Μm Beads Research Articles

Analysis of heat generation during the production of drug nanosuspensions in a wet stirred media mill

Although heat is generated during the wet stirred media milling of drug suspensions, leading to notable temperature rise, a comprehensive analysis of heat generation does not exist. Hence, we investigated the impact of stirrer speed, bead loading, and bead size at three levels on the evolution of suspension temperature at the mill outlet during the milling of fenofibrate. The particle sizes and viscosities of the milled suspensions and power were measured. Our results suggest that stirrer speed had the most significant impact on the temperature increase, followed by bead loading and bead size. Both the time when the temperature reached 22 °C and the temperature at 5 min of milling were strongly correlated with the power. Assessing the impacts of the process parameters on the temperature rise, cycle time, power, and median particle size holistically, an optimal milling process was identified: 3000 rpm with 50% loading of 200 or 400 µm beads. A power number correlation was established to calculate power at any milling condition which determines the heat generation rate. Overall, this study indicated the importance of developing a good understanding of heat generation during nanomilling for development of a robust milling process especially for thermally labile drugs.

Environmentally relevant concentrations of microplastics modulated the immune response and swimming activity, and impaired the development of marine medaka Oryzias melastigma larvae

Microplastics (MPs), due to their impacts on the ecosystem and their integration into the food web either through trophic transfer or ingestion directly from the ambient environment, are an emerging class of environmental contaminants posing a great threat to marine organisms. Most reports on the toxic effects of MPs exclusively focus on bioaccumulation, oxidative stress, pathological damage, and metabolic disturbance in fish. However, the collected information on fish immunity in response to MPs is poorly defined. In particular, little is known regarding mucosal immunity and the role of mucins. In this study, marine medaka (Oryzias melastigma) larvae were exposed to 6.0 µm beads of polystyrene microplastics (PS-MPs) at three environmentally relevant concentrations (102 particles/L, 104 particles/L, and 106 particles/L) for 14 days. The experiment was carried out to explore the developmental and behavioural indices, the transcriptional profiles of mucins, pro-inflammatory, immune, metabolism and antioxidant responses related genes, as well as the accumulation of PS-MPs in larvae. The results revealed that PS-MPs were observed in the gastrointestinal tract, with a concentration- and exposure time-dependent manner. No significant difference in the larval mortality was found between the treatment groups and the control, whereas the body length of larvae demonstrated a significant reduction at 106 particles/L on 14 days post-hatching. The swimming behaviour of the larvae became hyperactive under exposure to 104 and 106 particles/L PS-MPs. In addition, PS-MP exposure significantly up-regulated the mucin gene transcriptional levels of muc7-like and muc13-like, however down-regulated the mucin gene expression levels of heg1, muc2, muc5AC-like and muc13. The immune- and inflammation and metabolism-relevant genes (jak, stat-3, il-6, il-1β, tnf-а, ccl-11, nf-κb, and sod) were significantly induced by PS-MPs at 104 and 106 particles/L compared to the control. Taken together, this study suggests that PS-MPs induced inflammation response and might obstruct the immune functions and retarded the growth of the marine medaka larvae even at environmentally relevant concentrations.

Study on the expression of Nk-lysin from Nile tilapia (Oreochromis niloticus) in Pichia pastoris and its biological function

