Biodegradability and mechanical properties of polycaprolactone composites encapsulating phosphate-solubilizing bacterium Bacillus sp. PG01

This study examined the feasibility of using polycaprolactone (PCL) and its composites (with starch and/or clay) in encapsulating cells of phosphate-solubilizing bacteria (PSB) for the development of biodegradable and “controlled-release” bacterial fertilizer. The PSB used in this work was an indigenous Bacillus sp. PG01 isolate. The results show that the PG01 strain was able to degrade all the cell-loaded capsules made of PCL and PCL composites, resulting in a continual cell release. Morphology observation indicates that severe disruption of the capsule structure occurred after incubation for 15–20 days. The biodegradability of the capsules decreased in the order of PCL/starch (20 wt%) > PCL/starch (20 wt%)/cay (7 wt%) > PCL alone > PCL/clay (7 wt%). Similar trends were also observed for the decrease in tensile strength and elongation at break, suggesting strong connections between biodegradability and the mechanical properties. Addition of starch appeared to enhance the biodegradability of the capsules, whereas the clay-blended composites were less biodegradable. The amount and rate of cell release from cell-encapsulated PCL-based capsules were positively dependent on the biodegradability and on the decrease in the mechanical strength. Nevertheless, the pattern of cell release was quite similar for all types of capsules. The outcome of this work seems to suggest that by proper manipulation of composite compositions, the controlled release of the bacterial fertilizer (i.e., Bacillus sp. PG01 cells) might be achievable.     

Locally controlled release of basic fibroblast growth factor from multilayered capsules

Biodegradable multilayered capsules encapsulating basic fibroblast growth factor (bFGF) were developed as a cytokine release carrier for drug delivery systems. The multilayered hollow capsules were fabricated via the layer-by-layer (LbL) assembly of chitosan (CT) and dextran sulfate (Dex). The bFGF was encapsulated into the CT/Dex multilayered capsules by controlling the membrane permeability, and the local and sustained release of bFGF from the capsules was examined. At pH < 8.0, the capsule membrane tightened, and FITC-dextran ( Mw = 4000) could not enter the capsules. However, FITC-dextran ( M w = 250000) easily entered the capsules at pH > 8.0, which can be attributed to the electrostatic repulsion of Dex caused by the deprotonation of the amine group in CT. After treatment with acetic acid buffer (pH 5.6), FITC-dextran or bFGF was successfully encapsulated into the capsules. The amount of encapsulated bFGF was approximately 34 microg/1 mg of capsule. Initially, about 30% of the encapsulated bFGF was released in serum-free medium within a few hours, however, the release was sustained over 70 h. When the bFGF encapsulating capsules were added to cell culture medium (serum-free), the mouse L929 fibroblast cells proliferated well for 2 weeks as compared to cultures, where bFGF was added to the medium or where bFGF and empty hollow capsules were added separately. The proliferation is due to the local and sustained release of bFGF from the adsorbent capsule to the cell surface.     

Use of Atomic Force Microscopy and Transmission Electron Microscopy for Correlative Studies of Bacterial Capsules

Bacteria can possess an outermost assembly of polysaccharide molecules, a capsule, which is attached to their cell wall. We have used two complementary, high-resolution microscopy techniques, atomic force microscopy (AFM) and transmission electron microscopy (TEM), to study bacterial capsules of four different gram-negative bacterial strains: Escherichia coli K30, Pseudomonas aeruginosa FRD1, Shewanella oneidensis MR-4, and Geobacter sulfurreducens PCA. TEM analysis of bacterial cells using different preparative techniques (whole-cell mounts, conventional embeddings, and freeze-substitution) revealed capsules for some but not all of the strains. In contrast, the use of AFM allowed the unambiguous identification of the presence of capsules on all strains used in the present study, including those that were shown by TEM to be not encapsulated. In addition, the use of AFM phase imaging allowed the visualization of the bacterial cell within the capsule, with a depth sensitivity that decreased with increasing tapping frequency.     

