Phagotrophic phytoflagellates in microbial food webs

Phagotrophy by pigmented flagellates is known from the literature but has recently been rediscovered in the context of microbial food webs. Particle ingestion rates were found to be equivalent for pigmented and nonpigmented microflagellates in both field and laboratory studies. Ingestion rates of the chrysophytes Ochromonas danica, O. minuta, and Poterioochromonas malhamensis, the dinoflagellate Peridinium inconspicuum, and the cryptophytes Cryptomonas ovata and C. erosa were compared with those of two nonpigmented Monas species using 0.57 μm polystyrene beads as a food source. Ingestion rates were 0.31 to 3.17 beads/cell/h and filtration rates were 10⁻⁷ to 10⁻⁸ ml/cell/h with no detectable difference between pigmented and nonpigmented forms. Ingestion rates in unpigmented Monas species showed a linear increase with increasing particle concentration from 1.9 × 10⁶ to 1.6 × 10⁷ beads/ml. Light and DOC levels in the range of those encountered by phytoflagellates in the field also influenced laboratory measurements of bead ingestion by Poterioochromonas malhamensis. Ingestion rates decreased and photosynthesis increased over the natural PAR light range from 0 to 1800 microeinsteins/s/m². At 40 microeinsteins/s/m² maximum ingestion rates and high rates of photosynthesis occurred simultaneously. Ingestion rates decreased above 4 mgC/l supplied as glucose. DOC levels commonly occurring in Lake Oglethorpe range from 3.5 to 10.0 mgC/l. These studies suggest that mixotrophy, the trophic utilization of particulate food and dissolved organic matter as well as photosynthetically fixed organic matter, is a balanced process that can be regulated by environmental conditions. In field studies during a chrysophyte bloom, phytoflagellate grazing exceeded heterotrophic microflagellate grazing and constituted up to 55% of the bactivory of all microflagellates, ciliates, rotifers, and crustaceans combined. Neither bacterial abundance, light nor temperature were good predicters of grazing rates for the phagotrophic phytoflagellate association as a whole during this unstratified period. Phagotrophs are often most abundant at the metalimnetic plate during stratification.     

Short-duration low-direct-current electrical field treatment is a practical tool for considerably reducing counts of gram-negative bacteria entrapped in gel beads

Application of a direct-current electrical field for very short times can serve as a practical nonthermal procedure to reduce or modify the microbial distribution in gel beads. The viability of Escherichia coli and Serratia marcescens entrapped in alginate and agarose beads decreases as the field intensity and duration of electrical field increase.     

Feeding experiments on Penilia avirostris dana (cladocera: crustacea)

Feeding in the marine cladoceran, Penilia avirostris Dana, was investigated using plastic micronic beads as a controlled particle source. The range of bead size ingested by P. avirostris was largely limited to diameters of 20 μm and below, perhaps a competitive advantage in nature, was found that: (1) as the concentration of beads increased, the number of beads ingested increased: (2) as the number of P. avirostris increased, the number of beads ingested increased: and (3) the number of beads ingested also increased with body size. The size of P. avirostris, however, did not appear to limit the size of bead ingested, while larger sized animals may be better competitors for particles.     

Immobilization of the marine microalga Phaeodactylum tricornutum in alginate for in situ experiments: Bead stability and suitability

