Healing an ischial tuberosity pressure ulcer in a patient with neurogenic heterotopic ossification: a case report.

Neurogenic heterotopic ossification (NHO) is widely recognised as an aberrant bone formation in soft tissue following central nervous system injury. It is most frequently associated with pain and limited movement, especially in the hip. However, it may be neglected in patients with paraplegia with a pressure ulcer (PU). We report the case of an 18-year-old male patient who presented with a hard-to-heal ischial tuberosity PU and who had undergone three operations at other hospitals during the previous six months, which had failed to repair the PU. There was a history of paraplegia as a consequence of spinal cord injury two years previously. Computed tomography and three-dimensional reconstruction showed massive heterotopic ossification (HO) in the wound bed and around the right hip. Histological findings were consistent with a diagnosis of HO. The HO around the wound was completely excised, negative pressure wound therapy was used to promote granulation, and a gluteus maximus musculocutaneous flap was used to cover the wound. We conclude that for patients with paraplegia, with a hard-to-heal PU, it should be determined whether it is associated with NHO. Surgical resection of HO surrounding the wound and improving the microcirculation are critical for repair and reconstruction of these PUs.

Tyrosine kinase receptor ErbB4 in Advillin-positive neurons contributes to inflammatory pain hypersensitivity in mouse DRG.

Inflammatory pain is a common type of pathological pain. Although the dorsal root ganglion (DRG) is key to pathogenesis of inflammatory pain, the underlying specific molecular and cellular mechanisms remain unclear. In this study, we used mouse models of acute or chronic inflammatory pain, induced by formalin or complete Freund' s adjuvant (CFA), respectively, to explore whether tyrosine kinase receptor ErbB4 participates in the pathogenesis of inflammatory pain. Firstly, we found that both the expression of Neuregulin 1 (Nrg1) and phosphorylation of ErbB4 receptor were upregulated in DRG after inflammatory pain, implying the activation of ErbB4 in DRG. Using ErbB4-mutant mice, we found reduced pain sensitivity of mice when ErbB4 gene expression was largely ablated; furthermore, ErbB4 deletion decreased the inflammatory pain hypersensitivity of either formalin- or CFA-induced mouse models. Moreover, the pain sensitivity was reduced in mice with specific deletion of ErbB4 on advillin-positive neurons within DRG. Importantly, pain hypersensitivity also decreased in Advillin-Cre;ErbB4-/- cKO mice after formalin- or CFA-induced inflammatory pain. Finally, gene quantification differential expression analysis, using RNAseq technology in combination with GO and KEGG enrichment analysis, suggested that calcium signaling pathway possibly mediated the roles of ErbB4 on DRG sensory neurons in inflammatory pain models. Together, these results indicate that ErbB4 on advillin-positive sensory neurons enhances inflammatory pain sensitivity, providing new clues towards the pathogenic mechanisms of inflammatory pain.

Engineering Pichia pastoris with surface-display minicellulosomes for carboxymethyl cellulose hydrolysis and ethanol production.

BACKGROUNDS: Engineering yeast as a consolidated bioprocessing (CBP) microorganism by surface assembly of cellulosomes has been aggressively utilized for cellulosic ethanol production. However, most of the previous studies focused on Saccharomyces cerevisiae, achieving efficient conversion of phosphoric acid-swollen cellulose (PASC) or microcrystalline cellulose (Avicel) but not carboxymethyl cellulose (CMC) to ethanol, with an average titer below 2 g/L. RESULTS: Harnessing an ultra-high-affinity IM7/CL7 protein pair, here we describe a method to engineer Pichia pastoris with minicellulosomes by in vitro assembly of three recombinant cellulases including an endoglucanase (EG), an exoglucanase (CBH) and a ß-glucosidase (BGL), as well as a carbohydrate-binding module (CBM) on the cell surface. For the first time, the engineered yeasts enable efficient and direct conversion of CMC to bioethanol, observing an impressive ethanol titer of 5.1 g/L. CONCLUSIONS: The research promotes the application of P. pastoris as a CBP cell factory in cellulosic ethanol production and provides a promising platform for screening the cellulases from different species to construct surface-assembly celluosome.

A Highly Efficient Indirect P. pastoris Surface Display Method Based on the CL7/Im7 Ultra-High-Affinity System.

