Barium sulphate microparticles are taken up by three different cell types: HeLa, THP-1, and hMSC.

Cytotoxicity and cellular uptake of spherical barium sulphate microparticles (diameter 1 µm) were studied with three different cell lines, i.e. THP-1 cells (monocytes; model for a phagocytosing cell line), HeLa cells (epithelial cells; model for a non-phagocytosing cell line), and human mesenchymal stem cells (hMSCs; model for non-phagocytosing primary cells). Barium sulphate is a chemically and biologically inert solid which allows to distinguish two different processes, e.g. the particle uptake and potential adverse biological reactions. Barium sulphate microparticles were surface-coated by carboxymethylcellulose (CMC) which gave the particles a negative charge. Fluorescence was added by conjugating 6-aminofluorescein to CMC. The cytotoxicity of these microparticles was studied by the MTT test and a live/dead assay. The uptake was visualized by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). The particle uptake mechanism was quantified by flow cytometry with different endocytosis inhibitors in THP-1 and HeLa cells. The microparticles were easily taken up by all cell types, mostly by phagocytosis and micropinocytosis, within a few hours. STATEMENT OF SIGNIFICANCE: The interaction of particles and cells is of primary importance in nanomedicine, drug delivery, and nanotoxicology. It is commonly assumed that cells take up only nanoparticles unless they are able to phagocytosis. Here, we demonstrate with chemically and biologically inert microparticles of barium sulphate that even non-phagocytosing cells like HeLa and hMSCs take up microparticles to a considerable degree. This has considerable implication in biomaterials science, e.g. in case of abrasive debris and particulate degradation products from implants like endoprostheses.

Assessing discards in an illegal small-scale fishery using fisher-led reporting.

About a third of all marine fish in the world are caught in Small-Scale Fisheries (SSF). SSF are increasingly recognised as essential for food security and livelihoods for vulnerable and economically fragile communities globally. Although individual SSF vessels are usually perceived as having little impact on the ecosystem, the cumulative impact of gear type and number of vessels may be substantial. Bottom trawling is a common fishing method that can greatly influence the marine ecosystem by damaging the seafloor and generating high levels of discards. However, appropriate sampling coverage using on-board observer programmes to collect these data from SSF are rare, as they are expensive and pose logistical constraints. A mobile App was used to assess whether self-reporting by fishers could provide reliable fine-scale information on fishing effort and discards over time in an illegal shrimp trawling fishery in northern Peru. Maps depicting the spatial distribution of trawling effort and the proportion of discards from observers and fishers were compared using the Similarity in Means (SIM) Index, which ranges from 0 when spatial patterns differ completely to 1 when spatial patterns are very similar. High levels of agreement between spatio-temporal patterns of effort (SIM Index = 0.81) and discards (0.96) were found between fisher and observer maps. Moreover, far greater spatial coverage was accomplished by fishers, suggesting that self-reporting via an App represents a useful approach to collect reliable fisheries data as an initial step for effective monitoring and management of these fisheries. Supplementary Information: The online version contains supplementary material available at 10.1007/s11160-022-09708-9.

Advanced interatrial block predicts recurrence of embolic stroke of undetermined source. / El bloqueo interatrial avanzado predice recurrencia de infarto cerebral embólico de origen no determinado.

BACKGROUND: Advanced interatrial block (IAB) is an independent risk factor for ischaemic stroke. This study aimed to analyse whether advanced IAB predicts recurrence of embolic stroke of undetermined source (ESUS). METHODS: 104 patients with a confirmed diagnosis of ESUS were followed up for a median period of 15 months (interquartile range, 10-48). We recorded data on clinical variables, P-wave characteristics, and presence of IAB on the electrocardiogram. Electrocardiogram findings were interpreted by a blinded, centralised rater at (XXXX2). ESUS recurrence was the primary outcome variable. RESULTS: Median age was 47 years (range, 19-85); 50% of patients were women. IAB was detected in 36 patients (34.6%); IAB was partial in 29 cases (27.9%) and advanced in 7 (6.7%). Sixteen patients (15.4%) presented stroke recurrence; of these, 5 had partial and 4 had advanced IAB (P = .01; odds ratio [OR] = 9.44; 95% confidence interval [CI], 1.88-47.46; relative risk [RR] = 4.62; 95% CI, 2.01-10.61). Median P-wave duration was longer in patients with stroke recurrence (P = .009). The multivariate logistic regression analysis identified the following independent risk factors for stroke recurrence: advanced IAB (P < .001; OR = 10.86; 95% CI, 3.07-38.46), male sex (P = .028; OR = 4.6; 95% CI, 1.18-17.96), and age older than 50 years (P = .039; OR = 3.84; 95% CI, 1.06-13.88). In the Cox proportional hazards model, the risk variables identified were age older than 50 years (P = .002; hazard ratio, 7.04; 95% CI, 2.06-23.8) and P-wave duration (per ms) (P = .007; hazard ratio, 1.02; 95% CI, 1.01-1.04). CONCLUSIONS: Advanced IAB and age older than 50 years predict ESUS recurrence.