Nk-lysin, the compound of the natural killer cells (NK) and cytotoxic T lymphocytes (CTL), is an effective antimicrobial peptide that not only presents pathogen-killing activity but also plays an important role in immunomodulatory effect. To obtain large quantity of Nk-lysin from Nile tilapia (Oreochromis niloticus) for biological assays, a 366 bp cDNA sequence encoding the mature peptide of Nk-lysin was cloned into a secretion plasmid of pPIC9K, and a large-scale of the recombinant Nk-lysin was effectively produced via Pichia pastoris expression system. Antibacterial assay shown that the recombinant Nk-lysin obviously inhibited the growth of several pathogenic bacteria, including Vibrio cholerae (MIC = 31.25 μg/mL), Pseudomonas aeruginosa (MIC = 31.25 μg/mL), and Aeromonas hydrophila (MIC = 7.81 μg/mL) which is the main pathogen of aquatic fish, including Nile tilapia. Further studies revealed that recombinant Nk-lysin significantly enhanced the phagocytic activity of monocytes/macrophages (MO/MΦ) on microspheres (0.5 and 1.0 μm beads) and bioparticles (Streptococcus agalactiae and Aeromonas hydrophila), and reactive oxygen species (ROS), these activities might be mediated by the P2X7 receptor (P2X7R). Moreover, the increased expressions of pro-inflammatory factor in il-12 and ifn-γ genes and the decreased expressions of anti-inflammatory factor in il-10 and tgf-β genes were observed in MO/MΦ after recombinant Nk-lysin treatment significantly. Meanwhile, the expression of tlr9, myd88, jak1, and nf-κb genes were up-regulated significantly in Nk-lysin-treated MO/MΦ. Taken together, these results indicate that recombinant Nk-lysin of Nile tilapia processes the capacities on antibacterial and immunomodulation, and might be a promising antibiotic substitute. Furthermore, applying P. pastoris expression system to product bioactive Nk-lysin of Nile tilapia could be sufficient to meet the requirements for both research and aquacultural application.

Assessment of acute toxicity and developmental transformation impacts of polyethylene microbead exposure on larval daggerblade grass shrimp (Palaemon pugio)

Due to the ubiquity of microplastic contamination in coastal waters, there is potential for adverse impacts to organism development. One organism of interest is the daggerblade grass shrimp, Palaemon pugio, an ecologically important species in estuaries along the east coast of North America. We exposed larval grass shrimp to virgin polyethylene microbeads (35 and 58 µm) at a high (0.375 and 1.95 mg/L), medium (0.0375 and 0.195 mg/L), and a low concentration (0.00375 and 0.0195 mg/L), respectively for 23 days to assess mortality, transformation time from larval to juvenile stage, and weight. Average percent mortality was 3.7 to 4.8 times higher in the experimental treatments compared to controls. The greatest proportion of mortality was observed in the first 11 days. Median time for transformation ranged from 20.2 to 20.8 days. Shrimp exposed to the 35 µm beads in the high treatment (20.2 days) transformed significantly faster than the control shrimp (20.8 days). Although development was not delayed and size of the shrimp did not differ, the acute toxicity of microplastics on grass shrimp is a concern due to their role in energy cycling within tidal-creeks. These findings suggest potential population and community level effects following microplastic exposure.

Active holographic tweezers based on spiraling interdigitated transducers

Recently, our team demonstrated the selective manipulations of microparticles [Baudoin et al., Sci. Adv. 5, eaav1967 (2019)] and cells [Baudoin et al., Nat. Commun. 11, 4244 (2020)] in a standard microscopy environment with active holographic tweezers based on spiraling interdigitated transducers. In this talk, we will present (i) the ultra-high frequency (250 MHz) selective manipulation of 4 μm beads with nanoNewton forces and (ii) first results on the the 3D manipulation of particles and in particular their axial displacement without any motion of the transducer, by tuning the driving frequency [Gong et al., Phys. Rev. Appl. 16, 024034 (2021)]. We will also introduce a new mixed finite element/angular spectrum code, which enables to understand the underlying physic and opens perspectives to improve the tweezers design.

Reduced-Pressure Process for Fabricating Tea Tree Oil-Polyvinylpyrrolidone Electrospun Fibers.

Electrospun fibers containing tea tree oil (TTO) can be explored for practical applications due to the antimicrobial and anti-inflammatory activities of TTO. Considering that there are potentially toxic components in TTO, it is necessary to eliminate or reduce its content in the preparation process of TTO-doped electrospun fibers. In this work, electrospun TTO-PVP (polyvinylpyrrolidone) fibers containing an 18.18 wt.% decreased content of 1,8-Cineole were successfully fabricated by intense evaporation of a self-made reduced-pressure electrospinning (RP-ES) setup (as low as 94.4 kPa). In addition, such intense evaporation led to a morphology change, where a typical average fiber diameter increased from 0.831 to 1.148 μm, fewer and smaller beads in fibers, along with a rougher and grooves fiber surface. These morphology changes allowed Terpinen-4-ol to remain in the fiber for a more extended period. In addition, RP-ES proved the possibility for intense evaporation and continuous vapor removal by continuously environmental vacuum pumping of electrospinning.