Microalgae immobilization in alginate matrixes has been recently used to perform in situ experiments. However, the susceptibility of alginate matrixes to cation chelating agents and to antigelling cations, which can cause bead disruption or dissolution, is a major limitation for in situ exposures in estuarine and marine systems. The ultimate goal of this study was to produce alginate beads stable in seawater and suited for Phaeodactylum tricornutum growth. For this, different concentrations of alginate isolated from Macrocystis pyrifera (1.5, 1.9 and 2.3% [w/v]) and Laminaria hyperborea (4.0, 4.9 and 5.8% [w/v]), two concentrations of the hardening cations calcium and strontium (2.0 and 4.0% [w/v]), and the use of the polycation chitosan were investigated. Only beads found to be more stable after 16 days of exposure in seawater were inoculated with the microalga. P. tricornutum immobilized in beads prepared from 5.8% L. hyperborea alginate and in all beads in which a chitosan hardening treatment was applied showed a weak microalgal growth. Beads prepared using 4.9% of L. hyperborea alginate and a 4% (w/v) strontium solution were found to be the most stable and the most suitable for microalgal growth, and were exposed in the field, under natural fluctuating conditions of light and temperature. In situ growth rates of immobilized P. tricornutum cells demonstrated the potential of these beads for future use in in situ experiments in estuarine and marine systems.     

Osteogenic Differentiation of Human Mesenchymal Stem Cells in Mineralized Alginate Matrices

Mineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic cells in such materials will potentially generate biological bone grafts that may even further augment bone healing. Here, we studied osteogenic differentiation of human mesenchymal stem cells (MSC) in an alginate hydrogel system where the cells were co-immobilized with alkaline phosphatase (ALP) for gradual mineralization of the microenvironment. MSC were embedded in unmodified alginate beads and alginate beads mineralized with ALP to generate a polymer/hydroxyapatite scaffold mimicking the composition of bone. The initial scaffold mineralization induced further mineralization of the beads with nanosized particles, and scanning electron micrographs demonstrated presence of collagen in the mineralized and unmineralized alginate beads cultured in osteogenic medium. Cells in both types of beads sustained high viability and metabolic activity for the duration of the study (21 days) as evaluated by live/dead staining and alamar blue assay. MSC in beads induced to differentiate in osteogenic direction expressed higher mRNA levels of osteoblast-specific genes (RUNX2, COL1AI, SP7, BGLAP) than MSC in traditional cell cultures. Furthermore, cells differentiated in beads expressed both sclerostin (SOST) and dental matrix protein-1 (DMP1), markers for late osteoblasts/osteocytes. In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture. Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering.     

Enhanced efficacy of nitrifying biomass by modified PVA_SB entrapment technique

In this study, we developed a novel technique for preparing polyvinyl alcohol (PVA) hydrogel as an immobilizing matrix by the addition of sodium bicarbonate. This resulted in an increase in the specific surface area of PVA_sodium bicarbonate (PVA_SB) hydrogel beads to 65.23 m² g^?1 hydrogel beads, which was approximately 85 and 14 % higher than those of normal PVA and PVA_sodium alginate (PVA_SA) hydrogel beads, respectively. The D _e value of PVA_SB hydrogel beads was calculated as 7.49 × 10^?4 cm² s^?1, which was similar to the D _e of PVA_SA hydrogel beads but nearly 38 % higher than that of the normal PVA hydrogel beads. After immobilization with nitrifying biomass, the oxygen uptake rate and the ammonium oxidation rate of nitrifying biomass entrapped in PVA_SB hydrogel beads were determined to be 19.53 mg O₂ g MLVSS^?1 h^?1 and 10.59 mg N g MLVSS^?1 h^?1, which were 49 and 43 % higher than those of normal PVA hydrogel beads, respectively. Scanning electron microscopy observation of the PVA_SB hydrogel beads demonstrated relatively higher specific surface area and revealed loose microstructure that was considered to provide large spaces for microbial growth. This kind of structure was also considered beneficial for reducing mass transfer resistance and increasing pollutant uptake.     

Electrical potentials in the ectomycorrhizal fungus Laccaria bicolor after a rainfall event

We measured extracellular bioelectrical activities of the ectomycorrhizal basidiomycete Laccaria bicolor under field conditions to examine its response to environmental factors. Six fruit bodies of L. bicolor in a cluster, to which electrodes were attached, exhibited less electrical potentials at the beginning, probably due to the lack of precipitation for over a week. However, its electrical potential fluctuated after raining, sometimes over 100 mV. The electrical potential of the fruit bodies and its fluctuation were correlated with precipitation. Causality analysis of electrical potential after the rain showed electrical signal transport among fruit bodies, particularly between spatially close ones, with potential directionality. Our preliminary results bring a call for studies on fungal electrical potentials in a more ecological context under field conditions.     