Cell surface display systems for immobilization of peptides and proteins on the surface of cells have various applications, such as vaccine generation, protein engineering, bio-conversion and bio-adsorption. Though plenty of methods have been established in terms of traditional yeast surface display systems, the development of a universal display method with high efficiency remains a challenge. Here we report an indirect yeast surface display method by anchoring Im7 proteins on the surface of P. pastoris, achieving highly efficient display of target proteins, including fluorescence proteins (sfGFP and mCherry) or enzymes (human Arginase I), with a CL7 fusion tag through the ultra-high-affinity interaction between Im7 and CL7. This indirect P. pastoris surface display approach is highly efficient and provides a robust platform for displaying biomolecules.

LncRNA COL1A2-AS1 inhibits the scar fibroblasts proliferation via regulating miR-21/Smad7 pathway.

lncRNA COL1A2-AS1 (COL1A2 antisense RNA 1), a lncRNA overexpressed in hypertrophic scar, has been demonstrated to be involved in the hypertrophic scar formation. However, the mechanisms of lncRNA COL1A2-AS1 inhibiting the scar fibroblasts proliferation remains not well understood. In this study, we demonstrated that lncRNA COL1A2-AS1 was upregulated in hypertrophic scar tissue and fibroblasts, and suppressed fibroblasts proliferation by promoting Smad7 expression. Furthermore, we found that miR-21 was involved in lncRNA COL1A2-AS1-induced expression of Smad7, by which COL1A2-AS1 acted as endogenous sponge to adsorb miR-21 and in turn regulated Smad7 and a cascade of molecular to play a protective role in hypertrophic scar. In addition, overexpression of miR-21 attenuated COL1A2-AS1-mediated proliferation suppression of hypertrophic scar fibroblasts. In conclusion, our study demonstrated that COL1A2-AS1/miR-21/Smad pathway plays an important role in inhibiting hypertrophic scar formation, and suggested this novel pathway may be a new target for hypertrophic scar treatment.

Linear DNA vaccine prepared by large-scale PCR provides protective immunity against H1N1 influenza virus infection in mice.

Linear DNA vaccines provide effective vaccination. However, their application is limited by high cost and small scale of the conventional polymerase chain reaction (PCR) generally used to obtain sufficient amounts of DNA effective against epidemic diseases. In this study, a two-step, large-scale PCR was established using a low-cost DNA polymerase, RKOD, expressed in Pichia pastoris. Two linear DNA vaccines encoding influenza H1N1 hemagglutinin (HA) 1, LEC-HA, and PTO-LEC-HA (with phosphorothioate-modified primers), were produced by the two-step PCR. Protective effects of the vaccines were evaluated in a mouse model. BALB/c mice were immunized three times with the vaccines or a control DNA fragment. All immunized animals were challenged by intranasal administration of a lethal dose of influenza H1N1 virus 2 weeks after the last immunization. Sera of the immunized animals were tested for the presence of HA-specific antibodies, and the total IFN-γ responses induced by linear DNA vaccines were measured. The results showed that the DNA vaccines but not the control DNA induced strong antibody and IFN-γ responses. Additionally, the PTO-LEC-HA vaccine effectively protected the mice against the lethal homologous mouse-adapted virus, with a survival rate of 100% versus 70% in the LEC-HA-vaccinated group, showing that the PTO-LEC-HA vaccine was more effective than LEC-HA. In conclusion, the results indicated that the linear H1N1 HA-coding DNA vaccines induced significant immune responses and protected mice against a lethal virus challenge. Thus, the low-cost, two-step, large-scale PCR can be considered a potential tool for rapid manufacturing of linear DNA vaccines against emerging infectious diseases.

Expression and Characterization of the RKOD DNA Polymerase in Pichia pastoris.

The present study assessed high-level expression of the KOD DNA polymerase in Pichia pastoris. Thermococcus kodakaraensis KOD1 is a DNA polymerase that is widely used in PCR. The DNA coding sequence of KOD was optimized based on the codon usage bias of P. pastoris and synthesized by overlapping PCR, and the nonspecific DNA-binding protein Sso7d from the crenarchaeon Sulfolobus solfataricus was fused to the C-terminus of KOD. The resulting novel gene was cloned into a pHBM905A vector and introduced into P. pastoris GS115 for secretory expression. The yield of the target protein reached approximately 250 mg/l after a 6-d induction with 1% (v/v) methanol in shake flasks. This yield is much higher than those of other DNA polymerases expressed heterologously in Escherichia coli. The recombinant enzyme was purified, and its enzymatic features were studied. Its specific activity was 19,384 U/mg. The recombinant KOD expressed in P. pastoris exhibited excellent thermostability, extension rate and fidelity. Thus, this report provides a simple, efficient and economic approach to realize the production of a high-performance thermostable DNA polymerase on a large scale. This is the first report of the expression in yeast of a DNA polymerase for use in PCR.