In Situ Synthesis of Photoactive Polymers on a Living Cell Surface via Bio-Palladium Catalysis for Modulating Biological Functions.

Cell surface engineering with functional polymers is an effective strategy to modulate cell activity. Here, a bio-palladium catalyzed polymerization strategy was developed for in situ synthesis of conjugated polymers on living cell surfaces. Through Sonagashira polymerization, photoactive polyphenyleneethynylene (PPE) is synthesized on the cell surface via cell-generated bio-Pd catalyst. The in situ formed PPE is identified by excellent light-harvest capacity and blue fluorescence on the surfaces of E. coli and C. pyrenoidosa. Besides imaging microbes for tracing the polymerization process, PPE also exhibits enhanced antibacterial activity against E. coli. It can also augment the ATP synthesis of C. pyrenoidosa through enlarging the light absorption and accelerating the cyclic electron transport of the algae. With this bio-metal catalyzed polymerization method, functional polymers can be synthesized in situ on the living cell surface.

Natural mechanisms and artificial PEG-induced mechanism that repair traumatic damage to the plasmalemma in eukaryotes.

Eukaryotic tissues are composed of individual cells surrounded by a plasmalemma that consists of a phospholipid bilayer with hydrophobic heads that bind cell water. Bound-water creates a thermodynamic barrier that impedes the fusion of a plasmalemma with other membrane-bound intracellular structures or with the plasmalemma of adjacent cells. Plasmalemmal damage consisting of small or large holes or complete transections of a cell or axon results in calcium influx at the lesion site. Calcium activates fusogenic pathways that have been phylogenetically conserved and that lower thermodynamic barriers for fusion of membrane-bound structures. Calcium influx also activates phylogenetically conserved sealing mechanisms that mobilize the gradual accumulation and fusion of vesicles/membrane-bound structures that seal the damaged membrane. These naturally occurring sealing mechanisms for different cells vary based on the type of lesion, the type of cell, the proximity of intracellular membranous structures to the lesion and the relation to adjacent cells. The reliability of different measures to assess plasmalemmal sealing need be carefully considered for each cell type. Polyethylene glycol (PEG) bypasses calcium and naturally occurring fusogenic pathways to artificially fuse adjacent cells (PEG-fusion) or artificially seal transected axons (PEG-sealing). PEG-fusion techniques can also be used to rapidly rejoin the closely apposed, open ends of severed axons. PEG-fused axons do not (Wallerian) degenerate and PEG-fused nerve allografts are not immune-rejected, and enable behavioral recoveries not observed for any other clinical treatment. A better understanding of natural and artificial mechanisms that induce membrane fusion should provide better clinical treatment for many disorders involving plasmalemmal damage.

A Fissionable Artificial Eukaryote-like Cell Model.

The use of artificial cells has attracted considerable attention in various fields from biotechnology to medicine. Here, we develop a cell-sized vesicle-in-vesicle (VIV) structure containing a separate inner vesicle (IV) that can be loaded with DNA. We use polymerase chain reaction (PCR) to successfully amplify the amount of DNA confined to the IV. Subsequent osmotic stress-induced fission of a mother VIV into two daughter VIVs successfully divides the IV content while keeping it confined to the IV of the daughter VIVs. The fission rate was estimated to be ∼20% quantified by fluorescence microscope. Our VIV structure represents a step forward toward construction of an advanced, fissionable cell model.

Exocytosis of latex beads during the encystment of Acanthamoeba.

Cells of Acanthamoeba castellanii (Neff) are known to form mature cysts characterized by a cellulose-containing cell wall when transferred to a nonnutrient medium. Amebas which engulfed latex beads before encystment formed mature cysts essentially devoid of bead material. The encystment of bead-containing cells appeared to be similar to that of control cells since no important differences between the two were observed with respect to cellular levels of glycogen or protein, cellulose synthetase activity, the amount of cyst wall polysaccharide formed, or the percentage of cysts formed. Actinomycin D and cycloheximide inhibited encystment as well as bead expulsion. Ultrastructural analysis revealed that the beads, which initially were contained in phagocytic vesicles, were released from the cell by fusion of vesicular membranes with the plasma membrane. Exocytosis was observed in cells after 3 hr of encystment, with most of the beads being lost before cyst wall formation. Each bead-containing vesicle involved in expulsion was conspicuously demarcated by an area of concentrated cytoplasm, which was more homogeneously granular than the surrounding cytoplasm. Beads were not observed in the cytoplasm of mature cysts but were occasionally found in the cyst wall.