Chitosan/Polyethylene Glycol Beads Crosslinked with Tripolyphosphate and Glutaraldehyde for Gastrointestinal Drug Delivery

This study reports on the preparation of chitosan (CS)/polyethylene glycol (PEG) hydrogel beads using sodium diclofenac (DFNa) as a model drug. Following the optimization of the polymer to drug ratio, the chitosan beads were modified by ionic crosslinking with sodium tripolyphosphate (TPP). The CS/PEG/DFNa beads obtained from a (w/w/w) ratio of 1/0.5/0.5 with crosslinking in 10% (w/v) TPP at pH 6.0 for 30 min yielded excellent DFNa encapsulation levels with over 90% loading efficiency. The dissolution profile of DFNa from CS/PEG/DFNa beads demonstrated that this formulation was able to maintain a prolonged drug release for approximately 8 h. Among the formulations tested, the CS/PEG/DFNa (1/0.5/1 (w/w/w)) beads crosslinked with a combination of TPP (10% (w/v) for 30 min) and glutaraldehyde (GD) (5% (w/v)) were able to provide minimal DFNa release in the gastric and duodenal simulated fluids (pH 1.2 and 6.8, respectively) allowing for a principally gradual drug release over 24 h in the intestinal (jejunum and ileum) simulated fluid (pH 7.4). Thus, overall the CS/PEG beads crosslinked with TPP and GD look to be a promising and novel alternative gastrointestinal drug release system.     

Assessment of artificial substrates for evaluating groundwater microbial quality

Protection of groundwater resources requires the development of reliable ecological indicators. Microorganisms involved in ecological services or being associated with particular hosts or habitats could be used for this purpose. Nevertheless, their tracking remains limited because of sampling issues, and a lack of devices for their long term monitoring. In the present study, three artificial substrates (glass and clay beads, and gravel particles) were tested in terms of efficacy at favoring bacterial growth, and at capturing bacterial diversity of waters (i.e., groundwater, surface water and wastewater). Total proteins, total carbohydrates, dehydrogenase and hydrolytic activities were used to monitor biofilm development on these artificial substrates. Fingerprinting analyses based on rrs (16S rRNA) − rrl (23S rRNA) spacer analyses (ARISA) and next generation sequencing (NGS) of partial rrs DNA segments (V5-V6) were used to compare operating taxonomic units (OTUs), and infer bacterial genera trapped on these substrates. Glass beads were found less efficient than the other two artificial substrates at increasing protein contents and microbial activities (hydrolytic and dehydrogenase activities). ARISA showed a discrimination of bacterial communities developing on artificial substrates that was matching water types. An incubation period of 7 days allowed a reliable assessment of bacterial diversity. From this incubation period, around 75% of water genera with more than four V5-V6 rrs DNA sequences detected in a water type were recovered from biofilms growing on artificial substrates. Based on relative abundances of genera, clay beads and gravel particles were more efficient than glass beads to capture and obtain bacterial communities matching those of the initial waters. Between 45–67% of similarities were found for these artificial substrates while it was between 36 and 43% for glass beads. This study demonstrated clay beads and gravel particles as being efficient tools for capturing bacterial diversity and monitoring bacterial growth. Overall, clay beads appeared the best choice for field monitoring because of the ease of their size standardization in comparison with gravel particles.     

