Effect of mesenchymal stromal cells encapsulated within polyethylene glycol-collagen hydrogels formed in situ on alkali-burned corneas in an ex vivo organ culture model

Background aimsCorneal inflammation after alkali burns often results in vision loss due to corneal opacification and neovascularization. Mesenchymal stem cells (MSCs) and their secreted factors (secretome) have been studied for their anti-inflammatory and anti-angiogenic properties with encouraging results. However, topical instillation of MSCs or their secretome is often accompanied by issues related to delivery or rapid washout. Polyethylene glycol (PEG) and collagen are well-known biomaterials used extensively in scaffolds for tissue engineering. To effectively suppress alkaline burn-induced corneal injury, the authors proposed encapsulating MSCs within collagen gels cross-linked with multi-functional PEG-succinimidyl esters as a means to deliver the secretome of immobilized MSCs.MethodsHuman MSCs were added to a neutralized collagen solution and mixed with a solution of four-arm PEG-N-hydroxysuccinimide. An ex vivo organ culture was conducted using rabbit corneas injured by alkali burn. MSCs were encapsulated within PEG-collagen hydrogels and injected onto the wounded cornea immediately following alkali burn and washing. Photographs of the ocular surface were taken over a period of 7 days after the alkali burn and processed for immunohistochemical evaluation. Samples were split into three groups: injury without treatment, MSCs alone, and MSCs encapsulated within PEG-collagen hydrogels.ResultsAll corneas in ex vivo organ culture lost their transparency immediately after alkali burn, and only the groups treated with MSCs and MSCs encapsulated within PEG-collagen hydrogels recovered some transparency after 7 days. Immunohistochemical analysis revealed increased expression of vimentin in the anterior corneal stroma of the group without treatment indicative of fibrotic healing, whereas less stromal vimentin was detected in the group containing MSCs encapsulated within the PEG-collagen hydrogels.ConclusionsPEG-collagen hydrogels enable the encapsulation of viable MSCs capable of releasing secreted factors onto the ocular surface. Encapsulating MSCs within PEG-collagen hydrogels may be a promising method for delivering their therapeutic benefits in cases of ocular inflammatory diseases, such as alkali burn injuries.     

The Mechanism of Amino Acid Efflux from Seed Coats of Developing Pea Seeds as Revealed by Uptake Experiments

The uptake of amino acids by excised seed coat halves of developing seeds of pea (Pisum sativum L.) was characterized. The influx of L-valine and L-glutamic acid was proportional to their external concentration, with coefficients of proportionality (k) of 11.0 and 7.1 [mu]mol g-1 fresh weight min-1 M-1, respectively. The influx of L-lysine could be analyzed into a component with linear kinetics (k = 8.1 [mu]mol g-1 fresh weight min-1 M-1) and one with saturation kinetics (Michaelis constant = 6.5 mM), but the latter may have resulted from the mutual interaction between the influx of the cationic lysine and the membrane potential. The influx of the amino acids was not affected by 10 [mu]M carbonylcyanide m-chlorophenylhydrazone, but was inhibited by about 50% in the presence of 2.5 mM p-chloromercuribenzene sulfonic acid. Conservative estimates of the permeability coefficients of the plasma membrane of seed coat parenchyma cells for lysine, glutamic acid, and several neutral amino acids were all in the range of 4 x 10-7 cm s-1 to 9 x 10-7 cm s-1, which is 4 to 5 orders of magnitude greater than those reported for artificial lipid bilayers. It is concluded that nonselective pores constitute a pathway in the plasma membrane for passive transport of amino acids. It is argued that this pathway is also used for the efflux of endogenous amino acids, the process by which nitrogen becomes available for the embryo.     

Presence of hybridizing DNA sequences homologous to bovine acidic and basicβ-crystallins in all classes of vertebrates

Summary The eye lens-crystallins in cow and chicken are encoded by a family of at least six genes. In order to assess the distribution of the corresponding genes among other vertebrates we hybridized -crystallin sequences (A2, A3/A1, A4, B1, B2, B3), isolated from a bovine lens cDNA library, to Southern blots on whichEcoR1-digested chromosomal DNA was blotted from different vertebrate species. These included human, chimpanzee, calf, rat, pigeon, duck, monitor lizard, toad, trout, and lamprey. Positive hybridization signals were found in the representatives of virtually all classes of vertebrates. The basic B-crystallins gave hybridization signals in more species than the acidic A ones. In monitor lizard and toad the weakest hybridization signals for basic crystallin probes were found. For acidic crystallin probes the distribution pattern was more simple; among cold-blooded vertebrates a signal for A2 was found in trout and lamprey, for A4 in trout, and for A3/A1 only in toad. The results demonstrate that the duplications leading to the -crystallin gene family occurred before or during the earliest stages of vertebrate evolution.     