Advanced interatrial block predicts recurrence of embolic stroke of undetermined source.

BACKGROUND: Advanced interatrial block (IAB) is an independent risk factor for ischaemic stroke. This study aimed to analyse whether advanced IAB predicts recurrence of embolic stroke of undetermined source (ESUS). METHODS: 104 patients with a confirmed diagnosis of ESUS were followed up for a median period of 15 months (interquartile range, 10-48). We recorded data on clinical variables, P-wave characteristics, and presence of IAB on the electrocardiogram (ECG). ECG findings were interpreted by a blinded, centralised rater at (XXXX2). ESUS recurrence was the primary outcome variable. RESULTS: Median age was 47 years (range, 19-85); 50% of patients were women. IAB was detected in 36 patients (34.6%); IAB was partial in 29 cases (27.9%) and advanced in 7 (6.7%). Sixteen patients (15.4%) presented stroke recurrence; of these, 5 had partial and 4 had advanced IAB (P = .01; odds ratio [OR] = 9.44; 95% confidence interval [CI], 1.88-47.46; relative risk [RR] = 4.62; 95% CI, 2.01-10.61). Median P-wave duration was longer in patients with stroke recurrence (P = .009). The multivariate logistic regression analysis identified the following independent risk factors for stroke recurrence: advanced IAB (P < .001; OR = 10.86; 95% CI, 3.07-38.46), male sex (P = .028; OR = 4.6; 95% CI, 1.18-17.96), and age older than 50 years (P = .039; OR = 3.84; 95% CI, 1.06-13.88). In the Cox proportional hazards model, the risk variables identified were age older than 50 years (P = .002; hazard ratio, 7.04; 95% CI, 2.06-23.8) and P-wave duration (per ms) (P = .007; hazard ratio, 1.02; 95% CI, 1.01-1.04). CONCLUSIONS: Advanced IAB and age older than 50 years predict ESUS recurrence.

Cell-biological effects of zinc oxide spheres and rods from the nano- to the microscale at sub-toxic levels.

Zinc oxide particles were synthesized in various sizes and shapes, i.e., spheres of 40-nm, 200-nm, and 500-nm diameter and rods of 40∙100 nm2 and 100∙400 nm2 (all PVP-stabilized and well dispersed in water and cell culture medium). Crystallographically, the particles consisted of the hexagonal wurtzite phase with a primary crystallite size of 20 to 100 nm. The particles showed a slow dissolution in water and cell culture medium (both neutral; about 10% after 5 days) but dissolved within about 1 h in two different simulated lysosomal media (pH 4.5 to 4.8). Cells relevant for respiratory exposure (NR8383 rat alveolar macrophages) were exposed to these particles in vitro. Viability, apoptosis, and cell activation (generation of reactive oxygen species, ROS, release of cytokines) were investigated in an in vitro lung cell model with respect to the migration of inflammatory cells. All particle types were rapidly taken up by the cells, leading to an increased intracellular zinc ion concentration. The nanoparticles were more cytotoxic than the microparticles and comparable with dissolved zinc acetate. All particles induced cell apoptosis, unlike dissolved zinc acetate, indicating a particle-related mechanism. Microparticles induced a stronger formation of reactive oxygen species than smaller particles probably due to higher sedimentation (cell-to-particle contact) of microparticles in contrast to nanoparticles. The effect of particle types on the cytokine release was weak and mainly resulted in a decrease as shown by a protein microarray. In the particle-induced cell migration assay (PICMA), all particles had a lower effect than dissolved zinc acetate. In conclusion, the biological effects of zinc oxide particles in the sub-toxic range are caused by zinc ions after intracellular dissolution, by cell-to-particle contacts, and by the uptake of zinc oxide particles into cells. Graphical headlights • The cytotoxicity of zinc oxide particles is mainly due to the intracellular release of zinc ions. • The size and shape of zinc oxide micro- and nanoparticles has only small effects on lung cells in the sub-toxic range. • Zinc oxide particles are rapidly taken up by cells, regardless of their size and shape. • Zinc oxide particles rapidly dissolve after cellular uptake in endolysosomes.