Co-encapsulation of amyloglucosidase with starch and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> as basis for a long-lasting CO<Subscript>2</Subscript> release

CO₂ is known as a major attractant for many arthropod pests which can be exploited for pest control within novel attract-and-kill strategies. This study reports on the development of a slow-release system for CO₂ based on calcium alginate beads containing granular corn starch, amyloglucosidase and Saccharomyces cerevisiae. Our aim was to evaluate the conditions which influence the CO₂ release and to clarify the biochemical reactions taking place within the beads. The amyloglucosidase was immobilized with a high encapsulation efficiency of 87% in Ca-alginate beads supplemented with corn starch and S. cerevisiae biomass. The CO₂ release from the beads was shown to be significantly affected by the concentration of amyloglucosidase and corn starch within the beads as well as by the incubation temperature. Beads prepared with 0.1 amyloglucosidase units/g matrix solution led to a long-lasting CO₂ emission at temperatures between 6 and 25?°C. Starch degradation data correlated well with the CO₂ release from beads during incubation and scanning electron microscopy micrographs visualized the degradation of corn starch granules by the co-encapsulated amyloglucosidase. By implementing MALDI-ToF mass spectrometry imaging for the analysis of Ca-alginate beads, we verified that the encapsulated amyloglucosidase converts starch into glucose which is immediately consumed by S. cerevisiae cells. When applied into the soil, the beads increased the CO₂ concentration in soil significantly. Finally, we demonstrated that dried beads showed a CO₂ production in soil comparable to the moist beads. The long-lasting CO₂-releasing beads will pave the way towards novel attract-and-kill strategies in pest control.     

Rapid Immunocapture of Pseudomonas putida Cells from Lake Water by Using Bacterial Flagella

Monoclonal antibodies to Pseudomonas putida Paw340 cells were produced. In an enzyme-linked immunosorbent assay (ELISA) against whole bacterial cells, a hybridoma cell line termed MLV1 produced a monoclonal antibody that reacted with P. putida Paw340 but showed no cross-reaction with 100 medical isolates and 150 aquatic isolates. By ELISA, immunogold electron microscopy, and Western blot (immunoblot) analysis, MLV1 antibody was found to react with purified bacterial flagella. The surfaces of magnetic polystyrene beads were coated with MLV1 antibody. By mixing MLV1 antibody-coated beads with lake water samples containing the target P. putida host, bead-cell complexes which could be recovered by attraction towards a magnet were formed. Prevention of nonspecific attachment of cells to the beads required the incorporation of detergents in the isolation protocol. These detergents affected colony-forming ability; however, the cells remained intact for direct detection. When reisolated by standard cultural methods, approximately 20% of the initial target population was recovered. Since the beads and bead-cell complexes were recovered in a magnetic field, target bacteria were separated from other lake water organisms and from particulate material which was not attracted towards the magnet and were thereby enriched. This method may now provide a useful system for recovering recombinant bacteria selectively from environmental samples.     

Novel Monoclonal Antibody and Peptide Binders for Mycobacterium avium subsp. paratuberculosis and Their Application for Magnetic Separation

The generation of novel Mycobacterium avium subsp. paratuberculosis (MAP)-specific monoclonal antibodies and phage-display derived peptide binders, along with their application for the magnetic separation (MS) of MAP cells, is described. Our aim was to achieve even greater MAP capture capability than is possible with peptide-mediated magnetic separation (PMS) using a 50:50 mix of biotinylated-aMp3 and biotinylated-aMptD peptide-coated beads. Gamma-irradiated whole MAP cells and ethanol extracted antigens (EEA) from these cells were used to elicit an immune response and as phage-display biopanning targets. A range of novel binders was obtained and coated onto paramagnetic beads, both individually and in various combinations, for MS evaluation. IS900 PCR was employed after MS to provide quick results. Capture sensitivity was assessed using a range of MAP concentrations after which the most promising beads were tested for their specificity for MAP, by performing MS followed by culture using 10 other Mycobacterium species. Magnetic beads coated with the biotinylated EEA402 peptide demonstrated a greater level of MAP capture than the current PMS method, even when low numbers of MAP (<10 cfu/ml) were present; however these beads also captured a range of other mycobacteria and so lacked capture specificity. Magnetic beads coated with monoclonal antibodies 6G11 and 15D10 (used as a 50:50 mix or as dually coated beads) also demonstrated improved MAP capture relative to the current PMS method, but with little cross-reactivity to other Mycobacterium spp. Therefore, two new MS protocols are suggested, the application of which would be dependent upon the required endpoint. Biotinylated EEA402-coated beads could potentially be used with a MAP-specific PCR to ensure detection specificity, while beads coated with 6G11 and 15D10 monoclonal antibodies could be used with culture or the phage amplification assay.     

