from shaken platelet-rich plasma and from unshaken platelet rich plasma, and not to those induced in erythrocytes by exogenously added amphiphiles. These results are in favor of the hypothesis that a significant pool of nanovesicles in blood isolates is created during their harvesting. The identity, shape, size and composition of NVs in isolates strongly depend on the technology of their harvesting.

Effect of carbon black nanomaterial on biological membranes revealed by shape of human erythrocytes, platelets and phospholipid vesicles.

BACKGROUND: We studied the effect of carbon black (CB) agglomerated nanomaterial on biological membranes as revealed by shapes of human erythrocytes, platelets and giant phospholipid vesicles. Diluted human blood was incubated with CB nanomaterial and observed by different microscopic techniques. Giant unilamellar phospholipid vesicles (GUVs) created by electroformation were incubated with CB nanomaterial and observed by optical microscopy. Populations of erythrocytes and GUVs were analyzed: the effect of CB nanomaterial was assessed by the average number and distribution of erythrocyte shape types (discocytes, echinocytes, stomatocytes) and of vesicles in test suspensions, with respect to control suspensions. Ensembles of representative images were created and analyzed using computer aided image processing and statistical methods. In a population study, blood of 14 healthy human donors was incubated with CB nanomaterial. Blood cell parameters (concentration of different cell types, their volumes and distributions) were assessed. RESULTS: We found that CB nanomaterial formed micrometer-sized agglomerates in citrated and phosphate buffered saline, in diluted blood and in blood plasma. These agglomerates interacted with erythrocyte membranes but did not affect erythrocyte shape locally or globally. CB nanomaterial agglomerates were found to mediate attractive interaction between blood cells and to present seeds for formation of agglomerate - blood cells complexes. Distortion of disc shape of resting platelets due to incubation with CB nanomaterial was not observed. CB nanomaterial induced bursting of GUVs while the shape of the remaining vesicles was on the average more elongated than in control suspension, indicating indirect osmotic effects of CB nanomaterial. CONCLUSIONS: CB nanomaterial interacts with membranes of blood cells but does not have a direct effect on local or global membrane shape in physiological in vitro conditions. Blood cells and GUVs are convenient and ethically acceptable methods for the study of effects of various substances on biological membranes and therefrom derived effects on organisms.

Suppression of membrane vesiculation as anticoagulant and anti-metastatic mechanism. Role of stability of narrow necks.

Nanovesicles that are pinched off from biological membranes in the final stage of budding constitute a cell-cell communication system. Recent studies indicate that in vivo they are involved in blood clot formation and in cancer progression. The bud is connected to the mother membrane by a thin neck so it dwells close to the mother membrane. Using the electron microscopy we have observed in blood cells that adhesion between the membrane of the bud and of the mother cell in the vicinity of the neck took place and prevented the bud to pinch off from the mother vesicle. The same effect was observed in giant phospholipid vesicles (GPVs) due to attractive interaction between the bud and the mother vesicle mediated by the plasma protein beta-2-glycoprotein I. The stability of the neck is important for this process. By using Fourier method we analyzed thermal fluctuations of a GPV while a protrusion composed of beads connected by thin necks was spontaneously integrated into the mother GPV. Stepwise change of Fourier coefficients indicates an increased stability of necks which contributes to the retention of buds by the mother membrane and promotes anticoagulant and anti-metastatic mechanism by suppression of nanovesiculation.

A 32-month follow-up study of nanovesicle concentrations in blood from 12 patients with gastrointestinal stromal tumour treated with imatinib.

Clinical studies have indicated that the NV (nanovesicle) concentration in blood samples is a potential indicator of clinical status and can be used to follow the development of the disease. For 32 months, we monitored the effect of imatinib treatment on NV concentrations in blood samples from 12 patients with GIST (gastrointestinal stromal tumour). The NV concentration before the treatment increased with respect to control by a factor of 3.5 on average (range 2.6-9.2). The first week after initiation of the treatment, the NV concentration increased considerably, by a factor of 13 on average (range 5.9-21.2), whereas on average, after 1 month, it decreased to the level of the control and remained at that level for at least 1.5 years. Recent assessment (after 2.5 years) showed a somewhat increased NV concentration, by a factor of 2 on average (range 0.7-3.9). Low NV concentrations in blood samples during the treatment reflect a favourable effect of imatinib in these patients and no remission of the disease was hitherto observed.

Effect of engineered TiO2 and ZnO nanoparticles on erythrocytes, platelet-rich plasma and giant unilamelar phospholipid vesicles.

