Niche regulation of corneal epithelial stem cells at the limbus

Among all adult somatic stem cells,those of the corneal epithelium are unique in their exclusive location in a definedlimbai structure termed Palisades of Vogt.As a result,surgical engraftment oflimbal epithelial stem cells with or withoutex vivo expansion has long been practiced to restore sights in patients inflicted with limbal stem cell deficiency.Neverthe-less,compared to other stem cell examples,relatively little is known about the limbal niche,which is believed to play apivotal role in regulating self-renewal and fate decision of limbal epithelial stem cells.This review summarizes relevantliterature and formulates several key questions to guide future research into better understanding of the pathogenesis oflimbal stem cell deficiency and further improvement of the tissue engineering of the corneal epithelium by focusing onthe limbal niche.     

Using disaccharides to enhance in vitro and in vivo transgene expression mediated by a lipid-based gene delivery system

BACKGROUND: Lipid-based vectors have been widely applied to in vivo and in vitro gene delivery. Disaccharides can effectively stabilize lipid membranes. This study examined whether disaccharides could enhance the transgene expression mediated by lipid-based vectors. METHODS: Different disaccharides were incorporated into the vectors prepared with DOTAP/protamine/DNA (LPD) or with DNA/cationic liposomes containing DOTAP, DOTAP/Chol, DOTAP/DOPE, or DC-Chol/DOPE. The levels of transgene expression and internalized plasmid of CHO cells were represented by the percentages of GFP-positive cells and the fluorescence intensity of ethidium-monoazide covalently labeled plasmid, respectively. The vectors containing either cellobiose or trehalose were also intravenously injected into mouse tail vein to investigate the potentials of in vivo applications. RESULTS: For enhancing the transgene expression, cellobiose was found to be effective for all the vectors whereas maltose decreased the effectiveness of DOTAP/Chol liposomes and LPD. For the internalization of plasmid, most disaccharides were able to increase the cellular delivery of DOTAP, DOTAP/Chol, and DOTAP/DOPE liposomes, but caused decreases in the cellular entry of DC-Chol/DOPE liposomes. An approximately linear correlation between the internalized plasmid and the transgene expression was observed for all the treatments in this study. When the vectors were administered to mouse by intravenous injection, 10-fold and 3-fold increases in the luciferase expression of lung were observed for DOTAP liposomes containing 330 mM cellobiose and trehalose, respectively. CONCLUSIONS: This study showed that using trehalose and cellobiose with a lipid-based delivery system provides a straightforward approach to effectively enhance both in vitro and in vivo transgene expression.     

Development of poly(amino ester glycol urethane)/siRNA polyplexes for gene silencing

Nonviral vectors, with their low immunogenicity and lack of pathogenicity, offer significant promise for siRNA therapy with fewer safety concerns. Nonviral vectors were also preferred in most transient siRNA delivery due to their ease of preparation. Previously, we incorporated tertiary amines and polyethylene glycol (PEG) into poly(ester urethane) to synthesize a soluble poly(amino ester glycol urethane), PaE(G)U, as a novel DNA transfection reagent for transgene delivery. The aim of this study was to develop PaE(G)U/siRNA polyplexes for gene silencing. We characterized the properties of PaE(G)U/siRNA polyplexes and compared them with those of PaE(G)U/DNA polyplexes. Using the Alexa Fluor 488-labeled, nonsilencing control siRNA as the reporter, we visualized cellular uptake of PaE(G)U/siRNA polyplexes and optimized the mass ratio of PaE(G)U/siRNA for delivery at 80/1. At this ratio, the average diameter of polyplexes was 540 nm, which was significantly larger than the average diameter of PaE(G)U/DNA polyplexes at 155 nm for efficient DNA delivery. Using the optimized PaE(G)U/siRNA polyplexes, transient silencing of constitutive luciferase expression (up to 92%) was achieved in our recombinant human HT-1080 fibroblast model via anti-luciferase siRNA delivery. In conclusion, PaE(G)U/siRNA polyplexes were developed and optimized for cellular uptake to allow efficient gene silencing. Engineering of soluble biodegradable polymers to incorporate amino, ester, PEG, and urethane units in the backbone constitutes a useful approach for the future design of siRNA carriers.     

Stable chloroplast transformation in cabbage (<Emphasis Type="Italic">Brassica oleracea </Emphasis>L. var. <Emphasis Type="Italic">capitata </Emphasis>L.) by particle bombardment

The objectives of this research were first to isolate plastid gene sequences from cabbage (Brassica oleracea L. var. capitata L.), and to establish the chloroplast transformation technology of Brassica. A universal transformation vector (pASCC201) for Brassica chloroplast was constructed with trnV–rrn16S (left) and trnI–trnA–rrn23S (right) of the IR_A region as a recombination site for the transformed gene. In transforming plasmid pASCC201, a chimeric aadA gene was cloned between the rrn16S and rrn23S plastid gene borders. Expression of aadA confers resistance to spectinomycin and streptomycin antibiotics. The uidA gene was also inserted into the pASCC201 and transferred into the leaf cells of cabbage via particle gun mediated transformation. Regenerated plantlets were selected by 200 mg/l spectinomycin and streptomycin. After antibiotic selection, the regeneration percentage of the two cabbage cultivars was about 2.7–3.3%. The results of PCR testing and Southern blot analysis confirmed that the uidA and aadA genes were present in the chloroplast genome via homologously recombined. Northern blot hybridizations, immunoblotting and GUS histochemical assays indicated that the uidA gene were stable integrated into the chloroplast genome. Foreign protein was accumulated at 3.2–5.2% of the total soluble protein in transgenic mature leaves. These results suggest that the expression of a variety of foreign genes in the chloroplast genome will be a powerful tool for use in future studies.     

