Identification of small non-coding RNAs in the planarian Dugesia japonica via deep sequencing.

Freshwater planarian flatworm possesses an extraordinary ability to regenerate lost body parts after amputation; it is perfect organism model in regeneration and stem cell biology. Recently, small RNAs have been an increasing concern and studied in many aspects, including regeneration and stem cell biology, among others. In the current study, the large-scale cloning and sequencing of sRNAs from the intact and regenerative planarian Dugesia japonica are reported. Sequence analysis shows that sRNAs between 18nt and 40nt are mainly microRNAs and piRNAs. In addition, 209 conserved miRNAs and 12 novel miRNAs are identified. Especially, a better screening target method, negative-correlation relationship of miRNAs and mRNA, is adopted to improve target prediction accuracy. Similar to miRNAs, a diverse population of piRNAs and changes in the two samples are also listed. The present study is the first to report on the important role of sRNAs during planarian Dugesia japonica regeneration.

Differential expression of microRNA patterns in planarian normal and regenerative tissues.

MicroRNAs (miRNAs) are ~22-nt small non-coding RNAs that regulate the expression of specific target genes in many eukaryotes. miRNAs have been shown to play important roles in stem cell maintenance, cell fate determination, and differentiation. Planarians are capable of regenerating entire body plans from tiny fragments; this regenerative capacity is facilitated by a population of pluripotent stem cells known as neoblasts. Planarians have been a classic model system for the study of many aspects of stem cell biology. However, very limited knowledge on miRNA involved in this regulatory mechanism exists. This study profiles the expression of miRNAs in the normal and regenerative tissues of planarians using miRCURY LNA array technology. Thirteen miRNAs showed significant differences in expression between these two tissues. To further confirm our results, we examined the expression of two miRNAs by qRT-PCR. Results show that some known miRNAs may play key roles in the regulatory mechanisms of regeneration. Our findings can be utilized in future research on miRNA function.

Cloning and identification of microRNAs in earthworm (Eisenia fetida).

MicroRNAs (miRNAs) (noncoding RNAs of 20-25 nucleotides) play important roles in the post-transcriptional regulation of gene expression in various eukaryotes and prokaryotes. Piwi-interacting RNAs function by combining with PIWI proteins to regulate protein synthesis and to stabilize mRNA, the chromatin framework, and genome structure. This study investigates the role of miRNAs in regeneration. A scrDNA library was constructed, and 17 noncoding RNAs from Eisenia fetida (an optimal model for the study of earthworm regeneration) were cloned and characterized. In addition, reverse transcription polymerase chain reaction was performed to analyze the expression of four small RNAs during different developmental stages. The expression levels of these RNAs in regenerating tissue were higher than in normal tissue, and the expression patterns of these small RNAs were unique during development.

Modular design, expression and characterization of novel bifunctional mutants of fibrolase with combined platelet aggregation-inhibition and fibrinolytic activity.

Fibrolase is a non-hemorrhagic zinc metalloproteinase found in southern copperhead snake (Agkistrodon contortrix contortrix) venom that acts directly on fibrin clots and does not require plasminogen or any other blood-borne intermediate for activity. Chimeras of fibrolase with RGD peptides conferring antiplatelet activity have been synthesized covalently, but we describe a simpler, cheaper and less toxic method, using site-directed mutagensis. Fibrolase variants that constitute the arginine-glycine-aspartic acid (Arg-Gly-Asp, RGD) motif were constructed using site-directed mutagenesis. Chimeric genes of fibrolase were expressed in Escherichia coli to obtain the bifunctional chimeric molecule of fibrolase that can inhibit platelet aggregation. After refolding and purification, platelet-targeted thrombolysis and antiplatelet aggregation of the target chimeric protein were determined. The mutant RGD-F2, using the GPRGDWRMLG peptide to replace the TSVSHD sequence between sites 69 and 72, not only had almost the same catalytic ability as wild-type fibrolase but also a strong ability to inhibit platelet aggregation.

Co-overexpression of PpPDI enhances secretion of ancrod in Pichia pastoris.

Ancrod, a serine protease purified from the venom of Agkistrodon rhodostoma, is highly specific for fibrinogen. It causes anticoagulation by defibrinogenation and has been used as a therapeutic anticoagulant for the treatment of moderate to severe forms of peripheral arterial circulatory disorders in a variety of countries. The DNA of ancrod was amplified by recursive PCR with a yeast bias codon and cloned into the pGEM-T Easy vector. In order to achieve a high level secretion and a full activity expression of ancrod in Pichia pastoris (P. pastoris), the P. pastoris protein disulfide bond isomerase (PpPDI) was co-overexpressed in the strain. The secretion characteristics of ancrod with and without PpPDI were examined. With co-overexpression of PpPDI, the production of recombinant ancrod (rAncrod) was increased to 315 mg/L in the culture medium, which is twofold higher than the control strain carrying only the ancrod gene. Through purified by Ni²âº affinity chromatography and phenyl Sepharose column, the purity of rAncrod was found to be as high as 95.2%. The fibrinogenolytic and zymographic activities of the rAncrod were determined and found to be similar to that of the native protein. This improved expression system can facilitate further studies and the industrial production of ancrod.

Transcriptome profiling and digital gene expression by deep-sequencing in normal/regenerative tissues of planarian Dugesia japonica.

Planarians exhibit an extraordinary ability to regenerate lost body parts which is attributed to an abundance of pluripotent somatic stem cells called neoblasts. In this article, we report a transcriptome sequence of a Planaria subspecies Dugesia japonica derived by high-throughput sequencing. In addition, we researched transcriptome changes during different periods of regeneration by using a tag-based digital gene expression (DGE) system. Consequently, 11,913,548 transcriptome sequencing reads were obtained. Finally, these reads were eventually assembled into 37,218 unique unigenes. These assembled unigenes were annotated with various methods. Transcriptome changes during planarian regeneration were investigated by using a tag-based DGE system. We obtained a sequencing depth of more than 3.5million tags per sample and identified a large number of differentially expressed genes at various stages of regeneration. The results provide a fairly comprehensive molecular biology background to the research on planarian development, particularly with regard to its regeneration progress.