Cellulosic wall component produced by the golgi apparatus of Pleurochrysis scherffelii.

The Golgi apparatus of a marine chrysophycean alga Pleurochrysis scherffelii Pringsheim produces wall fragments (circular-to-ellipsoidal "scales") which are released to the periphery by an exocytotic process involving the fusion of cisternae and the plasma membrane. The cellulosic component of the scales is a complex network of fibrils (10 to 25 angstroms in diameter) that resist treatment with strong alkali. Untreated washed scales yield galactose, ribose, arabinose, and traces of glucose; alkali-purified scales yield much more glucose. The fibrillar scale constituent shows a positive iodine dichroism of the intact wall, a positive zinc chloride-iodine reaction, breakage sites characteristic of highly crystalline cellulose, and solubility in Schweizer's reagent.

Estimation of Cochlodinium polykrikoides growth potential using a dialysis membrane.

We developed a test to measure the growth potential of C. polykrikoides using a dialysis membrane and artificial seawater. Nitrite nitrogen and inorganic phosphorus in the medium were almost completely removed when the medium was dialyzed against artificial seawater for five or more 6-hour cycles using a dialysis membrane (Spectrum's Spectra/Por 7 Membrane) with a molecular-weight cut-off of 50,000, regardless of the presence of C. polykrikoides. The phytoplankton grew well even after dialysis. To estimate the growth potential of C. polykrikoides, a minimum initial concentration of > 100 cells/ml is required. Methods using short-term starvation culturing of C. polykrikoides to measure growth potential were determined to be ineffective; instead, controlled tests using artificial seawater are recommended. The dialysis membrane used in this study can also be employed to measure the algal growth potential of other phytoplankton species.

Supplemental diagnosis and molecular taxonomy of Myxobolus diaphanus (Fantham, Porter et Richardson, 1940) (Myxozoa) parasitizing Fundulus diaphanus (Cyprinodontiformes) in Nova Scotia, Canada.

Myxobolus diaphanus (Fantham, Porter et Richardson, 1940) was found in banded killifish Fundulus diaphanus (Lesueur) at several freshwater localities in Nova Scotia, including the type locality at the mouth of the Salmon River, Guysborough County. The new material, the first to be reported in 64 years, was used to supplement information on spore morphology, to document the site of development in the tissue, and to compare sequence data of the 18S rDNA to other studied myxobolids. Plasmodia with developed spores occurred in loose connective tissue of the head, the dermis (particularly in the roof of the mouth and at the base of fins), surface of the brain and ovary, muscle epimysium, and the submucosa of the intestine. Developed plasmodia containing spores were also found free in the lumen of the vena cava and within fluid-filled spaces of the skull, mandible and lower jaw. A phylogenetic analysis using 18S rDNA (878 bp) placed M. diaphanus in a terminal clade containing certain freshwater species of Henneguya, all of which occur in North America and have elongate spore bodies.

Effect of light and Triton X-100 on the size of protoplasts from Ulothrix gigas.

An electronic particle-size analyzer (Coulter Counter with Channelyzer) was used both to monitor the purity of large numbers of protoplasts during sequential steps in their isolation from Ulothrix gigas and also to detect changes in volume (swelling or growth) after incubation of purified protoplasts under various conditions. Protoplasts almost devoid of contaminating debris, but heterogenous in size, were obtained by pelleting partly-purified protoplasts through sucrose solutions. Pure protoplasts of discrete sizes were obtained by centrifuging these resuspended protoplasts on iso-osmotic mannitol/sucrose gradients. The protoplasts were shown to enlarge (swell) and then burst when transferred to increasingly hypotonic solutions or when incubated in Triton X-100. The rate of protoplast enlargement (growth) was also monitored on the Coulter Counter and differences in size of large populations were discernible within 2 h in protoplasts incubated under low light, and growth rates of such protoplasts could be assessed for up to 36 h. The Coulter Counter provides a rapid, reproducible and sensitive method for measuring the size distribution of large numbers of protoplasts. This, combined with the techniques described here for the isolation of protoplasts from U. gigas in high yield and purity, provides a system eminently suitable for investigating a wide array of physiological and developmental processes in plant protoplasts.