β-glucan mitigation on toxicological effects in monocytes/macrophages of Nile tilapia (Oreochromis niloticus) following copper exposure

The protective effect of β-glucan against toxicological effects caused by copper oxide nanoparticles (Cu NPs) and copper ions (Cu ions) were studied in monocytes/macrophages (MO/MФ) of Nile tilapia (Oreochromis niloticus). Our results demonstrated that CuO NPs and Cu ions exposure aroused strong oxidative lesion in MO/MФ by detection of cellular reactive oxygen species (ROS) and reduced glutathione (GSH), as well as identification of several antioxidant-related cytokines. Meanwhile, the serious pro-inflammatory responses were accompanied during the processes of oxidative lesion by TNFα, IL-1β, and IL-6 genes validation. Copper induced MO/MФ underwent apoptosis through mitochondrial signaling pathway by mitochondrial membrane potential (ΔΨm) detection and Bax, Bcl-2, Cyt-c, Apaf-1, Caspase 9, Caspase 3 genes validation. Furthermore, the phagocytic abilities were inhibition in MO/MФ by evaluation of microspheres (0.5 and 1.0 μm beads) and bioparticles (S. agalactiae and A. hydrophila) uptake and LPS-induced NO production. However, β-glucan might participate in immunomodulation through C-type lectin receptor (CLR) and complement receptor 3 (CR3) to suppress pro-inflammatory responses, thereby revered all the copper induced aforementioned adverse effects in MO/MΦ. Taken together, our results provide insights on the mechanisms through β-glucan administration to mitigate toxicological effects of CuO NPs and Cu ions exposure to the MO/MΦ, which will benefit aspects related to fish farming and aquaculture production.

Polystyrene microplastics induce gut microbiome and metabolome changes in Javanese medaka fish (Oryzias javanicus Bleeker, 1854)

Microplastics (MPs) have become emerging pollutants of public health concern, due to their impact on aqua-terrestrial ecosystems and integration into the food web, with evidence of human exposure and unrevealed health implications. There is a paucity of information regarding the effects of MPs exposure on the gut system using metagenomic and metabolomic approaches. In this study, Javanese medaka fish was exposed to 5 µm beads of polystyrene microplastics (PS-MPs) suspensions, at concentrations of 100 μg/L (MP-LOW), 500 μg/L (MP-MED), and 1000 μg/L (MP-HIGH), for a duration of 21 days, and evaluated for gut microbiome and metabolome responses. The results revealed a significant reduction (p < 0.05) in richness and diversity of the gut microbiome in the MP-HIGH group, and identification of 7 bacterial genera as differential features by the Linear discriminant analysis Effect Size (LEfSe). The gut metabolic profile revealed upregulation of 9 metabolites related to energy metabolism, via tricarboxylic acid cycle (TCA), creatine pathway, and urea cycle, as determined by the pathway analysis. Furthermore, positive correlation was found between the genus Aeromonas and glucose, lactate, and creatine metabolites. The study revealed that PS-MPs exposure resulted in altered bacterial microbiome and metabolic disorder related to energy metabolism. It further provided additional data on gut bacterial genera and metabolites associated with MPs toxicity in aquatic organism, which will inevitably enable its future health risks assessment in animals and possibly humans.

Controlling Droplet Marangoni Flows to Improve Microscopy-Based TB Diagnosis.