Piezoelectric 3-D Fibrous Poly(3-hydroxybutyrate)-Based Scaffolds Ultrasound-Mineralized with Calcium Carbonate for Bone Tissue Engineering: Inorganic Phase Formation, Osteoblast Cell Adhesion, and Proliferation.

Elaboration of novel biocomposites providing simultaneously both biodegradability and stimulated bone tissue repair is essential for regenerative medicine. In particular, piezoelectric biocomposites are attractive because of a possibility to electrically stimulate cell response. In the present study, novel CaCO3-mineralized piezoelectric biodegradable scaffolds based on two polymers, poly[( R)3-hydroxybutyrate] (PHB) and poly[3-hydroxybutyrate- co-3-hydroxyvalerate] (PHBV), are presented. Mineralization of the scaffold surface is carried out by the in situ synthesis of CaCO3 in the vaterite and calcite polymorphs using ultrasound (U/S). Comparative characterization of PHB and PHBV scaffolds demonstrated an impact of the porosity and surface charge on the mineralization in a dynamic mechanical system, as no essential distinction was observed in wettability, structure, and surface chemical compositions. A significantly higher (4.3 times) piezoelectric charge and a higher porosity (∼15%) lead to a more homogenous CaCO3 growth in 3-D fibrous structures and result in a two times higher relative mass increase for PHB scaffolds compared to that for PHBV. This also increases the local ion concentration incurred upon mineralization under U/S-generated dynamic mechanical conditions. The modification of the wettability for PHB and PHBV scaffolds from hydrophobic (nonmineralized fibers) to superhydrophilic (mineralized fibers) led to a pronounced apatite-forming behavior of scaffolds in a simulated body fluid. In turn, this results in the formation of a dense monolayer of well-distributed and proliferated osteoblast cells along the fibers. CaCO3-mineralized PHBV surfaces had a higher osteoblast cell adhesion and proliferation assigned to a higher amount of CaCO3 on the surface compared to that on PHB scaffolds, as incurred from micro-computed tomography (µCT). Importantly, a cell viability study confirmed biocompatibility of all the scaffolds. Thus, hybrid biocomposites based on the piezoelectric PHB polymers represent an effective scaffold platform functionalized by an inorganic phase and stimulating the growth of the bone tissue.

Biomimetic fabrication of mineralized composite films of nanosilver loaded native fibrillar collagen and chitosan.

Silver nanoparticles loaded fibrillar collagen-chitosan matrix (CC) was prepared by biomimetic approach by blending silver nanoparticles (tAgNPs), collagen fibril and chitosan hydrogel followed by cross-linking and biomineralization. Electron micrograph showed that the surface of the composites exhibited native fibrillar morphology of collagen and their cross-section revealed layer-like arrangement of native fibrillar collagen. The mineralized composites exhibited surface mineralization of calcium phosphates incorporated with magnesium. FT-IR ATR analysis revealed the uniform blending of collagen and chitosan without any chemical interaction between them. XRD analysis showed incorporation of silver nanoparticles and lamellar structure of collagen and chitosan. The mechanical property of the dry composite film showed increase in tensile strength with the addition of chitosan and raised to 4.6 fold in M-CC4 composite. The incorporation of chitosan in M-CC3 led to 2.2 fold increase in mineralization as confirmed by the TGA analysis. Contact angle analysis revealed the hydrophilic nature of the composite. Hemolysis analysis of the composites verified the hemocompatible nature of composites with hemolysis < 5%. MTT assay for the composites was carried by seeding MG-63 cells and indicated cell viability > 80%. Antibacterial activity analysis showed the percent growth inhibition of about 27% and 37% for S. aureus and E. coli respectively. The prepared composite would possess silver nanoparticles loaded collagen fibril in the native state and the formed biomineral will be similar to the bone mineral. Hence the fabricated composite -could be used as a biomaterial for bone tissue engineering applications.