Encapsulation of R. planticola Rs-2 from alginate-starch-bentonite and its controlled release and swelling behavior under simulated soil conditions

The plant growth-promoting bacteria (PGPR) Raoultella planticola Rs-2 was encapsulated with the various blends of alginate, starch, and bentonite for development of controlled-release formulations. The stability and release characteristics of these different capsule formulations were evaluated. The entrapment efficiency of Rs-2 in the beads (capsules) was more than 99%. The diameter of dry beads ranged from 0.98 to 1.41 mm. The bacteria release efficiency, swelling ratio, and biodegradability of the different bead formulations were enhanced by increasing the starch or alginate contents, but were impeded by higher bentonite content. The release kinetics of viable cells from capsules and the swelling ratio of capsules were studied in simulated soil media of varying temperature, moisture, pH, and salt content. The release of loaded Rs-2 cells and swelling of capsules are greatly affected by moisture, temperature, pH and salt content of the release medium. The release of viable Rs-2 cells from capsules was positively associated with the swelling properties of the capsules. The release of Rs-2 cells occurred through a Case II diffusion mechanism. In summary, this work indicates that alginate-starch-bentonite blends are a viable option for the development of efficient controlled-release formulations of Rs-2 biofertilizer, and which could have a promising application in natural field conditions.     

The permeability of EUDRAGIT RL and HEMA-MMA microcapsules to glucose and inulin

Measurement of the rate of glucose diffusion from EUDGRAGIT RL and HEMA-MMA microcapsules coupled with a Thiele modulus/Biot number analysis of the glucose utilization rate suggests that pancreatic islets and CHO (Chinese hamster ovary) cells (at moderate to high cell densities) should not be adversely affected by the diffusion restrictions associated with these capsule membranes. The mass transfer coefficients for glucose at 20 degrees C were of the same order of magnitude for both capsules, based on release measurements: approximately 5 x 10(-6) cm/s for EUDRAGIT RL and approximately 2 x 10(-6) for HEMA-MMA. Inulin release from EUDRAGIT RL was slower than for glucose (mass transfer coefficient 14 +/- 4 x 10(-8) cm/s). The Thiele moduli were much less than 1, either for a single islet at the center of a capsule or CHO cells uniformly distributed throughout a capsule at 10(-6) cells/ mL, so that diffusion restrictions within the cells in EUDRAGIT RL or 800 mum HEMA-MMA capsules should be negligible. The ratio of external to internal diffusion resistance (Biot number) was less than 1, so that at most, only a small diffusion effect on glucose utilization should be expected (i.e., the overall effectiveness factors were greater than 0.8). These calculations were consistent with experimental observation of encapsulated islet behavior but not fully with CHO cell behavior. Permeability restricted cell viability and growth is potentially a major limitation of encapsulated cells; further analysis is warranted.     

Effects of phosphate-solubilizing bacteria application on soil phosphorus availability in coal mining subsidence area in Shanxi

The effect of phosphate-solubilizing bacteria (PSB) application on phosphorus (P) availability in reclaimed soil in coal mining subsidence region was investigated. Seven treatments were carried out including control, chicken manure (CM), PSB, PSB + tricalcium phosphate (TCP), CM?+?TCP, PSB?+?ground phosphate rock (GPR) and CM?+?GPR. The results showed soil Olsen-P concentration and phosphatase level as well as the yield of pakchoi (Brassica chinensis L.) were significantly higher in PSB application treatments compared to the corresponding CM application treatments. Soil phosphatase, invertase and urease contents were increased most significantly in PSB treatment, 1.18-, 1.31- and 2.32-fold higher than those in the control, respectively. Soil Ca₂-P, Ca₈-P, Fe-P and Al-P concentrations exhibited the greatest increases in PSB?+?TCP treatment, while occluded-P showed minor changes in different treatments. Application of PSB fertilizer reduced the transformation of Olsen-P to Ca₁₀-P, thus increasing P availability in reclaimed soil of coal mining subsidence area.     

