Augmented Dried versus Cryopreserved Amniotic Membrane as an Ocular Surface Dressing

Purpose

Dried amniotic membrane (AM) can be a useful therapeutic adjunct in ophthalmic surgery and possesses logistical advantages over cryopreserved AM. Differences in preservation techniques can significantly influence the biochemical composition and physical properties of AM, potentially affecting clinical efficacy. This study was established to investigate the biochemical and structural effects of drying AM in the absence and presence of saccharide lyoprotectants and its biocompatibility compared to cryopreserved material.

Methods

AM was cryopreserved or dried with and without pre-treatment with trehalose or raffinose and the antioxidant epigallocatechin (EGCG). Structural and visual comparisons were assessed using electron microscopy. Localisation, expression and release of AM biological factors were determined using immunoassays and immunofluorescence. The biocompatibility of the AM preparations co-cultured with corneal epithelial cell (CEC) or keratocyte monolayers were assessed using cell proliferation, cytotoxicity, apoptosis and migration assays.

Results

Drying devitalised AM epithelium, but less than cryopreservation and cellular damage was reduced in dried AM pre-treated with trehalose or raffinose. Dried AM alone, and with trehalose or raffinose showed greater factor retention efficiencies and bioavailability compared to cryopreserved AM and demonstrated a more sustained biochemical factor time release in vitro. Cellular health assays showed that dried AM with trehalose or raffinose are compatible and superior substrates compared to cryopreserved AM for primary CEC expansion, with increased proliferation and reduced LDH and caspase-3 levels. This concept was supported by improved wound healing in an immortalised human CEC line (hiCEC) co-cultured with dried and trehalose or raffinose membranes, compared to cryopreserved and fresh AM.

Conclusions

Our modified preservation process and our resultant optimised dried AM has enhanced structural properties and biochemical stability and is a superior substrate to conventional cryopreserved AM. In addition this product is stable and easily transportable allowing it to be globally wide reaching for use in clinical and military sectors.     

The Snakelike Chain Character of Unstructured RNA

In the absence of base-pairing and tertiary structure, ribonucleic acid (RNA) assumes a random-walk conformation, modulated by the electrostatic self-repulsion of the charged, flexible backbone. This behavior is often modeled as a Kratky-Porod “wormlike chain” (WLC) with a Barrat-Joanny scale-dependent persistence length. In this study we report measurements of the end-to-end extension of poly(U) RNA under 0.1 to 10 pN applied force and observe two distinct elastic-response regimes: a low-force, power-law regime characteristic of a chain of swollen blobs on long length scales and a high-force, salt-valence-dependent regime consistent with ion-stabilized crumpling on short length scales. This short-scale structure is additionally supported by force- and salt-dependent quantification of the RNA ion atmosphere composition, which shows that ions are liberated under stretching; the number of ions liberated increases with increasing bulk salt concentration. Both this result and the observation of two elastic-response regimes directly contradict the WLC model, which predicts a single elastic regime across all forces and, when accounting for scale-dependent persistence length, the opposite trend in ion release with salt concentration. We conclude that RNA is better described as a “snakelike chain,” characterized by smooth bending on long length scales and ion-stabilized crumpling on short length scales. In monovalent salt, these two regimes are separated by a characteristic length that scales with the Debye screening length, highlighting the determining importance of electrostatics in RNA conformation.     

Liver-type fatty acid binding protein interacts with hepatocyte nuclear factor 4α

Hepatocyte nuclear factor 4α (HNF4α) regulates liver type fatty acid binding protein (L-FABP) gene expression. Conversely as shown herein, L-FABP structurally and functionally also interacts with HNF4α. Fluorescence resonance energy transfer (FRET) between Cy3-HNF4α (donor) and Cy5-L-FABP (acceptor) as well as FRET microscopy detected L-FABP in close proximity (∼80 Å) to HNF4α, binding with high affinity K_d ∼250–300 nM. Circular dichroism (CD) determined that the HNF4α/L-FABP interaction altered protein secondary structure. Finally, L-FABP potentiated transactivation of HNF4α in COS7 cells. Taken together, these data suggest that L-FABP provides a signaling path to HNF4α activation in the nucleus.     

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors.The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.     

Breakdown of low-level total petroleum hydrocarbons (TPH) in contaminated soil using grasses and willows

A phytoremediation study targeting low-level total petroleum hydrocarbons (TPH) was conducted using cool- and warm-season grasses and willows (Salix species) grown in pots filled with contaminated sandy soil from the New Haven Rail Yard, CT. Efficiencies of the TPH degradation were assessed in a 90-day experiment using 20–8.7–16.6 N-P-K water-soluble fertilizer and fertilizer with molasses amendments to enhance phytoremediation. Plant biomass, TPH concentrations, and indigenous microbes quantified with colony-forming units (CFU), were assessed at the end of the study. Switchgrass grown with soil amendments produced the highest aboveground biomass. Bacterial CFU's were in orders of magnitude significantly higher in willows with soil amendments compared to vegetated treatments with no amendments. The greatest reduction in TPH occurred in all vegetated treatments with fertilizer (66–75%) and fertilizer/molasses (65–74%), followed sequentially by vegetated treatments without amendments, unvegetated treatments with amendments, and unvegetated treatments with no amendment. Phytoremediation of low-level TPH contamination was most efficient where fertilization was in combination with plant species. The same level of remediation was achievable through the addition of grasses and/or willow combinations without amendment, or by fertilization of sandy soil.     