Evaluation of the role of Disabled-2 in nerve growth factor-mediated neurite outgrowth and cellular signalling

Disabled-2 (DAB2) is an adapter protein that plays a key role in cell proliferation and differentiation. We reported here that DAB2 is expressed in various regions of rat central nervous system and is most abundant in the olfactory bulb. The up-regulation of DAB2 upon 5,7-dihydroxytryptamine-induced spinal cord lesion implicates that DAB2 may participate in the regulation of neuronal plasticity. To investigate DAB2 function in the regulation of neurite outgrowth, the rat p59 and p82 form of DAB2 was individually and stably expressed in the PC12 cells. Both p59 and p82 inhibited nerve growth factor (NGF)-induced neurite outgrowth concomitantly with a decrease in the expression of neuron-specific cytoskeleton protein beta-tubulin III. To unveil the molecular mechanism of DAB2 in NGF signaling, we found that RhoA-GTPase activity was up-regulated in DAB2 stable lines whereas the Ras/MAPK and PI3-kinase/Akt signaling was not affected. The inhibitory effect of DAB2 on NGF-mediated neurite outgrowth was reversed by the pretreatment of Rho-kinase (ROCK) inhibitor Y27632, implicating that DAB2 modulates RhoA/ROCK signaling. Together, this study defines a role of DAB2 in the control of neuronal plasticity and demonstrates for the first time that DAB2 is a negative regulator in NGF-mediated neurite outgrowth.     

Degradation of polyethylene succinate (PES) by a new thermophilic <Emphasis Type="Italic">Microbispora</Emphasis> strain

Thermophilic actinomycetes were isolated from sediment of the Chingshuei hot spring in north Taiwan, and the strain HS 45-1 was selected from colonies which formed distinct clear zones on agar plate with emulsified polyethylene succinate (PES). The film of PES disappeared within 6 days in liquid cultures at 50°C. The strain HS 45-1 was also able to degrade poly (ε-carpolactone) (PCL) and poly (3-hydroxybutyrate) (PHB) films completely within 6 days in liquid cultures. Basing on the results of phynotypic characteristics, phylogenetic studies and DNA-DNA hybridization, strain HS 45-1 should be assigned to Micorbispora rosea subsp. taiwanensis.     

Identification of a hypothetical protein of plant pathogenic Xanthomonas campestris as a novel beta-galactosidase

Xc17L, a lactose-utilizing mutant of Xanthomonas campestris pv. campestris previously isolated by mutagenesis with nitrous acid, displays a level of beta-galactosidase 3.5-fold higher than that in the parental Xc17. In this study, the gene encoding the enzyme displaying a higher specific activity in Xc17L was inactivated by mini-Tn5 transposition.Sequencing revealed that the product (579 aa, 63.5 kDa) of this gene, designated galD, was previously annotated to encode a hypothetical protein on the genome. Mutation of the gene by marker exchange, complementation test and Western blot analysis together confirmed that galD is indeed the gene involved in beta-galactosidase elevation in Xc17L. With only the N-terminal region possessing similarity to the known beta-galactosidases and partially conserved consensus motif, GalD is recognized as a member of the glycosyl hydrolase family 35. Insertion with GmOmega, which causes polar effects, into the upstream genes followed by Western blotting showed that galD is cotranscribed with the upstream genes and expressed constitutively. Mutation in galD causes no significant changes including pathogenicity in the bacterium.     

Prokaryotic assemblages and metagenomes in pelagic zones of the South China Sea

Background

Prokaryotic microbes, the most abundant organisms in the ocean, are remarkably diverse. Despite numerous studies of marine prokaryotes, the zonation of their communities in pelagic zones has been poorly delineated. By exploiting the persistent stratification of the South China Sea (SCS), we performed a 2-year, large spatial scale (10, 100, 1000, and 3000 m) survey, which included a pilot study in 2006 and comprehensive sampling in 2007, to investigate the biological zonation of bacteria and archaea using 16S rRNA tag and shotgun metagenome sequencing.

Results

Alphaproteobacteria dominated the bacterial community in the surface SCS, where the abundance of Betaproteobacteria was seemingly associated with climatic activity. Gammaproteobacteria thrived in the deep SCS, where a noticeable amount of Cyanobacteria were also detected. Marine Groups II and III Euryarchaeota were predominant in the archaeal communities in the surface and deep SCS, respectively. Bacterial diversity was higher than archaeal diversity at all sampling depths in the SCS, and peaked at mid-depths, agreeing with the diversity pattern found in global water columns. Metagenomic analysis not only showed differential %GC values and genome sizes between the surface and deep SCS, but also demonstrated depth-dependent metabolic potentials, such as cobalamin biosynthesis at 10 m, osmoregulation at 100 m, signal transduction at 1000 m, and plasmid and phage replication at 3000 m. When compared with other oceans, urease at 10 m and both exonuclease and permease at 3000 m were more abundant in the SCS. Finally, enriched genes associated with nutrient assimilation in the sea surface and transposase in the deep-sea metagenomes exemplified the functional zonation in global oceans.

Conclusions

Prokaryotic communities in the SCS stratified with depth, with maximal bacterial diversity at mid-depth, in accordance with global water columns. The SCS had functional zonation among depths and endemically enriched metabolic potentials at the study site, in contrast to other oceans.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1434-3) contains supplementary material, which is available to authorized users.