Effect of Drug Loading Method on Drug Content and Drug Release from Calcium Pectinate Gel Beads

Drug-loaded calcium pectinate gel (CaPG) beads were prepared by either mixing, absorption, or swelling method. The effects of drug loading method as well as the drug loading factors (i.e., drug concentration, soaking time in drug solution, type of solvent) on drug content and drug release were investigated. The amount of drug uptake (i.e., drug content) into CaPG beads increased as the initial drug concentration increased and varied depending on the loading method. The in vitro release studies in 0.1 N hydrochloric acid (HCl) and pH 6.8 buffer indicated that the drug loading method affected drug release and release parameter, time for 50% of drug release (T ₅₀). The mixing method provided a faster drug release and lower T ₅₀ than the absorption method and swelling method, respectively. This is probably due to higher drug content in CaPG beads. The increased concentration of drug in soaking solution and soaking time resulted in higher drug content and thus faster drug release (lower in T ₅₀ values). When using 0.1 N HCl as solvent for soaking instead of water, the drug release was slower owing to the increase in molecular tortuosity of CaPG beads. The drug release was also affected by pH of the release medium in which drug release in 0.1 N HCl was faster than in pH 6.8 buffer.     

Coimmobilization of Nitrosomonas europaea and Paracoccus denitrificans cells using polyelectrolyte complex-stabilized calcium alginate gel

Calcium alginate gel (CAG) that withstands phosphate ions in the medium was prepared by reinforcing a network structure of the gel with a polyelectrolyte complex (PEC) consisting of potassium poly(vinyl alcohol) sulfate and trimethylammonium glycol chitosan iodide. The PEC-stabilized CAG beads were used as a supporting matrix for the coimmobilization of Nitrosomonas europaea ATCC 25978 cells and Paracoccus denitrificans IFO 12442 cells. The coimmobilized cells were aerobically cultured on a medium containing 3 mM of phosphate ions, using (NH₄)₂SO₄ as a substrate and ethanol as a carbon source. Ammonia was consumed without forming nitrite, indicating the concurrence of nitrification and denitrification in the same system. No breakage of the gel beads was observed during the cultivation. Repeated aerobic cultivation using a column packed with beads of coimmobilized cells had stable initial activity for at least one month.     

A simplified and efficient system for separating proteins by preparative polyacrylamide electrophoresis. With notes on horse heart myoglobin

A simplified but effective method for preparative polyacrylamide gel electrophoresis is presented, suitable for single runs of ∼15 mg of protein, which allows: (1) a gel casting shaped and cooled so that proteins do not assume curvature as they migrate down the column; (2) direct contact of the bottom of the gel with buffer; (3) use of reduced electrical field since anode and cathode are spaced only 9.5 cm apart; (4) increased stability of the anodal face of the gel, by employing a dilute running buffer and consequently a still lower current (7 to 8 mA at 150 V); (5) obtaining successive effluents in separate Visking sacs, at appropriate time intervals, and removal of proteins in absence of an electrical field; (6) rinsing of the column between collections; (7) advance planning of the actual times of collection, calculated from the rates of migration of proteins of interest relative to the time of passage of (yellow) “myoglobin standard” determined in analytical columns run under the same conditions.     