BACKGROUND: Massive industrial production of engineered nanoparticles poses questions about health risks to living beings. In order to understand the underlying mechanisms, we studied the effects of TiO2 and ZnO agglomerated engineered nanoparticles (EPs) on erythrocytes, platelet-rich plasma and on suspensions of giant unilamelar phospholipid vesicles. RESULTS: Washed erythrocytes, platelet-rich plasma and suspensions of giant unilamelar phospholipid vesicles were incubated with samples of EPs. These samples were observed by different microscopic techniques. We found that TiO2 and ZnO EPs adhered to the membrane of washed human and canine erythrocytes. TiO2 and ZnO EPs induced coalescence of human erythrocytes. Addition of TiO2 and ZnO EPs to platelet-rich plasma caused activation of human platelets after 24 hours and 3 hours, respectively, while in canine erythrocytes, activation of platelets due to ZnO EPs occurred already after 1 hour. To assess the effect of EPs on a representative sample of giant unilamelar phospholipid vesicles, analysis of the recorded populations was improved by applying the principles of statistical physics. TiO2 EPs did not induce any notable effect on giant unilamelar phospholipid vesicles within 50 minutes of incubation, while ZnO EPs induced a decrease in the number of giant unilamelar phospholipid vesicles that was statistically significant (p < 0,001) already after 20 minutes of incubation. CONCLUSIONS: These results indicate that TiO2 and ZnO EPs cause erythrocyte aggregation and could be potentially prothrombogenic, while ZnO could also cause membrane rupture.

Nanoparticles isolated from blood: a reflection of vesiculability of blood cells during the isolation process.

BACKGROUND: Shedding of nanoparticles from the cell membrane is a common process in all cells. These nanoparticles are present in body fluids and can be harvested by isolation. To collect circulating nanoparticles from blood, a standard procedure consisting of repeated centrifugation and washing is applied to the blood samples. Nanoparticles can also be shed from blood cells during the isolation process, so it is unclear whether nanoparticles found in the isolated material are present in blood at sampling or if are they created from the blood cells during the isolation process. We addressed this question by determination of the morphology and identity of nanoparticles harvested from blood. METHODS: The isolates were visualized by scanning electron microscopy, analyzed by flow cytometry, and nanoparticle shapes were determined theoretically. RESULTS: The average size of nanoparticles was about 300 nm, and numerous residual blood cells were found in the isolates. The shapes of nanoparticles corresponded to the theoretical shapes obtained by minimization of the membrane free energy, indicating that these nanoparticles can be identified as vesicles. The concentration and size of nanoparticles in blood isolates was sensitive to the temperature during isolation. We demonstrated that at lower temperatures, the nanoparticle concentration was higher, while the nanoparticles were on average smaller. CONCLUSION: These results indicate that a large pool of nanoparticles is produced after blood sampling. The shapes of deformed blood cells found in the isolates indicate how fragmentation of blood cells may take place. The results show that the contents of isolates reflect the properties of blood cells and their interaction with the surrounding solution (rather than representing only nanoparticles present in blood at sampling) which differ in different diseases and may therefore present a relevant clinical parameter.

Post-prandial rise of microvesicles in peripheral blood of healthy human donors.

BACKGROUND: Microvesicles isolated from body fluids are membrane - enclosed fragments of cell interior which carry information on the status of the organism. It is yet unclear how metabolism affects the number and composition of microvesicles in isolates from the peripheral blood. AIM: To study the post - prandial effect on microvesicles in isolates from the peripheral blood of 21 healthy donors, in relation to blood cholesterol and blood glucose concentrations. RESULTS: The average number of microvesicles in the isolates increased 5 hours post - prandially by 52%; the increase was statistically significant (p = 0.01) with the power P = 0.68, while the average total blood cholesterol concentration, average low density lipoprotein cholesterol concentration (LDL-C) and average high density lipoprotein cholesterol concentration (HDL-C) all remained within 2% of their fasting values. We found an 11% increase in triglycerides (p = 0.12) and a 6% decrease in blood glucose (p < 0.01, P = 0.74). The post - prandial number of microvesicles negatively correlated with the post - fasting total cholesterol concentration (r = - 0.46, p = 0.035) while the difference in the number of microvesicles in the isolates between post - prandial and post - fasting states negatively correlated with the respective difference in blood glucose concentration (r = - 0.39, p = 0.05). CONCLUSIONS: In a population of healthy human subjects the number of microvesicles in isolates from peripheral blood increased in the post - prandial state. The increase in the number of microvesicles was affected by the fasting concentration of cholesterol and correlated with the decrease in blood glucose.

Isolated microvesicles from peripheral blood and body fluids as observed by scanning electron microscope.

Microvesicles are sub-micron structures shed from the cell membrane in a final step of the budding process. After being released into the microenvironment they are free to move and carry signaling molecules to distant cells, thereby they represent a communication system within the body. Since all cells shed microvesicles, it can be expected that they will be found in different body fluids. The potential diagnostic value of microvesicles has been suggested, however, a standardized protocol for isolation has not yet been agreed upon. It is unclear what is the content of the isolates and whether the isolated microvesicles were present in vivo or-have they been created within the isolation procedure. To present evidence in this direction, in this work we focus on the visualization of the material obtained by the microvesicle isolation procedure. We present scanning electronic microscope images of microvesicles isolated from blood, ascites, pleural fluid, cerebrospinal fluid, postoperative drainage fluid and chyloid fluid acquired from human and animal patients. Vesicular structures sized from 1microm downto 50nm are present in isolates of all considered body fluids, however, the populations differ in size and shape reflecting also the composition of the corresponding sediments. Isolates of microvesicles contain numerous cells which indicates that methods of isolation and determination of the number of microvesicles in the peripheral blood are to be elaborated and improved.