In developing countries, the most common diagnostic method for tuberculosis (TB) is microscopic examination sputum smears. Current assessment requires time-intensive inspection across the microscope slide area, and this contributes to its poor diagnostic sensitivity of ≈50%. Spatially concentrating TB bacteria in a smaller area is one potential approach to improve visual detection and potentially increase sensitivity. We hypothesized that a combination of magnetic concentration and induced droplet Marangoni flow would spatially concentrate Mycobacterium tuberculosis on the slide surface by preferential deposition of beads and TB–bead complexes in the center of an evaporating droplet. To this end, slide substrate and droplet solvent thermal conductivities and solvent surface tension, variables known to impact microfluidic flow patterns in evaporating droplets, were varied to select the most appropriate slide surface coating. Optimization in a model system used goniometry, optical coherence tomography, and microscope images of the final deposition pattern to observe the droplet flows and maximize central deposition of 1 μm fluorescent polystyrene particles and 200 nm nanoparticles (NPs) in 2 μL droplets. Rain-X® polysiloxane glass coating was identified as the best substrate material, with a PBS-Tween droplet solvent. The use of smaller, 200 nm magnetic NPs instead of larger 1 μm beads allowed for bright field imaging of bacteria. Using these optimized components, we compared standard smear methods to the Marangoni-based spatial concentration system, which was paired with magnetic enrichment using iron oxide NPs, isolating M. bovis BCG (BCG) from samples containing 0 and 103 to 106 bacilli/mL. Compared to standard smear preparation, paired analysis demonstrated a combined volumetric and spatial sample enrichment of 100-fold. With further refinement, this magnetic/Marangoni flow concentration approach is expected to improve whole-pathogen microscopy-based diagnosis of TB and other infectious diseases.

Granular bed filtration of a liquid aerosol

Liquid aerosols are found in many industrial sectors, particularly in the mechanical industry (oil mists). Granular bed (GB) filtration could be an interesting means for the removal of these liquid aerosols, due to their high storage capacity, robustness and low maintenance requirements. The aim of the study was to establish the filtration performance (filtration efficiency, pressure drop) of a fixed granular bed during filtration and its clogging by a liquid aerosol. Three grades of sand, obtained by sieving 100–200, 200–355 and 355–500 μm and glass beads of 140 μm diameter were used as the granular medium. The evolution of filtration efficiency and pressure drop of the GB was monitored during clogging with a DEHS aerosol of MMAD = 0.7 μm. Our measurements show that the filtration performance of granular beds during clogging was very similar to that observed for fibrous media. The fraction of the GB pore volume occupied by liquid when the drainage plateau is reached was between 28 and 33%. The GB reaches a drainage stage whose pressure drop can be correctly predicted using a model described for fibrous media.

Effect of bead size and doxorubicin loading on tumor cellular injury after transarterial embolization and chemoembolization in a rat model of hepatocellular carcinoma

Embolic agents used in transarterial embolization for intermediate stage hepatocellular carcinoma reduce blood flow into tumors and can deliver anticancer drugs. Tumor blood supply can be interrupted using doxorubicin-eluting beads (DEB-TACE) or non-loaded beads (TAE) of different calibers. In this preclinical study, we characterized the extent of remaining stressed tumor cells after treatment, hypoxia within the surviving tumor regions, and inflammatory immune cell infiltrates after embolization with 40-60 or 70-150 μm with non-loaded or doxorubicin-loaded beads at 3 and 7 days after treatment. TAE-treated tumors had more stressed and surviving tumor cells after 3 days, irrespective of bead size, compared with DEB-TACE-treated tumors. Hypoxic stress of residual cells increased after treatment with 70-150 μm beads without or with doxorubicin. Treatment with DEB-TACE of 70-150 μm resulted in increased inflammation and proliferation in the adjacent parenchyma. Inflammatory cell infiltrates were reduced at the periphery of tumors treated with 40-60 μm DEB-TACE.

