Cloning, characterization, and heterologous expression of a candidate Hirudin gene from the salivary gland transcriptome of Hirudo nipponia.

Hirudin is a pharmacologically active substance in leeches with potent blood anticoagulation properties. Although recombinant hirudin production isolated from Hirudo medicinalis Linnaeus and Hirudinaria manillensis Lesson is known, to our knowledge, this study is the first to report recombinant hirudin expression and production from Hirudo nipponia Whitman. Thus, the present study aimed to clone and characterize the full-length cDNA of a candidate hirudin gene (c16237_g1), which is localized on the salivary gland transcriptome of H. nipponia, and further evaluate its recombinant production using a eukaryotic expression system. The 489-bp cDNA possessed several properties of the hirudin "core" motifs associated with binding to the thrombin catalytic pocket. A fusion expression vector (pPIC9K-hirudin) was constructed and successfully transformed into Pichia pastoris strain GS115 via electroporation. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and western blot analysis confirmed hirudin expression. The recombinant protein was expressed with a yield of 6.68 mg/L culture. Mass spectrometry analysis further confirmed target protein expression. The concentration and antithrombin activity of purified hirudin were 1.67 mg/mL and 14,000 ATU/mL, respectively. These findings provide a basis for further elucidating the molecular anticoagulation mechanism of hirudin, and address China's growing market demand for engineered H. nipponia-derived hirudin and hirudin-based drugs.

Transcriptomic analysis of the salivary gland of medicinal leech Hirudo nipponia.

Hirudo nipponia (known as Shui Zhi in Chinese) is a well-known Chinese medicine with numerous active ingredients in its body, especially in its saliva. This native Chinese blood-sucking leech has been used for therapeutic purposes since before 100 AD. Modern Chinese physicians use it for a wide range of diseases. Genomic data and molecular information about the pharmacologically active substances produced by this medicinal leech are presently unavailable despite this organism's medicinal importance. In this study, we performed transcriptome profiling of the salivary glands of medicinal leech H. nipponia using the Illumina platform. In total, 84,657,362 clean reads were assembled into 50,535 unigenes. The obtained unigenes were compared to public databases. Furthermore, a unigene sequence similarity search and comparisons with the whole transcriptome of medical leech were performed to identify potential proteins. Finally, more than 21 genes were predicted to be involved in anticoagulatory, antithrombotic, antibacterial, anti-inflammatory and antitumor processes, which might play important roles in the treatment of various diseases. This study is the first analysis of a sialotranscriptome in H. nipponia. The transcriptome profile will shed light on its genetic background and provide a useful tool to deepen our understanding of the medical value of H. nipponia.

The effect of four types of artificial nerve graft structures on the repair of 10-mm rat sciatic nerve gap.

Investigating the effect of four types of artificial nerve graft (ANG) structures on rat sciatic nerve defect repair will aid future ANG designs. In this study, fibroin fibers and polylactic acid were used to prepare four ANGs with differing structures: nerve conduit with micron-sized pores (Conduit with pore group), nerve conduit without micron-sized pores (Conduit group), nerve scaffold comprising Conduit with pore group material plus silk fibers (Scaffold with pore group), and nerve scaffold comprising Conduit group material plus silk fibers (Scaffold group). ANGs or autologous nerves (Autologous group) were implanted into 10 mm rat sciatic nerve defects (n = 50 per group). Twenty weeks after nerve grafting, the time required to retract the surgical limb from the hot water was ranked as follows: Conduit with pore group > Scaffold with pore group > Conduit group > Scaffold group > Autologous group. The static sciatic index was ranked in descending order: Autologous group > Scaffold group > Conduit group > Scaffold with pore group > Conduit with pore group. Immunofluorescence staining identified significant differences in the distribution and number of axons, Schwann cells, and fibroblasts. These findings indicate that ANGs with micron-sized pores had a negative impact on the repair of peripheral nerve defects, while internal microchannels were beneficial. © 2017 The Authors. Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3077-3085, 2017.