X-ray powder diffraction to analyse bimetallic core-shell nanoparticles (gold and palladium; 7-8 nm).

A comparative X-ray powder diffraction study on poly(N-vinyl pyrrolidone) (PVP)-stabilized palladium and gold nanoparticles and bimetallic Pd-Au nanoparticles (both types of core-shell nanostructures) was performed. The average diameter of Au and Pd nanoparticles was 5 to 6 nm. The two types of core-shell particles had a core diameter of 5 to 6 nm and an overall diameter of 7 to 8 nm, i.e. a shell thickness of 1 to 2 nm. X-ray powder diffraction on a laboratory instrument was able to distinguish between a physical mixture of gold and palladium nanoparticles and bimetallic core-shell nanoparticles. It was also possible to separate the core from the shell in both kinds of bimetallic core-shell nanoparticles due to the different domain size and because it was known which metal was in the core and which was in the shell. The spherical particles were synthesized by reduction with glucose in aqueous media. After purification by multiple centrifugation steps, the particles were characterized with respect to their structural, colloid-chemical, and spectroscopic properties, i.e. particle size, morphology, and internal elemental distribution. Dynamic light scattering (DLS), differential centrifugal sedimentation (DCS), atomic absorption spectroscopy (AAS), ultraviolet-visible spectroscopy (UV-vis), high-angle annular dark field imaging (HAADF), and energy-dispersed X-ray spectroscopy (EDX) were applied for particle characterization.

A systematic electron microscopic study on the uptake of barium sulphate nano-, submicro-, microparticles by bone marrow-derived phagocytosing cells.

Nanoparticles can act as transporters for synthetic molecules and biomolecules into cells, also in immunology. Antigen-presenting cells like dendritic cells are important targets for immunotherapy in nanomedicine. Therefore, we have used primary murine bone marrow-derived phagocytosing cells (bmPCs), i.e. dendritic cells and macrophages, to study their interaction with spherical barium sulphate particles of different size (40 nm, 420 nm, and 1 µm) and to follow their uptake pathway. Barium sulphate is chemically and biologically inert (no dissolution, no catalytic effects), i.e. we can separate the particle uptake effect from potential biological reactions. The colloidal stabilization of the nanoparticles was achieved by a layer of carboxymethylcellulose (CMC) which is biologically inert and gives the particles a negative zeta potential (i.e. charge). The particles were made fluorescent by conjugating 6-aminofluoresceine to CMC. Their uptake was visualized by flow cytometry, confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and correlative light and electron microscopy (CLEM). Barium sulphate particles of all sizes were readily taken up by dendritic cells and even more by macrophages, with the uptake increasing with time and particle concentration. They were mainly localized inside phagosomes, heterophagosomes, and in the case of nanoparticles also in the nearby cytosol. No particles were found in the nucleus. In nanomedicine, inorganic nanoparticles from the nanometer to the micrometer size are therefore well suited as transporters of biomolecules, including antigens, into dendritic cells and macrophages. The presented model system may also serve to describe the aseptic loosening of endoprostheses caused by abrasive wear of inert particles and the subsequent cell reaction, a question which relates to the field of nanotoxicology. STATEMENT OF SIGNIFICANCE: The interaction of particles and cells is at the heart of nanomedicine and nanotoxicology, including abrasive wear from endoprostheses. It also comprises the immunological reaction to different kinds of nanomaterials, triggered by an immune response, e.g. by antigen-presenting cells. However, it is often difficult to separate the particle effect from a chemical or biochemical reaction to particles or their cargo. We show how chemically inert barium sulphate particles with three different sizes (nano, sub-micro, and micro) interact with relevant immune cells (primary dendritic cells and macrophages). Particles of all three sizes are readily taken up into both cell types by phagocytosis, but the uptake by macrophages is significantly more prominent than that by dendritic cells. The cells take up particles until they are virtually stuffed, but without direct adverse effect. The uptake increases with time and particle concentration. Thus, we have an ideal model system to follow particles into and inside cells without the side effect of a chemical particle effect, e.g. by degradation or ion release.

