Strategies for promoting tendon-bone healing: Current status and prospects

Tendon-bone insertion (TBI) injuries are common, primarily involving the rotator cuff (RC) and anterior cruciate ligament (ACL). At present, repair surgery and reconstructive surgery are the main treatments, and the main factor determining the curative effect of surgery is postoperative tendon-bone healing, which requires the stable combination of the transplanted tendon and the bone tunnel to ensure the stability of the joint. Fibrocartilage and bone formation are the main physiological processes in the bone marrow tract. Therefore, therapeutic measures conducive to these processes are likely to be applied clinically to promote tendon-bone healing. In recent years, biomaterials and compounds, stem cells, cell factors, platelet-rich plasma, exosomes, physical therapy, and other technologies have been widely used in the study of promoting tendon-bone healing. This review provides a comprehensive summary of strategies used to promote tendon-bone healing and analyses relevant preclinical and clinical studies. The potential application value of these strategies in promoting tendon-bone healing was also discussed

Transcriptional engineering for improved microbial cell performances in biofuel production

Escherichia coli and Saccharomyces cerevisiae are widely used to produce different kinds of compounds because they have several advantages over other organisms, such as short growing period, well-known genetics and metabolism, and availability of genetic manipulation tools. Biofuels have attracted researchers’ attention as they are cleaner and more sustainable compared with fossil fuels. When using Escherichia coli or Saccharomyces cerevisiae for biofuel production, one major challenge is that biofuels are highly toxic to them. Various strain engineering methods have been applied to improve microbes’ performances under stress conditions. Transcriptional engineering can be used as an alternative strain engineering method, as it is less time-consuming and labor-intensive than classical strain engineering, and does not require comprehensive genetic and metabolic information as compared with rational metabolic engineering approach. In this study, transcriptional engineering of cAMP receptor protein (CRP) and transcription factor IIB (TFIIB) was employed to improve E. coli strain tolerance towards isobutanol and S. cerevisiae strain oxidative stress resistance, respectively. The mechanism and applications of CRP engineering to elicit improved cell performances were also discussed. For transcriptional engineering, error-prone PCR and saturation mutagenesis were used to introduce mutations into CRP or TFIIB. The constructed random mutagenesis libraries were then subcultured in medium supplemented with certain stressors. After 3-5 rounds of enrichment, strains with improved traits were isolated and their growth were verified. Other characteristics of the obtained mutants were also investigated, such as their cross-tolerance towards other stresses, transcription profiles and intracellular reactive oxygen species level. Specifically, the isobutanol tolerant IB2 of E. coli had improved growth rates (0.18 h-1) than the control (0.05 h-1) in the presence of 1.2% isobutanol. When challenged with 1% isobutanol, IB2 had as many as 308 genes with altered expression level, with the major functional groups of genes related to acid resistance, nitrate reduction, flagella and fimbrial activity, and sulfate reduction and transportation. The oxidative stress resistant M1 (A330S) of S. cerevisiae had significantly increased growth rate (0.423 h-1) than the control (completely inhibited) under 8mM H2O2 stress. M1 also had cross-tolerance towards 1.5M NaCl, higher survival rate under 10mM H2O2, and higher catalase activity than the control, but the final ethanol production didn’t show any improvement.Doctor of Philosophy (SCBE

Enhancing E. coli isobutanol tolerance through engineering its global transcription factor cAMP receptor protein (CRP)

The limited isobutanol tolerance of Escherichia coli is a major drawback during fermentative isobutanol production. Different from classical strain engineering approaches, this work was initiated to improve E. coli isobutanol tolerance from its transcriptional level by engineering its global transcription factor cAMP receptor protein (CRP). Random mutagenesis libraries were generated by error-prone PCR of crp, and the libraries were subjected to isobutanol stress for selection. Variant IB2 (S179P, H199R) was isolated and exhibited much better growth (0.18 h−1) than the control (0.05 h−1) in 1.2% (v/v) isobutanol (9.6 g/L). Genome-wide DNA microarray analysis revealed that 58 and 308 genes in IB2 had differential expression (>2-fold, p < 0.05) in the absence and presence of 1% (v/v) isobutanol, respectively. When challenged with isobutanol, genes related to acid resistance (gadABCE, hdeABD), nitrate reduction (narUZYWV), flagella and fimbrial activity (lfhA, yehB, ycgR, fimCDF), and sulfate reduction and transportation (cysIJH, cysC, cysN) were the major functional groups that were up-regulated, whereas most of the down-regulated genes were enzyme (tnaA) and transporters (proVWX, manXYZ). As demonstrated by single-gene knockout experiments, gadX, nirB, rhaS, hdeB, and ybaS were found associated with strain isobutanol resistance. The intracellular reactive oxygen species (ROS) level in IB2 was only half of that of the control when facing stress, indicating that IB2 can withstand toxic isobutanol much better than the control. Biotechnol. Biotechnol