Cell activity during peripheral nerve defect repair process using a nerve scaffold.

Peripheral nerve defects, but not artificial nerves, are repaired by endogenous cells. We examined cell activity during the repair process in the presence of autologous nerves and artificial preparations in order to guide future artificial nerve fabrication. PLGA tubes, nerve scaffolds comprising a PLGA tube plus 6,000 fibroin fibers, or autologous nerves were implanted into 10 mm rat sciatic nerve defects (n = 60 per group). Over a period of 1-20 weeks after nerve grafting, sections were stained and imaged to distinguish the cell types present and we quantified the recovery of motor and sensory function in the surgically implanted limb. We observed a decreasing trend in inflammatory cell and fibroblast counts over time which ranked in magnitude as: (PLGA group > nerve scaffold > autologous nerve> sham) and an opposite trend in Schwann cell counts. Differences in withdrawal time from hot water and static sciatic index (SSI) indicated that, after repair, sensory and motor function were best in the sham group, followed by the autologous group, the nerve scaffold group, and the PLGA group. These findings indicate that the inflammatory reaction is significant in the first two weeks after nerve grafting, followed by the rebirth of fibroblasts and Schwann cells, which guide axon regeneration. This inflammatory response was a fundamental stage of peripheral defect repair, but a weaker inflammatory response corresponded to better recovery of sensorimotor functional.

A honeycomb composite of mollusca shell matrix and calcium alginate.

A honeycomb composite is useful to carry cells for application in bone, cartilage, skin, and soft tissue regenerative therapies. To fabricate a composite, and expand the application of mollusca shells as well as improve preparing methods of calcium alginate in tissue engineering research, Anodonta woodiana shell powder was mixed with sodium alginate at varying mass ratios to obtain a gel mixture. The mixture was frozen and treated with dilute hydrochloric acid to generate a shell matrix/calcium alginate composite. Calcium carbonate served as the control. The composite was transplanted subcutaneously into rats. At 7, 14, 42, and 70 days after transplantation, frozen sections were stained with hematoxylin and eosin, followed by DAPI, ß-actin, and collagen type-I immunofluorescence staining, and observed using laser confocal microscopy. The composite featured a honeycomb structure. The control and composite samples displayed significantly different mechanical properties. The water absorption rate of the composite and control group were respectively 205-496% and 417-586%. The composite (mass ratio of 5:5) showed good biological safety over a 70-day period; the subcutaneous structure of the samples was maintained and the degradation rate was lower than that of the control samples. Freezing the gel mixture afforded control over chemical reaction rates. Given these results, the composite is a promising honeycomb scaffold for tissue engineering.

Silk structure and degradation.

To investigate the structure of silk and its degradation properties, we have monitored the structure of silk using scanning electron microscopy and frozen sections. Raw silk and degummed raw silk were immersed in four types of degradation solutions for 156 d to observe their degradation properties. The subcutaneous implants in rats were removed after 7, 14, 56, 84, 129, and 145 d for frozen sectioning and subsequent staining with hematoxylin and eosin (H.E.), DAPI, Beta-actin and Collagen I immunofluorescence staining. The in vitro weight loss ratio of raw silk and degummed raw silk in water, PBS, DMEM and DMEM containing 10% FBS (F-DMEM) were, respectively, 14%/11%, 12.5%/12.9%, 11.1%/14.3%, 8.8%/11.6%. Silk began to degrade after 7 d subcutaneous implantation and after 145 d non-degraded silk was still observed. These findings suggest the immunogenicity of fibroin and sericin had no essential difference. In the process of in vitro degradation of silk, the role of the enzyme is not significant. The in vivo degradation of silk is related to phagocytotic activity and fibroblasts may be involved in this process to secrete collagen. This study also shows the developing process of cocoons and raw silk.