Isolation of retinal progenitor and stem cells from the porcine eye

Purpose: Retinal progenitor cells (RPCs) and retinal stem cells (RSCs) from rodents and humans have been isolated and characterized in vitro. Transplantation experiments have confirmed their potential as tools for cell replacement in retinal degenerative diseases. The pig represents an ideal pre-clinical animal model to study the impact of transplantation because of the similarity of its eye to the human eye. However, little is known about porcine RPCs and RSCs. We aimed to identify and characterize in vitro RPCs and RSCs from porcine ocular tissues. Methods: Cells from different subregions of embryonic, postnatal and adult porcine eyes were grown in suspension sphere culture in serum-free medium containing basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Growth curves and BrdU incorporation assays were performed to establish the proliferative capacity of isolated porcine retina-derived RPCs and ciliary epithelium (CE)-derived RSCs. Self-renewal potential was investigated by subsphere formation assays. Changes in gene expression were assayed by reverse transcription polymerase chain reaction (RT-PCR) at different passages in culture. Finally, differentiation was induced by addition of serum to the cultures and expression of markers for retinal cell types was detected by immunohistochemical staining with specific antibodies. Results: Dissociated cells from embryonic retina and CE at different postnatal ages generated primary nestin- and Pax6-immunoreactive neurosphere colonies in vitro in numbers that decreased with age. Embryonic and postnatal retina-derived RPCs and young CE-derived RSCs displayed self-renewal capacity, generating secondary neurosphere colonies. However, their self-renewal and proliferation capacity gradually decreased and they became more committed to differentiated states with subsequent passages. The expansion capacity of RPCs and RSCs was higher when they were maintained in monolayer culture. Porcine RPCs and RSCs could be induced to differentiate in vitro to express markers of retinal neurons and glia. Conclusions: Porcine retina and CE contain RPCs and RSCs which are undifferentiated, self-renewing and multipotent and which show characteristics similar to their human counterparts. Therefore, the pig could be a useful source of cells to further investigate the cell biology of RPCs and RSCs and it could be used as a non-primate large animal model for pre-clinical studies on stem cell-based approaches to regenerative medicine in the retina.

The antimicrobial effects of root canal irrigation and medication

The role of microorganisms in the development and maintenance of pulpal and periapical inflammation have been well documented. The success of root canal treatment largely depends on the elimination of microbial contamination from the root canal system. Although mechanical instrumentation of root canals can reduce bacterial population, effective elimination of bacteria cannot be achieved without the use of antimicrobial root canal irrigation and medication. This review will discuss the antimicrobial effects of the known root canal irrigants and medicaments and explore future developments in the field. © 2007 Mosby, Inc. All rights reserved.

A simplified protocol for routine total DNA isolation from salmonid fishes

An efficient total DNA isolation protocol, suitable for routine population genetic screening purposes is described. This phenol based extraction can utilize fresh, frozen or ethanol preserved tissues.

Multivariate prediction of clarified butter composition using Raman spectroscopy

Raman spectroscopy has been used to predict the abundance of the FA in clarified butterfat that was obtained from dairy cows fed a range of levels of rapeseed oil in their diet. Partial least squares regression of the Raman spectra against FA compositions obtained by GC showed good prediction for the five major (abundance >5%) FA with R-2=0.74-0.92 and a root mean SE of prediction (RMSEP) that was 5-7% of the mean. In general, the prediction accuracy fell with decreasing abundance in the sample, but the RMSEP was 1.25%. The Raman method has the best prediction ability for unsaturated FA (R-2=0.85-0.92), and in particular trans unsaturated FA (best-predicted FA was 18:1 tDelta9). This enhancement was attributed to the isolation of the unsaturated modes from the saturated modes and the significantly higher spectral response of unsaturated bonds compared with saturated bonds. Raman spectra of the melted butter samples could also be used to predict bulk parameters calculated from standard analyzes, such as iodine value (R-2=0.80) and solid fat content at low temperature (R-2=0.87). For solid fat contents determined at higher temperatures, the prediction ability was significantly reduced (R-2=0.42), and this decrease in performance was attributed to the smaller range of values in solid fat content at the higher temperatures. Finally, although the prediction errors for the abundances of each of the FA in a given sample are much larger with Raman than with full GC analysis, the accuracy is acceptably high for quality control applications. This, combined with the fact that Raman spectra can be obtained with no sample preparation and with 60-s data collection times, means that high-throughput, on-line Raman analysis of butter samples should be possible.

Isolation and preliminary biological assessment of AADGAPLIRFamide and SVPGVLRFamide from Caenorhabditis elegans

To date, 9 FMRFamide-related peptides (FaRPs) have been structurally characterised from Caenorhabditis elegans. Radioimmunometrical screening of an ethanolic extract of C. elegans revealed the presence of two additional FaRPs that were purified by reverse-phase HPLC and subjected to Edman degradation analysis and gas-phase sequencing. Unequivocal primary structures for the two FaRPs were determined as Ala-Ala-Asp-Gly-Ala-Pro-Leu-Ile-Arg-Phe-NH2 and Ser-Val-Pro-Gly-Val-Leu-Arg-Phe-NH2. Using MALDI-TOF mass. spectrometry, the molecular masses of the peptides were found to be 1032 Da (MH) and 875 Da (MH)(+), respectively. Two copies of AADGAPLIRFamide are predicted to be encoded on the precursor gene termed flp-13, while one copy of SVPGVLRFamide is located on flp-18. Synthetic replicates of the peptides were tested on Ascaris suum somatic muscle to assess bioactivity. ADDGAPLIRFamide had inhibitory effects on A. suum muscle strips, which occurred over a range of concentrations from a threshold for activity of 10 nM to 10 muM. SVPGVLRFamide was excitatory on A. suum somatic musculature from a threshold concentration for activity of 1 nM to 10 muM. The inhibitory and excitatory effects of AADGAPLIRFamide and SVPGVLRFamide, respectively, were the same for dorsal and ventral muscle strips as well as innervated and denervated preparations, suggesting that these physiological effects are not nerve cord dependent. Addition of ADDGAPLIRFamide (10 muM) to muscle strips preincubated in high-K+ and -Ca2+-free medium resulted in a normal inhibitory response. Peptide addition to muscle strips preincubated in Cl--free medium showed no inhibitory response, suggesting that the inhibitory response of the peptide may be chloride mediated. A normal excitatory response was noted following the addition of 10 muM SVPGVLRFamide to muscle strips preincubated in high-K+, Ca2+- and Cl--free media. (C) 2001 Academic Press.