Thermally induced gelable polymer networks for living cell encapsulation

We report the encapsulation of MIN6 cells, a pancreatic beta-cell line, using thermally induced gelable materials. This strategy uses aqueous solvent and mild temperatures during encapsulation, thereby minimizing adverse effects on cell function and viability. Using a 2:1 mixture of PNIPAAm-PEG-PNIPAAm tri-block copolymer and PNIPAAm homopolymer that exhibit reversible sol-to-gel transition at approximately 30 degrees C, gels were formed that exhibit mechanical integrity, and are stable in H(2)O, PBS and complete DMEM with negligible mass loss at 37 degrees C for 60 days. MTT assays showed undetectable cytotoxicity of the polymers towards MIN6 cells. A simple microencapsulation process was developed using vertical co-extrusion and a 37 degrees C capsule collection bath containing a paraffin layer above DMEM. Spherical capsules with diameters ranging from 500 to 900 microm were formed. SEM images of freeze-dried capsules with PBS as the core solution showed homogenous gel capsule membranes. Confocal microscopy revealed that the encapsulated cells tended to form small aggregates over 5 days, and staining for live and dead cells showed high viability post-encapsulation. A static glucose challenge with day-5 cultured microencapsulated cells exhibited glucose-dependent insulin secretion comparable to controls of free MIN6 cells grown in monolayers. These results demonstrate the potential use of these thermo-responsive polymers as cell encapsulation membranes.     

Prospects for genomic selection in conifer breeding: a simulation study of Cryptomeria japonica

Genomic selection (GS) can be a powerful technology in conifer breeding because conifers have long generation intervals, protracted evaluation times, and high costs of breeding inputs. To elucidate the potential of GS for conifer breeding, we simulated 60-year breeding programs in Cryptomeria japonica with and without GS. In conifers, the rapid decay of linkage disequilibrium (LD) can constitute a severe barrier to application of GS. For overcoming that barrier, we proposed an idea to leverage a seed orchard system, which has been used commonly in conifers, because some degree of LD exists in progenies derived from the limited number of elite trees in a seed orchard. The base population used for simulations consisted of progenies from 25 elite trees. Results show that GS breeding (GSB) done without model updating outperformed phenotypic selection breeding (PSB) during the first 30 years, but the genetic gain achieved over the 60 years was smaller in GSB than in PSB. However, GSB with model updating outperformed PSB over the 60 years. The genetic gain achieved over the 60 years of GSB with model updating was nearly twice that of PSB. Advantages of GSB over PSB prevailed, even for a low heritability polygenic trait. The number of markers necessary for efficient GS was a realistic level (e.g., one in every 1 cM), although higher marker density engendered higher accuracy of selection. These results suggest that GS can be useful in C. japonica breeding. Updating of the prediction model was, however, indispensable for attaining the large genetic gain.     

A Hybrid System Using Both Promoter Activation and Gene Amplification for Establishing Exogenous Protein Hyper-Producing Cell Lines

We previously developed a promoter-activated production (PAP) system using amplified ras oncogene to activate the cytomegalovirus (CMV) promoter controlling the foreign gene in mammalian cells. CHO cells were demonstrated to be suitable for the PAP system. Here, we show that very high-level production of a recombinant protein was achieved when the human CMV promoter was inserted into a glutamine synthetase (GS) minigene expression plasmid, pEE14. A highly productive host CHO cell line, ras clone I containing amplified ras oncogene, was further transfected with the plasmid expressing both hIL-6 gene and GS minigene, and selected with methionine sulphoximine. We were able to establish a hIL-6 hyper-producing cell line, D29, which exhibited a peak productivity rate of approximately 40 μg hIL-6 10^?6 cells day^?1 through a combination of the PAP system and the GS gene amplification system. The cellular productivity of D29 cells was about 13-fold higher than control hIL-6-producing cells derived from CHO cells whose hIL-6 gene was amplified by the GS gene amplification system, and about 5-fold higher than the I13 cells established by the PAP system, which contains amplified ras oncogene and non-amplified hIL-6 gene. When D29 cells were cultured for a month, an accumulation rate of approximately 80 μg hIL-6 ml^?1 per 3 days was achieved on the 9th day. These results indicate that this PAP and GS hybrid system enables the efficient and rapid establishment of recombinant protein hyper-producing cell lines.     