Design and Evaluation of a Millifluidic Insulator-Based Dielectrophoresis (DEP) Retention Device to Separate Bacteria from Tap Water

Water is a basic necessity critical to the survival of all living beings. However, many people around the world do not have consistent access to uncontaminated drinking water. Traditional water treatment methods, such as filtration and disinfection, require physical or chemical disinfectants and are prone to fouling. Dielectrophoresis (DEP) enables a system whereby polarized bioparticles exhibit lateral movement under the influence of applied, non-uniform electric fields. A single-stage, continuous flow, millimeter-sized DEP device was designed and fabricated to remove Escherichia coli K12 from contaminated tap water. Glass beads were used to alter the electric field distribution and create zones of high electric field to trap bacterial cells. The effect of varied voltages, flow rates and bead sizes on the removal efficiency was studied. The highest removal efficiency of E. coli K12 was 99.9%, with the device set at 60 V, a flow rate of 1.0 mL/min and a 200 µm bead size. Higher applied voltages, slower flow rates, and smaller bead sizes led to an increased reduction in bacteria. An optimized macro-scale system—with multiple stages of DEP—could be suitable for commercial use and would be an effective method of removing pathogens from polluted tap water.

Nanonewton scale adhesion force measurements on biotinylated microbeads with a robotic micropipette

Binding force between biomolecules has a crucial role in most biological processes. Receptor-ligand interactions transmit physical forces and signals simultaneously. Previously, we employed a robotic micropipette both in live cell and microbead adhesion studies to explore the adhesion force of biomolecules such as cell surface receptors including specific integrins on immune cells. Here we apply standard computational fluid dynamics simulations to reveal the detailed physical background of the flow generated by the micropipette when probing microbead adhesion on functionalized surfaces. Measuring the aspiration pressure needed to pick up the biotinylated 10 μm beads on avidin coated surfaces and converting it to a hydrodynamic lifting force on the basis of simulations, we found an unbinding force of 12 ± 2 nN, when targeting the beads manually; robotic targeting resulted in 9 ± 4 nN (mean ± SD). We measured and simulated the effect of the targeting offset, when the microbead was out of the axis (off-axis)of the micropipette. According to the simulations, the higher offset resulted in a higher lifting force acting on the bead. Considering this effect, we could readily correct the impact of the targeting offset to renormalize the experimental data. Horizontal force and torque also appeared in simulations in case of a targeting offset. Surprisingly, simulations show that the lifting force acting on the bead reaches a maximum at a flow rate of ~ 5 μl/s if the targeting offset is not very high (<5 μm). Further increasing the flow rate decreases the lifting force. We attribute this effect to the spherical geometry of the bead. We predict that higher flow rates cannot increase the hydrodynamic lifting force acting on the precisely targeted microbead, setting a fundamental force limit (16 nN in our setup) for manipulating microbeads with a micropipette perpendicular to the supporting surface. In order to extend the force range, we propose the offset targeting of microbeads.

High-Speed Lens-Free Holographic Sensing of Protein Molecules Using Quantitative Agglutination Assays.

Accurate, cost-effective, easy-to-use, and point-of-care sensors for protein biomarker levels are important for disease diagnostics. A cost-effective and compact readout approach that has been used for several diagnostic applications is lens-free holographic microscopy, which provides an ultralarge field of view and submicron resolution when it is coupled with pixel super-resolution techniques. Despite its potential as a diagnostic technique, lens-free microscopy has not previously been applied to quantitative protein molecule sensing in solution, which can simplify sensing protocols and ultimately enable measurements of binding kinetics in physiological conditions. Here, we sense interferon-γ (an immune system biomarker) and NeutrAvidin molecules in solution by combining lens-free microscopy with a one-step bead-based agglutination assay, enabled by a custom high-speed light-emitting diode (LED) array and automated image processing routines. We call this a quantitative large-area binding (QLAB) sensor. The high-speed light source provides, for the first time, pixel super-resolved imaging of >104 2 μm beads in solution undergoing Brownian motion, without significant motion blur. The automated image processing routines enable the counting of individual beads and clusters, providing a quantitative sensor readout that depends on both bead and analyte concentrations. Fits to the chemical binding theory are provided. For NeutrAvidin, we find a limit of detection (LOD) of <27 ng/mL (450 pM) and a dynamic range of 2-4 orders of magnitude. For mouse interferon-γ, the LOD is <3 ng/mL (200 pM) and the dynamic range is at least 4 orders of magnitude. The QLAB sensor holds promise for point-of-care applications in low-resource communities and where protocol simplicity is important.