Kinetics of chemotaxis, cytokine, and chemokine release of NR8383 macrophages after exposure to inflammatory and inert granular insoluble particles.

Accumulation of macrophages and neutrophil granulocytes in the lung are key events in the inflammatory response to inhaled particles. The present study aims at the time course of chemotaxis in vitro in response to the challenge of various biopersistent particles and its functional relation to the transcription of inflammatory mediators. NR8383 rat alveolar macrophages were challenged with particles of coarse quartz, barium sulfate, and nanosized silica for one, four, and 16h and with coarse and nanosized titanium dioxide particles (rutile and anatase) for 16h only. The cell supernatants were used to investigate the chemotaxis of unexposed NR8383 macrophages. The transcription of inflammatory mediators in cells exposed to quartz, silica, and barium sulfate was analyzed by quantitative real-time PCR. Challenge with quartz, silica, and rutile particles induced significant chemotaxis of unexposed NR8383 macrophages. Chemotaxis caused by quartz and silica was accompanied by an elevated transcription of CCL3, CCL4, CXCL1, CXCL3, and TNFα. Quartz exposure showed an earlier onset of both effects compared to the nanosized silica. The strength of this response roughly paralleled the cytotoxic effects. Barium sulfate and anatase did not induce chemotaxis and barium sulfate as well caused no elevated transcription. In conclusion, NR8383 macrophages respond to the challenge with inflammatory particles with the release of chemotactic compounds that act on unexposed macrophages. The kinetics of the response differs between the various particles.

EXPERIMENTAL-MORPHOLOGICAL SUBSTANTIATION OF EXPEDIENCY TO USE THE SKIN GLUE "DERMABOND" FOR POSTOPERATIVE WOUND CLOSURE.

We aimed to investigate the morphological features of healing of postoperative wounds in the early stages of reparative process in the experiment, depending on the used type of the wound closure. It is proved that the nature and type of the scar depends on the processes that occur in the wound at the early postoperative stage, which in turn greatly affects the form of suture material used. The experiment included 20 male rats, weighing 180-200 g. All rats were anesthetized by a single intraperitoneal injection of sodium thiopental. After the shaving operative field, 2 cm full-thickness incision wound was made on the anterior surface of the abdomen in the longitudinal direction. As suture material for wound closure in the 1st experimental group (10 rats) we used surgical filament "Polyamide 4-0¼. In the 2nd experimental group (10 rats) wounds were closured by using skin glue "Dermabond". According from our experiment, the usage of skin glue creates better conditions for wound healing. Thus, to achieve a more aesthetic scar, we recommend applying skin glue instead of using nodal joints.

Nanostructure of wet-chemically prepared, polymer-stabilized silver-gold nanoalloys (6 nm) over the entire composition range.

Bimetallic silver-gold nanoparticles were prepared by co-reduction using citrate and tannic acid in aqueous solution and colloidally stabilized with poly(N-vinylpyrrolidone) (PVP). The full composition range of silver : gold from 0 : 100 to 100 : 0 (n : n) was prepared with steps of 10 mol%. The nanoparticles were spherical, monodispersed, and had a diameter of ∼6 nm, except for Ag : Au 90 : 10 nanoparticles and pure Ag nanoparticles which were slightly larger. The size of the nanoalloys was determined by differential centrifugal sedimentation (DCS) and transmission electron microscopy (TEM). By means of X-ray powder diffraction (XRD) together with Rietveld refinement, precise lattice parameters, crystallite size and microstrain were determined. Scanning transmission electron microscopy (STEM) combined with energy-dispersive X-ray spectroscopy (EDX) and electron energy loss spectroscopy (EELS) showed that the particles consisted of a gold-rich core and a silver-rich shell. XRD and DCS indicated that the nanoparticles were not twinned, except for pure Ag and Ag : Au 90 : 10, although different domains were visible in the TEM. A remarkable negative deviation from Vegard's linear rule of alloy mixtures was observed (isotropic contraction of the cubic unit cell with a minimum at a 50 : 50 composition). This effect was also found for Ag:Au bulk alloys, but it was much more pronounced for the nanoalloys. Notably, it was much less pronounced for pure silver and gold nanoparticles. The microstrain was increased along with the contraction of the unit cell with a broad maximum at a 50 : 50 composition. The synthesis is based on aqueous solvents and can be easily scaled up to a yield of several mg of a well dispersed nanoalloy with application potential due to its tuneable antibacterial action (silver) and its optical properties for bioimaging.