Transmission of a bacterial consortium in Eisenia fetida egg capsules

The earthworm Eisenia fetida harbours Verminephrobacter eiseniae within their excretory nephridia. This symbiont is transferred from the parent into the egg capsules where the cells are acquired by the developing earthworm in a series of recruitment steps. Previous studies defined V. eiseniae as the most abundant cell type in the egg capsules, leaving approximately 30% of the bacteria unidentified and of unknown origin. The study presented here used terminal restriction fragment length polymorphism analysis together with cloning and sequencing of 16S rRNA genes to define the composition of the bacterial consortium in E. fetida egg capsules from early to late development. Newly formed capsules of E. fetida contained three bacterial types, a novel Microbacteriaceae member, a Flexibacteriaceae member and the previously described V. eiseniae. Fluorescent in situ hybridization (FISH) using specific and general rRNA probes demonstrated that the bacteria are abundant during early development, colonize the embryo and appear in the adult nephridia. As the capsules mature, Herbaspirillum spp. become abundant although they were not detected within the adult worm. These divergent taxa could serve distinct functions in both the adult earthworm and in the egg capsule to influence the competitive ability of earthworms within the soil community.     

Implantable encapsulated cytosine deaminase having 5-fluorocytosine-deaminating activity

A cell extract of Escherichia coli was found to contain a cytosine deaminase that can stoichiometrically deaminate 5-fluorocytosine to 5-fluorouracil. It was partially purified and aseptically encapsulated in semipermeable cellulose tubes. These capsules, each containing 0.20 U, were implanted under the skin of rats. After a month the capsules were taken out, and found to contain 0.025 ± 0.011 U per capsule (half-life of 10 ± 2 days) (mean ± S.D., n = 6).This fact provided us with an idea for a new approach to the chemotherapy of cancer with the combined use of 5-fluorocytosine administered orally and cytosine deaminase capsule implanted locally.     

Immunohistochemical Localization of GFAP and Glutamate Regulatory Proteins in Chick Retina and Their Levels of Expressions in Altered Photoperiods

Moderate to intense light is reported to damage the chick retina, which is cone dominated. Light damage alters neurotransmitter pools, such as those of glutamate. Glutamate level in the retina is regulated by glutamate–aspartate transporter (GLAST) and glutamine synthetase (GS). We examined immunolocalization patterns and the expression levels of both markers and of glial fibrillary acidic protein (GFAP, a marker of neuronal stress) in chick retina exposed to 2000 lux under 12-h light:12-h dark (12L:12D; normal photoperiod), 18L:6D (prolonged photoperiod), and 24L:0D (constant light) at post-hatch day 30. Retinal damage (increased death of photoreceptors and inner retinal neurons and Müller cell hypertrophy) and GFAP expression in Müller cells were maximal in 24L:0D condition compared to that seen in 12L:12D and 18L:6D conditions. GS was present in Müller cells and GLAST expressed in Müller cell processes and photoreceptor inner segments. GLAST expression was decreased in 24L:0D condition, and the expression levels between 12L:12D and 18L:6D, though increased marginally, were statistically insignificant. Similar was the case with GS expression that significantly decreased in 24L:0D condition. Our previous study with chicks exposed to 2000 lux reported increased retinal glutamate level in 24L:0D condition. The present results indicate that constant light induces decreased expressions of GLAST and GS, a condition that might aggravate glutamate-mediated neurotoxicity and delay neuroprotection in a cone-dominated retina.     

Transmission of Nephridial Bacteria of the Earthworm Eisenia fetida

The lumbricid earthworms (annelid family Lumbricidae) harbor gram-negative bacteria in their excretory organs, the nephridia. Comparative 16S rRNA gene sequencing of bacteria associated with the nephridia of several earthworm species has shown that each species of worm harbors a distinct bacterial species and that the bacteria from different species form a monophyletic cluster within the genus Acidovorax, suggesting that there is a specific association resulting from radiation from a common bacterial ancestor. Previous microscopy and culture studies revealed the presence of bacteria within the egg capsules and on the surface of embryos but did not demonstrate that the bacteria within the egg capsule were the same bacteria that colonized the nephridia. We present evidence, based on curing experiments, in situ hybridizations with Acidovorax-specific probes, and 16S rRNA gene sequence analysis, that the egg capsules contain high numbers of the bacterial symbiont and that juveniles are colonized during development within the egg capsule. Studies exposing aposymbiotic hatchlings to colonized adults and their bedding material suggested that juvenile earthworms do not readily acquire bacteria from the soil after hatching but must be colonized during development by bacteria deposited in the egg capsule. Whether this is due to the developmental stage of the host or the physiological state of the symbiont remains to be investigated.     