Direct demonstration of the flexibility of the glycosylated proline-threonine linker in the Cellulomonas fimi Xylanase Cex through NMR spectroscopic analysis

The modular xylanase Cex (or CfXyn10A) from Cellulomonas fimi consists of an N-terminal catalytic domain and a C-terminal cellulose-binding domain, joined by a glycosylated proline-threonine (PT) linker. To characterize the conformation and dynamics of the Cex linker and the consequences of its modification, we have used NMR spectroscopy to study full-length Cex in its nonglycosylated ( approximately 47 kDa) and glycosylated ( approximately 51 kDa) forms. The PT linker lacks any predominant structure in either form as indicated by random coil amide chemical shifts. Furthermore, heteronuclear (1)H-(15)N nuclear Overhauser effect relaxation measurements demonstrate that the linker is flexible on the ns-to-ps time scale and that glycosylation partially dampens this flexibility. The catalytic and cellulose-binding domains also exhibit identical amide chemical shifts whether in isolation or in the context of either unmodified or glycosylated full-length Cex. Therefore, there are no noncovalent interactions between the two domains of Cex or between either domain and the linker. This conclusion is supported by the distinct (15)N relaxation properties of the two domains, as well as their differential alignment within a magnetic field by Pf1 phage particles. These data demonstrate that the PT linker is a flexible tether, joining the structurally independent catalytic and cellulose-binding domains of Cex in an ensemble of conformations; however, more extended forms may predominate because of restrictions imparted by the alternating proline residues. This supports the postulate that the binding-domain anchors Cex to the surface of cellulose, whereas the linker provides flexibility for the catalytic domain to hydrolyze nearby hemicellulose (xylan) chains.     

Spontaneous entrapment of polynucleotides upon electrostatic interaction with ethanol-destabilized cationic liposomes

This study describes the effect of ethanol and the presence of poly(ethylene) glycol (PEG) lipids on the interaction of nucleotide-based polyelectrolytes with cationic liposomes. It is shown that preformed large unilamellar vesicles (LUVs) containing a cationic lipid and a PEG coating can be induced to entrap polynucleotides such as antisense oligonucleotides and plasmid DNA in the presence of ethanol. The interaction of the cationic liposomes with the polynucleotides leads to the formation of multilamellar liposomes ranging in size from 70 to 120 nm, only slightly bigger than the parent LUVs from which they originated. The degree of lamellarity as well as the size and polydispersity of the liposomes formed increases with increasing polynucleotide-to-lipid ratio. A direct correlation between the entrapment efficiency and the membrane-destabilizing effect of ethanol was observed. Although the morphology of the liposomes is still preserved at the ethanol concentrations used for entrapment (25-40%, v/v), entrapped low-molecular-weight solutes leak rapidly. In addition, lipids can flip-flop across the membrane and exchange rapidly between liposomes. Furthermore, there are indications that the interaction of the polynucleotides with the cationic liposomes in ethanol leads to formation of polynucleotide-cationic lipid domains, which act as adhesion points between liposomes. It is suggested that the spreading of this contact area leads to expulsion of PEG-ceramide and triggers processes that result in the formation of multilamellar systems with internalized polynucleotides. The high entrapment efficiencies achieved at high polyelectrolyte-to-lipid ratios and the small size and neutral character of these novel liposomal systems are of utility for liposomal delivery of macromolecular drugs.     

Thapsigargin and dimethyl sulfoxide activate medium P(i)<-->HOH oxygen exchange catalyzed by sarcoplasmic reticulum Ca2+-ATPase

Thapsigargin is a potent inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase. It binds the Ca(2+)-free E2 conformation in the picomolar range, supposedly resulting in a largely catalytically inactive species. We now find that thapsigargin has little effect on medium P(i) <--> HOH oxygen exchange and that this activity is greatly stimulated (up to 30-fold) in the presence of 30% (v/v) Me(2)SO. Assuming a simple two-step mechanism, we have evaluated the effect of thapsigargin and Me(2)SO on the four rate constants governing the reaction of P(i) with Ca(2+)-ATPase. The principal effect of thapsigargin alone is to stimulate EP hydrolysis (k(-2)), whereas that of Me(2)SO is to greatly retard P(i) dissociation (k(-1)), accounting for its well known effect on increasing the apparent affinity for P(i). These effects persist when the agents are used in combination and substantially account for the activated oxygen exchange (v(exchange) = k(-2)[EP]). Kinetic simulations show that the overall rate constant for the formation of EP is very fast (approximately 300 s(-1)) when the exchange is maximal. Thapsigargin greatly stabilizes Ca(2+)-ATPase against denaturation in detergent in the absence of Ca(2+), as revealed by glutaraldehyde cross-linking, suggesting that the membrane helices lock together. It seems that the reactions at the phosphorylation site, associated with the activated exchange reaction, are occurring without much movement of the transport site helices, and we suggest that they may be associated solely with an occluded H+ state.