The search for an 111In labeled agent for the solid component of gastric emptying

¹¹¹In-labeled solid meal was prepared by chelation of ¹¹¹In with Chelex resin bead. The effect of grinding of normal Chelex bead on ¹¹¹In chelation and retention in solid meal was evaluated in an in vitro system. The Chelex resin beads were ground in a mortar-pestle to form ground Chelex resin beads. Fine particles were removed by resuspension in distilled water and centrifugation (1000 g). One hundred to 150 μCi of ¹¹¹In chloride was diluted with 0.1 N HC1 and mixed with 1 g of Chelex resin beads. Unbound ¹¹¹In was removed by centrifugation (1000 g). The ¹¹¹In-labeled Chelex resin beads were mixed with fresh egg and ¹¹¹In-labeled solid meal was prepared by heating until solid. The meals were digested with HCl-pepsin (1.2mg/mL of pepsin in 0.1 N HC1) for 4 h in a stirrer-bath (37 °C). Aliquots were collected at intervals for determination of ¹¹¹In loss from ¹¹¹In-labeled solid meal.These results suggest that ¹¹¹In Chelex resin beads were retained in solid meals at a higher level than normal Chelex resin beads and other ¹¹¹In-tracers.     

Detecting,Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H₂O₂ production, respectively.     

Preparation and characterization of mixed-mode magnetic adsorbent with p-amino-benzamidine ligand: Operated in a magnetically stabilized fluidized bed reactor for purification of trypsin from bovine pancreas

The magnetic beads were synthesized using glycidylmethacrylate (GMA) and methylmethacrylate (MMA) monomers. A multimodal ligand (i.e., p-amino-benzamidine) was covalently immobilized onto magnetic beads after glutaraldehyde activation, and consequently used for purification of the trypsin from bovine pancreas. The p-amino-benzamidine ligand immobilized magnetic beads were characterized by FTIR, VSM, SEM, and analytical methods. Trypsin adsorption experiments were investigated under different experimental conditions (i.e., medium pH, initial trypsin concentration, temperature, and ionic strength) in a batch system. Maximum trypsin adsorption capacity was found to be 75.9 ± 2.6 mg/g beads. Adsorbed trypsin was eluted by using (0.1 M acetate buffer, pH 3.0) with a 97% recovery. The purification factor of trypsin from crude pancreas extract was 8.7 folds. The purity of the eluted trypsin from p-amino-benzamidine functionalized magnetic beads was determined as 86% by HPLC. The method developed in this report was successfully applied for purification of the trypsin from crude pancreas extract in a magnetically stabilized fluidized bed reactor.     

PHAGOCYTOSIS BY THE CELLULAR SLIME MOLD POLYSPHONDYLIUM PALLIDUM DURING GROWTH AND DEVELOPMENT

The phagocytic ability of amoebae of the cellular slime mold Polysphondylium pallidum, grown in shaken suspension, was examined. An established quantitative assay of the uptake of polystyrene (PS) beads was shown to be valid for this organism. The kinetics of phagocytosis were determined, and estimates of the concentration of PS beads necessary to achieve half-maximal phagocytic velocity (K_p), as well as the maximal velocity itself (V_p^max), were made. Comparison with previously published data on Acanthamoeba and guinea pig leukocytes suggested that the P. pallidum amoebae had the lowest K_p, while the leukocytes had the highest V_p^max. Beads approximately 1 µm in diameter appeared to be the optimal size for ingestion. Simultaneously with phagocytosis, comparable numbers of beads accumulated at the cell surface; this accumulation did not occur when phagocytosis was inhibited. Phagocytosis was depressed by protein in the medium, by increased osmolarity, and by inhibitors of aerobic metabolism. Starvation-initiated development, leading to encystment, was shown to affect the capacity of the cells to phagocytize, mainly by progressively decreasing the time span over which the cells ingested particles at a constant initial rate.