The polysaccharide capsule of the pathogenic fungus Cryptococcus neoformans enlarges by distal growth and is rearranged during budding

The capsule of Cryptococcus neoformans can undergo dramatic enlargement, a phenomenon associated with virulence. A prior study that used Ab to the capsule as a marker for older capsular material concluded that capsule growth involved the intermixing of new and old capsular material with displacement of older capsular polysaccharide towards the surface. Here we have revisited that question using complement (C), which binds to capsular polysaccharide covalently, and cannot redistribute by dissociation and binding at different sites. The experimental approach involved binding of C to cells with small capsules, inducing capsule growth, and following the location of C relative to the cell wall as the capsule enlarged. C remained close to the cell wall during capsule growth, indicating that capsule enlargement occurred by addition of new polysaccharide near the capsule edge. This conclusion was confirmed by an independent method that employed radioactive metabolic labelling of newly synthesized capsule with 3H-mannose followed by gradual capsular stripping with gamma-radiation. Capsule growth proceeded to a certain size, which was a function of cell size, and was not degraded when the cells were transferred to a non-inducing medium. During budding, an opening appeared in the capsule of the mother cell that permitted the nascent bud to separate. Scanning EM suggested that a physical separation formed between the capsules of the mother and daughter cells during budding, which may avoid mixture between both capsules. Our results indicate that C. neoformans capsular enlargement also occurs by apical growth and that budding results in capsular rearrangements.     

Occurrence of phosphate-solubilizing bacteria in some Iraqi soils

The phosphate-solubilizing bacteria (PSB) were enumerated in 52 soil samples collected from agricultural areas at Baghdad. The results revealed that more than 90% of the samples were inhabited with indigenous PSB. The number varied and ranged from 0.012–28.4×10⁴ cell g^–1 soil. The correlations between PSB counts and electrical conductivity, available phosphorus, cation-exchange capacity, soil moisture, organic matter and pH were insignificant. Both abundance and numbers of PSB were more pronounced in descending order under vegetables, legumes, grasses, cereal and orchard trees.     

Supramolecular organization in streptococcal pericellular capsules is based on hyaluronan tertiary structures

Supramolecular organization involving a polyanionic glycan in a bacterial capsule (hyaluronan, HA, in streptococcal capsules) is revealed, for the first time, by electron-histochemical methodology previously used to elucidate ultrastructure in extracellular matrix. Capsular HA filaments thereby revealed closely resemble aligned linear structures demonstrated by similar technology in HA solution. These parallel arrays, spontaneously formed, are based on HA tertiary structures (beta sheet-like) which are stabilized by hydrophobic and hydrogen bonds. HA tertiary structures in aqueous solutions resist shear stress as shown by rheo-NMR. Thus, supramolecular HA wrapping covering many cells probably stabilizes chains of bacteria. Streptococci possibly templated the ordered structures since eukaryotic B6 cell HA did not produce similarly organized envelopes. Supramolecular organization in streptococcal and pneumococcal capsules are compared. Their glycans are very similar but the potential for HA-like tertiary structures is not present in the pneumococcal type 3 polysaccharide and chains of cells are not formed to the same extent by pneumococci. We suggest that the streptococcal capsule exemplifies a simple extracellular matrix analogous to those in animal connective tissues, which contain glycans (chondroitin, keratan, and dermochondan sulfates) of the HA family, capable of undergoing aggregation to similar tertiary structures.     