A method for producing microbe-free Amyloodinium ocellatum (Brown) with Percoll.

A method for collecting microbe-free Amyloodinium ocellatum from infected red drum (Sciaenops ocellatus) is described. Surface contamination of trophonts removed from fish by osmotic shock was reduced by washing the preparation with antibiotic containing saltwater and Percoll density gradient purification. The resulting tomont preparation was free of external contamination. Sterile, infective dinospores excysted from the tomonts after 72 h incubation at 26 degrees C in antibiotic containing 30 ppt saltwater.

Three new freshwater species of centrohelid heliozoans: Acanthocystis crescenta sp. nov., A. kirilli sp. nov., and Choanocystis minima sp. nov.

Three new species of centrohelid heliozoans Acanthocystis crescenta, A. kirilli, and Choanocystis minima from two freshwater lakes of Valamo Island (North-Western Russia) were studied using light- and scanning electron microscopy. The main apomorphy of A. kirilli are slightly branched radial scales. Acanthocystis crescenta has characteristic radial scales with crescent-like structures on their tips. Cell diameter of these two species does not exceed 10 microm. Radial scales of C. minima have truncated tips and lack apical teeth; plate scales of this species are oviform, without lateral notches or central rib. The latter organism is the smallest heliozoan species known; the diameter of its body is about 3 microm. The possible domination of small heliozoans in the cryptic diversity of centrohelids is discussed.

Laboratory screening of coating libraries for algal adhesion.

Coatings libraries achieved through a combinatorial chemistry approach, which may generate tens to hundreds of formulations, can be deposited in an array of 12 patches, each approximately 9 cm(2), on 10 x 20 cm primed aluminum panels. However, existing methods to quantify algal biomass on coatings are unsuitable for this type of array format. This paper describes an algorithm modelled on a probability distribution that quantifies the area of surface covered by a green alga from digital images. The method allows coatings with potential fouling-release properties to be down-selected for further evaluation. The use of the algorithm is illustrated by a set of eight siloxane-polyurethane coatings made using organofunctional poly(dimethylsiloxane) (PDMS) and poly(epsilon-caprolactone)-PDMS-poly(epsilon-caprolactone) (PCL-PDMS-PCL) triblock copolymers along with four PDMS standards which were deposited on one panel. Six replicate panels were seeded with Ulva zoospores which grew into sporelings (small plants) that completely covered the surface. The ease of removal of the Ulva sporeling biofilms was determined by automated water jetting at six different impact pressures. The coverage of the biofilm on the twelve individual formulations after jet washing was quantified from the green colour of digital images. The data are discussed in relation to the composition of the coatings.

Effects of agitation on the microalgae Phaeodactylum tricornutum and Porphyridium cruentum.

The effect of mechanical agitation on the microalgae Phaeodactylum tricornutum and Porphyridium cruentum was investigated in aerated continuous cultures with and without the added shear protectant Pluronic F68. Damage to cells was quantified through a decrease in the steady state concentration of the biomass in the photobioreactor. For a given aeration rate, the steady state biomass concentration rose with increasing rate of mechanical agitation until an upper limit on agitation speed was reached. This maximum tolerable agitation speed depended on the microalgal species. Further increase in agitation speed caused a decline in the steady state concentration of the biomass. An impeller tip speed of >1.56 m s(-1) damaged P. tricornutum in aerated culture. In contrast, the damage threshold tip speed for P. cruentum was between 2.45 and 2.89 m s(-1). Mechanical agitation was not the direct cause of cell damage. Damage occurred because of the rupture of small gas bubbles at the surface of the culture, but mechanical agitation was instrumental in generating the bubbles that ultimately damaged the cells. Pluronic F68 protected the cells against damage and increased the steady state concentration of the biomass relative to operation without the additive. The protective effect of Pluronic was concentration-dependent over the concentration range of 0.01-0.10% w/v.