Reverse Combined Microflotation of Fine Magnetite from a Mixture with Glass Beads

Magnetite is an essential iron-bearing mineral. The primary method of magnetite ore beneficiation involves successive steps of crushing, grinding, and magnetic separation. Reverse cationic flotation is used at the final stage to remove silicate and aluminosilicate impurities from the magnetite concentrate and reduce silica content to 1–3%, depending on metallurgical processing route (electrometallurgy, direct iron reduction). In view of the stringent demands of the magnetite concentrate grade, before flotation, the ore is currently routinely ground down to a particle size below 35 µm, and magnetite particles are ground to a size below 10 µm. This significantly reduces the efficiency of flotation and increases iron loss in the tailings due to the hydraulic report in froth being up to 15–25%. Combined microflotation (CMF) looks to be a promising method of increasing fine-particle flotation efficiency, as it uses relatively small amounts of microbubbles alongside conventional coarse bubbles. Microbubbles act as flotation carriers, collecting gangue particles on their surface, which then coarse bubbles float. The purpose of this study is to explore the effectiveness of CMF for processing a model mixture that contained magnetite particles smaller than 10 µm and glass beads (Ballotini) below 37 µm in size when the initial iron content in the mixture was 63.76%. Commercial reagent Lilaflot 821M was used as both collector and frother. The flotation procedure, which included the introduction of 15 g/t of the collector before the start of flotation, and the addition of 5 g/t of the collector in combination with a microbubble dose of 0.018 m3/t 6 min after starting flotation, ensured an increase in the concentrate grade to 67.63% Fe and iron recovery of 91.16%.

Guided Electrokinetic Assembly of Polystyrene Microbeads onto Photopatterned Carbon Electrode Arrays

Assembly of microdevices from constituent parts currently relies on slow serial steps via direct assembly processes such as pick-and-place operations. Template Electrokinetic Assembly (TEA), a guided, noncontact assembly process, is presented in this work as a promising alternative to serial assembly processes. To characterize the process and its implementation of electrokinetic, dielectrophoretic, and electro-osmotic phenomena, we conducted studies to examine the assembly of polymer microparticles at specific locations on glassy carbon interdigitated electrode arrays (IDEAs). The IDEAs are coated with a layer of lithographically patterned resist, so that when an AC electric field is applied to the IDEA, microparticles suspended in the aqueous solution are attracted to the open regions of the electrodes not covered by photoresist. Interplay between AC electro-osmosis and dielectrophoretic forces guides 1 and 5 μm diameter polystyrene beads to assemble in regions, or "wells", uncovered by photoresist atop the electrodes. It was discovered that AC electro-osmosis under an applied frequency of 1 kHz is sufficient to effectively agglomerate 1 μm beads in the wells, whereas a stepwise process involving the application of a 1 MHz signal, followed by a 1 kHz signal, is required for the positioning of 5 μm beads, which are mainly affected by dielectrophoretic forces. Permanent entrapment of the microparticles is then demonstrated via the electropolymerization process of the conducting polymer polypyrrole.

Efficient Chain Formation of Magnetic Particles in Elastomers with Limited Space

The magnetic response of the storage modulus for bimodal magnetic elastomers containing magnetic particles with a diameter of 7.0 μm and plastic beads with a diameter of 200 μm were investigated by varying the volume fraction of plastic beads up to 0.60 while keeping the volume fraction of the magnetic particles at 0.10. The storage modulus at 0 mT for monomodal magnetic elastomers was 1.4 × 104 Pa, and it slightly increased with the volume fraction of plastic beads up to 0.6. The storage modulus at 500 mT for bimodal magnetic elastomers at volume fractions below 0.25 was constant, which was equal to that for the monomodal one (=7.9 × 104 Pa). At volume fractions of 0.25–0.40, the storage modulus significantly increased with the volume fraction, showing a percolation behavior. At volume fractions of 0.40-0.60, the storage modulus was constant at 2.0 × 105 Pa, independently of the volume fraction. These results indicate that the enhanced increase in the storage modulus was caused by the chain formation of the magnetic particles in vacancies made of plastic beads.