An improved method of microencapsulation and its evaluation to protect Lactobacillus spp. in simulated gastric conditions

An improved method of microencapsulation was developed to increase the efficacy of capsules in protecting the encapsulated bacteria under simulated gastric conditions. Lactobacillus acidophilus CSCC 2400 was encapsulated in calcium alginate and tested for its survival in simulated gastric conditions. The effects of different capsule sizes (200, 450, 1000 microm), different sodium alginate concentrations (0.75%, 1%, 1.5%, 1.8% and 2% w/v) and different concentrations of calcium chloride (0.1, 0.2, 1.0 M) on the viability of encapsulated bacteria were investigated. The viability of the cells in the microcapsules increased with an increase in alginate capsule size and gel concentration. There was no significant difference (p>0.05) in the viability of encapsulated cells when the concentration of calcium chloride was increased. Increase in cell load during encapsulation increased the number of bacterial survivors at the end of 3-h incubation in simulated gastric conditions. Hardening the capsule in calcium chloride solution for a longer time (8 h) had no impact on increasing the viability of encapsulated bacteria in a simulated gastric environment. The release of encapsulated cells at different phosphate buffer concentrations was also studied. When encapsulated L. acidophilus CSCC 2400 and L. acidophilus CSCC 2409 were subjected to low pH (pH 2) and high bile concentration (1.0% bile) under optimal encapsulation conditions (1.8% (w/v) alginate, 10(9) CFU/ml, 30 min hardening in 0.1 M CaCl(2) and capsule size 450 microm), there was a significant increase (p<0.05) in viable cell counts, compared to the free cells under similar conditions. Thus the encapsulation method described in this study may be effectively used to protect the lactobacillus from adverse gastric conditions.     

Mitochondrial toxicity detected in a health product with a boar spermatozoan bioassay

Seaweed and organic alfalfa capsules sold as "health promoting" products had repeatedly caused emesis in a consumer. Using the boar spermatozoan bioassay, the capsule contents were found to contain a toxic substance that inhibited boar sperm motility and depolarised mitochondria at low exposure concentrations of 10 microg/ml. The capsule also contained high amounts (10(5)-10(7) cfu/g), of endospore-forming bacteria and Streptomyces-like bacteria. Bacteria from the capsule produced toxic substances when cultured in the laboratory. Three different toxic responses were provoked in the spermatozoa exposed to extracts from the Streptomyces-like isolates: a) hyperpolarisation of the plasma membrane and depolarisation of the mitochondria; b) depolarisation of mitochondria similar to that caused by the capsule content extract; and c) motility inhibition, with no observed change of any cytosolic transmembrane potential. Membrane potential changes in the sperm cells exposed to the bacterial extracts were similar to those provoked by exposure to valinomycin and bafilomycin A1, to nigericin, and to oligomycin and ionomycin, respectively. Extracts prepared from Bacillus isolated from the capsule non-specifically depolarised all the cellular transmembrane potentials. The results demonstrate the potential value of a cell toxicity assay with boar spermatozoa for detecting hazardous substances in products intended for human consumption, without whole-animal exposure or using fetal calf serum for cell cultures.     

Calculations on O2 transfer in capsules with animal cells for the determination of maximum capsule size without O2 limitation

The size of the capsules without O2 limitation and maximum allowable capsule size for different cell densities were calculated by the observable modulus (modified Thiele modulus). When hybridoma (ATCC HB-8852) at 2.5 3 × 105cells/ ml was encapsulated and grown, the cell density reached to 2.6 × 107cells/ml in the 7th day of incubation. The average diameter of the capsule was 2.1mm. The maximum cell density obtained from experiment agreed with the calculated cell density for the given size of the capsules. © Rapid Science Ltd. 1998     

Preparation of artificial seeds using cell aggregates from horseradish hairy roots encapsulated in alginate gel with paraffin coat

Cell aggregates (CA) derived from horseradish hairy roots were used as inclusion materials for artificial seeds by entrapping them in alginate gel capsules. Regeneration of adventitious roots from the encapsulated CA was suppressed by covering the capsules with thin paraffin coats throughout storage of the capsules. The CA in coated capsules stored at 25°C up to 60 d retained root regeneration potential comparable to that of CA which were not subjected to storage. Plantlets were formed from the roots emerging from the CA in the capsules cultured under light condition.