Metabolic engineering of Mannheimia succiniciproducens for malic acid production using dimethylsulfoxide as an electron acceptor

Microbial production of various TCA intermediates and related chemicals through the reductive TCA cycle has been of great interest. However, rumen bacteria that naturally possess strong reductive TCA cycle have been rarely studied to produce these chemicals, except for succinic acid, due to their dependence on fumarate reduction to transport electrons for ATP synthesis. In this study, malic acid (MA), a dicarboxylic acid of industrial importance, was selected as a target chemical for mass production using Mannheimia succiniciproducens, a rumen bacterium possessing a strong reductive branch of the TCA cycle. The metabolic pathway was reconstructed by eliminating fumarase to prevent MA conversion to fumarate. The respiration system of M. succiniciproducens was reconstructed by introducing the Actinobacillus succinogenes dimethylsulfoxide (DMSO) reductase to improve cell growth using DMSO as an electron acceptor. Also, the cell membrane was engineered by employing Pseudomonas aeruginosa cis-trans isomerase to enhance MA tolerance. High inoculum fed-batch fermentation of the final engineered strain produced 61 g/L of MA with an overall productivity of 2.27 g/L/h, which is the highest MA productivity reported to date. The systems metabolic engineering strategies reported in this study will be useful for developing anaerobic bioprocesses for the production of various industrially important chemicals.     

A Stable Thoracic Hox Code and Epimorphosis Characterize Posterior Regeneration in Capitella teleta

Regeneration, the ability to replace lost tissues and body parts following traumatic injury, occurs widely throughout the animal tree of life. Regeneration occurs either by remodeling of pre-existing tissues, through addition of new cells by cell division, or a combination of both. We describe a staging system for posterior regeneration in the annelid, Capitella teleta, and use the C. teleta Hox gene code as markers of regional identity for regenerating tissue along the anterior-posterior axis. Following amputation of different posterior regions of the animal, a blastema forms and by two days, proliferating cells are detected by EdU incorporation, demonstrating that epimorphosis occurs during posterior regeneration of C. teleta. Neurites rapidly extend into the blastema, and gradually become organized into discrete nerves before new ganglia appear approximately seven days after amputation. In situ hybridization shows that seven of the ten Hox genes examined are expressed in the blastema, suggesting roles in patterning the newly forming tissue, although neither spatial nor temporal co-linearity was detected. We hypothesized that following amputation, Hox gene expression in pre-existing segments would be re-organized to scale, and the remaining fragment would express the complete suite of Hox genes. Surprisingly, most Hox genes display stable expression patterns in the ganglia of pre-existing tissue following amputation at multiple axial positions, indicating general stability of segmental identity. However, the three Hox genes, CapI-lox4, CapI-lox2 and CapI-Post2, each shift its anterior expression boundary by one segment, and each shift includes a subset of cells in the ganglia. This expression shift depends upon the axial position of the amputation. In C. teleta, thoracic segments exhibit stable positional identity with limited morphallaxis, in contrast with the extensive body remodeling that occurs during regeneration of some other annelids, planarians and acoel flatworms.     

The UreEF fusion protein provides a soluble and functional form of the UreF urease accessory protein

Four accessory proteins (UreD, UreE, UreF, and UreG) are typically required to form the nickel-containing active site in the urease apoprotein (UreABC). Among the accessory proteins, UreD and UreF have been elusive targets for biochemical and structural characterization because they are not overproduced as soluble proteins. Using the best-studied urease system, in which the Klebsiella aerogenes genes are expressed in Escherichia coli, a translational fusion of ureE and ureF was generated. The UreEF fusion protein was overproduced as a soluble protein with a convenient tag involving the His-rich region of UreE. The fusion protein was able to form a UreD(EF)G-UreABC complex and to activate urease in vivo, and it interacted with UreD-UreABC in vitro to form a UreD(EF)-UreABC complex. While the UreF portion of UreEF is fully functional, the fusion significantly affected the role of the UreE portion by interrupting its dimerization and altering its metal binding properties compared to those of the wild-type UreE. Analysis of a series of UreEF deletion mutants revealed that the C terminus of UreF is required to form the UreD(EF)G-UreABC complex, while the N terminus of UreF is essential for activation of urease.