Microslit on a chip: A simplified filter to capture circulating tumor cells enlarged with microbeads.

Microchips are widely used to separate circulating tumor cells (CTCs) from whole blood by virtues of sophisticated manipulation for microparticles. Here, we present a chip with an 8 μm high and 27.9 mm wide slit to capture cancer cells bound to 3 μm beads. Apart from a higher purity and recovery rate, the slit design allows for simplified fabrication, easy cell imaging, less clogging, lower chamber pressure and, therefore, higher throughput. The beads were conjugated with anti-epithelial cell adhesion molecules (anti-EpCAM) to selectively bind to breast cancer cells (MCF-7) used to spike the whole blood. The diameter of the cell-bead construct was in average 23.1 μm, making them separable from other cells in the blood. As a result, the cancer cells were separated from 5 mL of whole blood with a purity of 52.0% and a recovery rate of 91.1%, and also we confirmed that the device can be applicable to clinical samples of human breast cancer patients. The simple design with microslit, by eliminating any high-aspect ratio features, is expected to reduce possible defects on the chip and, therefore, more suitable for mass production without false separation outputs.

Particle and bacteria uptake by Japanese flounder (Paralichthys olivaceus) red blood cells: Size dependence and pathway specificity

Red blood cells (RBCs) are traditionally considered non-professional phagocytes functioning predominately in oxygen transport. In the present study, we examined the ability of Japanese flounder (Paralichthys olivaceus), a teleost species with important economic values, RBCs to uptake inorganic particles and bacteria in different size/form, as well as the involving endocytic pathways. We found that flounder RBCs exhibited relatively high uptake/attachment capacities for 0.1 μm–1.0 μm (diameter) latex beads, but not for 2.0 μm beads. For the 0.1 μm beads, the uptake/attachment was executed through macropinocytosis and caveolae-mediated pathway, while for 0.5 μm and 1.0 μm beads, the uptake/attachment depended primarily on macropinocytosis and partially on the caveolin-mediated pathway. In addition to latex beads, flounder RBCs also exhibited an apparent capacity to engulf both live and inactivated bacteria. For live bacteria, the endocytosis was clathrin-mediated, while for inactivated bacteria, clathrin- as well as non-clathrin-mediated endocytosis were involved. Taken together, these results demonstrated that teleost RBCs possess particle uptake/attachment and bacteria phagocytosis capacities via different pathways that depend on the physical size and biological nature of the engulfed objects.

Platelet P-selectin triggers rapid surface exposure of tissue factor in monocytes

Tissue factor (TF) plays a central role in haemostasis and thrombosis. Following vascular damage, vessel wall TF initiates the extrinsic coagulation cascade. TF can also be exposed by monocytes. Inflammatory or infectious stimuli trigger synthesis of new TF protein by monocytes over the course of hours. It has also been suggested that monocytes can expose TF within minutes when stimulated by activated platelets. Here, we have confirmed that monocytes rapidly expose TF in whole blood and further demonstrate that platelet P-selectin exposure is necessary and sufficient. Monocyte TF exposure increased within five minutes in response to platelet activation by PAR1-AP, PAR4-AP or CRP-XL. PAR1-AP did not trigger TF exposure on isolated monocytes unless platelets were also present. In whole blood, PAR1-AP-triggered TF exposure required P-selectin and PGSL-1. In isolated monocytes, although soluble recombinant P-selectin had no effect, P-selectin coupled to 2 µm beads triggered TF exposure. Cycloheximide did not affect rapid TF exposure, indicating that de novo protein synthesis was not required. These data show that P-selectin on activated platelets rapidly triggers TF exposure on monocytes. This may represent a mechanism by which platelets and monocytes rapidly contribute to intravascular coagulation.