Hospital arrival time and functional outcome after acute ischaemic stroke: results from the PREMIER study.

INTRODUCTION: Information regarding hospital arrival times after acute ischaemic stroke (AIS) has mainly been gathered from countries with specialised stroke units. Little data from emerging nations is available. We aim to identify factors associated with achieving hospital arrival times of less than 1, 3, and 6 hours, and analyse how arrival times are related to functional outcomes after AIS. METHODS: We analysed data from patients with AIS included in the PREMIER study (Primer Registro Mexicano de Isquemia Cerebral) which defined time from symptom onset to hospital arrival. The functional prognosis at 30 days and at 3, 6, and 12 months was evaluated using the modified Rankin Scale. RESULTS: Among 1096 patients with AIS, 61 (6%) arrived in <1 hour, 250 (23%) in <3 hours, and 464 (42%) in <6 hours. The factors associated with very early (<1 hour) arrival were family history of ischemic heart disease and personal history of migraines; in <3 hours: age 40-69 years, family history of hypertension, personal history of dyslipidaemia and ischaemic heart disease, and care in a private hospital; in <6 hours: migraine, previous stroke, ischaemic heart disease, care in a private hospital, and family history of hypertension. Delayed hospital arrival was associated with lacunar stroke and alcoholism. Only 2.4% of patients underwent thrombolysis. Regardless of whether or not thrombolysis was performed, arrival time in <3 hours was associated with lower mortality at 3 and 6 months, and with fewer in-hospital complications. CONCLUSIONS: A high percentage of patients had short hospital arrival times; however, less than 3% underwent thrombolysis. Although many factors were associated with early hospital arrival, it is a priority to identify in-hospital barriers to performing thrombolysis.

The dissolution and biological effects of silver nanoparticles in biological media.

Silver ions and silver nanoparticles have a well-known biological effect that typically occurs in biological or environmental media of complex composition. Silver nanoparticles release silver ions if oxidizing species like molecular oxygen or hydrogen peroxide are present. The presence of glucose as a model for reducing sugars has only a small effect on the dissolution rate. In the presence of chloride ions, precipitation of silver chloride nanoparticles occurs. At physiological salt concentrations, no precipitation of silver phosphate occurs as the precipitation of silver chloride always occurs first. If the surface of a silver nanoparticle is passivated by cysteine, the dissolution is quantitatively inhibited. Upon immersion of silver nanoparticles in pure water for 8 months, leading to about 50% dissolution, no change in the surface was observed by transmission electron microscopy. A model for the dissolution was derived from immersion and dissolution experiments in different media and from high-resolution transmission electron microscopy. A literature survey on the available data on the dissolution of silver nanoparticles showed that only qualitative trends can be identified as the nature of the nanoparticles and of the immersion medium are practically never comparable. The dissolution effects were confirmed by cell culture experiments (human mesenchymal stem cells and neutrophil granulocytes) where silver nanoparticles that were stored under argon had a clearly lower cytotoxicity than those stored under air. They also led to a less formation of reactive oxygen species (ROS). This underscores that silver ions are the toxic species.

Study of structure of calcium phosphate materials by means of electron spin resonance.

Structure of γ-irradiated calcium phosphates synthesized in different conditions was studied by means of ESR. It has been shown that amorphous and nanocrystalline calcium phosphates have qualitatively different ESR spectra. The new paramagnetic center in hydroxyapatite (HA) has been found. It consists of four protons of water molecules located in the parallelogram vertexes and the free electron formed in HA by γ-irradiation. This center is thermally stable up to 300 °C, when the water molecules release from the HA crystal lattice.