Development of 3D hydrogel culture systems with on-demand cell separation

Recently there has been an increased interest in the effects of paracrine signaling between groups of cells, particularly in the context of better understanding how stem cells contribute to tissue repair. Most current 3D co-culture methods lack the ability to effectively separate two cell populations after the culture period, which is important for simultaneously analyzing the reciprocal effects of each cell type on the other. Here, we detail the development of a 3D hydrogel co-culture system that allows us to culture different cell types for up to 7 days and subsequently separate and isolate the different cell populations using enzyme-sensitive glues. Separable 3D co-culture laminates were prepared by laminating PEG-based hydrogels with enzyme-degradable hydrogel adhesives. Encapsulated cell populations exhibited good segregation with well-defined interfaces. Furthermore, constructs can be separated on-demand upon addition of the appropriate enzyme, while cell viability remains high throughout the culture period, even after laminate separation. This platform offers great potential for a variety of basic cell signaling studies as the incorporation of an enzyme-sensitive adhesive interface allows the on-demand separation of individual cell populations for immediate analysis or further culture to examine persistence of co-culture effects and paracrine signaling on cell populations. See accompanying commentary by Danielle R. Bogdanowicz and Helen H. Lu DOI: 10.1002/biot.201300054     

Preparation of Infectious Cell-Free Herpes-Type Virus Associated with Marek''s Disease

Cell-free transmission of the herpes-type virus of Marek's disease was obtained by demineralized water treatment of infected chick embryo fibroblasts.     

Lectinhistochemical study of cell surface alterations in mouse embryos exposed to low radiation doses

Summary The peroxidase-coupled Dolichos biflorus agglutinin (DBA) served as a marker for cell surface alterations in embryonic mouse tissues exposed to low-dose radiation during the early organogenesis (day 9 post conception). In unirradiated embryos, DBA bound selectively to various organ primordia, depending on their differentiation state. The auditory vesicles and the developing blood vessels were the only tissues staining strongly with the lectin. The vascular endothelia also showed the highest radiosensitivity, expressed by the maximal reaction already at 12.5 cGy. Marked surface changes as well were registered in the basal part of the Rathke's pocket and in the roof of the diencephalon. After exposure to 25 cGy, a transient amplification of the reaction as compared with 12.5 cGy occurred firstly in the Rathke's pocket, then in the infundibulum and in auditory vesicles. The most distinct effects were achieved with 50 cGy. Remarkable is the prompt rise of the DBA-affinity in a narrow area of the myelencephalon, and subsequently also in the roof of the diencephalon. Furthermore, the infundibulum and the Rathke's pocket, both anlagen of the pituitary gland, bound heavily DBA during the entire examination period of 24 h. The present study demonstrated the oustanding suitability of theDolichos biflorus agglutinin for histochemical detection of subtle cellular alterations by small radiation doses.     

Purification and Host Specificity of Predatory Halobacteriovorax Isolates from Seawater

Halobacteriovorax (formerly Bacteriovorax) is a small predatory bacterium found in the marine environment and modulates bacterial pathogens in shellfish. Four strains of Halobacteriovorax originally isolated in Vibrio parahaemolyticus O3:K6 host cells were separated from their prey by an enrichment-filtration-dilution technique for specificity testing in other bacteria. This technique was essential, since 0.45-μm filtration alone was unable to remove infectious Vibrio minicells, as determined by scanning electron microscopy and cultural methods. Purified Halobacteriovorax strains were screened for predation against other V. parahaemolyticus strains and against Vibrio vulnificus, Vibrio alginolyticus, Escherichia coli O157:H7, and Salmonella enterica serovar Typhimurium DT104, all potential threats to seafood safety. They showed high host specificity and were predatory only against strains of V. parahaemolyticus. In addition, strains of Halobacteriovorax that were predatory for E. coli O157:H7 and S. Typhimurium DT104 were isolated from a tidal river at 5 ppt salinity. In a modified plaque assay agar, they killed their respective prey over a broad range of salinities (5 to 30 ppt). Plaques became smaller as the salinity levels rose, suggesting that the lower salinities were optimal for the predators'' replication. These species also showed broader host specificity, infectious against each other''s original hosts as well as against V. parahaemolyticus strains. In summary, this study characterized strains of Halobacteriovorax which may be considered for use in the development of broad-based biocontrol technologies to enhance the safety of commercially marketed shellfish and other foods.     

Psb27, a photosystem II assembly protein,enables quenching of excess light energy during its participation in the PSII lifecycle

Photosynthesis Research - Photosystem II (PSII), the enzyme responsible for oxidizing water into molecular oxygen, undergoes a complex lifecycle during which multiple assembly proteins transiently...     

A simple radioassay for dihydrofolate synthetase activity in Escherichia coli and its application to an inhibition study of new pteroate analogs

A simple radioactive assay system is elaborated for the measurement of dihyrofolate synthetase activity in Escherichia coli. It is also applicable to Neisseria gonorrhoeae and N. meningitidis extracts. Eight oxidized and reduced pteroate analogs have been examined for inhibitory activity. The most active inhibitor was dihydrohomopteroic acid followed by dihydro-10-thiopteroic acid, dihydrofolic acid, and dihydroisopteroic acid. The enzyme appears to be incapable of binding with substrate and any of the inhibitors in their oxidized forms.