Evaluation of intestinal protozoan morphology in human fecal specimens preserved in EcoFix: comparison of Wheatley's trichrome stain and EcoStain.

As a result of disposal problems related to the use of mercury compounds, many laboratories have switched from mercuric chloride-based Schaudinn's and polyvinyl alcohol (PVA) stool preservatives to other, non-mercury-based preservatives. A comparison of organism recoveries and morphologies of the intestinal protozoa was undertaken with PVA containing the EcoFix zinc-based Schaudinn's preservative (Meridian Diagnostics, Inc.); both Wheatley's modification of Gomori's trichrome stain (WT) and EcoStain (ES) were used to stain 51 human fecal specimens. Morphology, clarity of nuclear and cytoplasmic detail, overall color differences, and the ease or difficulty in detecting intestinal protozoa in fecal debris were assessed for the two permanent stained smears. Overall, organism morphology of the intestinal protozoa stained with WT and that of protozoa stained with ES were not equal in nuclear and cytoplasmic detail or range of color. However, the same organisms were identified in stained fecal smears with either WT or ES, with the exception of situations in which organism numbers were characterized as rare. Included were 67 protozoan challenges (number of organisms): Entamoeba histolytica-Entamoeba dispar (5), Entamoeba coli (9), Entamoeba hartmanni (6), Endolimax nana (12), Iodamoeba bütschlii (8), Blastocystis hominis (19), Giardia lamblia (6), Dientamoeba fragilis (2), yeast (2), and leukocytes (2). Five specimens were negative for parasites but contained fecal debris that was compared for morphologic detail and color range. The ES produces a more gray-green monotone with very little pink or red tone; contrast among the various colors is less than that seen with WT. Stain intensity for all organisms was acceptable, and there were no problems with stain deposition. The quality of the protozoan morphology with ES was often comparable to that with WT (36 of 67 [53.7%]) and, in some cases, better (24 of 67 [35.8%]). Organisms on the WT-stained smear exhibited better morphology in a few instances (4 of 67 [6%]), and in three instances, there were discrepant organism numbers.

Evaluation of commercially available preservatives for laboratory detection of helminths and protozoa in human fecal specimens.

Formalin and mercuric chloride-based low-viscosity polyvinyl alcohol (LV-PVA) are widely used by most diagnostic parasitology laboratories for preservation of helminth eggs and protozoan cysts and trophozoites in fecal specimens. Concerns about the toxicity of formalin and the difficulty of disposal of LV-PVA are powerful incentives to use alternate preservatives. Such alternatives have been marketed by several companies and are often presented as one-vial, non-mercuric chloride fixatives that aim at performing the same role as formalin and PVA combined. We compared five, one-vial commercial preservatives, two from Meridian Diagnostics, Inc. (Ecofix and sodium acetate-acetic acid-formalin), and one each from Scientific Device Laboratories, Inc. (Parasafe), Alpha Tec Systems, Inc. (Proto-fix), and Streck Laboratories, Inc. (STF), with 10% formalin and LV-PVA. Fecal specimens obtained from patients in a Brazilian hospital were aliquoted within 12 h of collection into the seven preservatives mentioned above and were processed after 1 month at the Centers for Disease Control and Prevention. Direct and concentrated permanent smears as well as concentrates for 20 positive specimens (a total of 259 processed samples) were prepared, stained according to the manufacturers' instructions, examined, and graded. Positive specimens contained one or more parasites with stages consisting of eggs, larvae, cysts, and a few trophozoites of Giardia intestinalis. Criteria for assessment of the preservatives included the quality of the diagnostic characteristics of helminth eggs, protozoan cysts, and trophozoites, ease of use, and cost. Acceptable alternatives to formalin for wet preparations were found. Ecofix was found to be comparable to the traditional "gold standard" LV-PVA for the visualization of protozoa in permanent stained smears. This study suggests that more acceptable alternatives to the traditional formalin and LV-PVA exist.