Single-plasmid systems based on CRISPR-Cas9 for gene editing in Lactococcus lactis.

Lactococcus lactis is a food-grade lactic acid bacterial species that is widely used in food and medical industries. Due to its relatively small genome and simple metabolism, L. lactis is commonly engineered to produce large quantities of recombinant proteins. The most common single-gene knockout strategy in L. lactis involves RecA-dependent homologous double-crossover recombination, which is relatively time-consuming and laborious. In this study, a precise and efficient genome-editing plasmid for L. lactis NZ9000 genome engineering, pLL, was established based on clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology. By studying the effects of different single guide RNA (sgRNA) promoters, the efficiency of gene deletion was optimized. For LLNZ_02045 (ldh), gene deletion efficiency of up to 50% was achieved. Effective sequential gene deletion of LLNZ_11240 (upp) and LLNZ_04580 (upp1) was also demonstrated using this tool. Additionally, the gene that encodes for uracil phosphoribosyltransferase was identified using this system. Similar robust gene deletion efficiencies of sgRNA that targeted different regions of a single gene suggested that gene deletion was not affected by the location of sgRNA binding. Thus, our study established a new gene-editing tool that may allow further investigation and understanding of the L. lactis NZ9000 genome.

An increase in cell membrane permeability in the in situ extractive fermentation improves the production of antroquinonol from Antrodia camphorata S-29.

The goals of this study were to increase the production of antroquinonol (AQ) and to elucidate the response mechanism of the cell membrane during the in situ extractive fermentation (ISEF) of Antrodia camphorata S-29. Through ISEF, the concentration of AQ reached a maximum of 146.1 ± 2.8 mg/L, which was approximately (7.4 ± 0.1)-fold that of the control (coenzyme Q0-induced fermentation). Transcriptome sequencing showed that four genes (FAD2, fabG, SCD, and FAS1) related to fatty acid biosynthesis were upregulated. FAD2 and SCD may regulate the increase in oleic acid (C18:1) and linoleic acid (C18:2) in the cell membrane of A. camphorata S-29, resulting in an increase in cell membrane permeability. AQ was successfully transferred to the n-tetradecane phase through the cell membrane, reducing product feedback inhibition and improving the production of AQ from A. camphorata S-29.

Comparison of high- and low-viscosity cement in the treatment of vertebral compression fractures: A systematic review and meta-analysis.

BACKGROUND: High-viscosity cement (HVC) has been gradually applied in percutaneous vertebroplasty (PVP) and percutaneous kyphoplasty (PKP). Although HVC has been reported to reduce cement leakage, different opinions exist. To assess the complications of HVC in cement leakage in the treatment of vertebral compression fractures and to evaluate the clinical effect of HVC compared with low-viscosity cement (LVC). METHODS: EMBASE, PubMed, Science Direct, Google Scholar and Cochrane Library databases were comprehensively searched from their inception to August 2017. Two researchers independently searched for articles and reviewed all retrieved studies. Forest plots were used to illustrate the results. The Q-test and I statistic were employed to evaluate between-study heterogeneity. Potential publication bias was assessed by funnel plot. RESULTS: HVC reduced the occurrence of cement leakage (risk ratio (RR) = 0.38, 95% confidence interval (CI) = 0.29 to 0.51, P < 0.00001), especially in the disc space (RR = 0.45, 95% CI = 0.45 to 0.80, P = 0.007) and the vein (RR = 0.54, 95% CI = 0.35 to 0.85, P = 0.008) but not in the intraspinal space (RR = 0.48, 95% CI = 0.19 to 1.23, P = 0.13) or the paravertebral area (RR = 0.63, 95% CI = 0.32 to 1.22, P = 0.17). No significant differences in the visual analogue scale (VAS), Oswestry Disability Index (ODI), injected cement volume or adjacent vertebral fracture were noted between HVC and LVC (P > 0.05). CONCLUSION: Compared with LVC, HVC results in a reduced incidence of cement leakage for the treatment of vertebral compression fractures, especially in the disc space and vein but not in the intraspinal space or the paravertebral area. In addition, HVC yields the same satisfactory clinical effect as LVC.

Ultra-early injection of low-viscosity cement in vertebroplasty procedure for treating osteoporotic vertebral compression fractures: A retrospective cohort study.

OBJECTIVE: To evaluate the clinical effect of ultra-early injection (before the phase of "tooth-paste-like") of low-viscosity cement in percutaneous vertebroplasty (PVP) for treating osteoporotic vertebral compression fractures (OVCFs). METHODS: Two hundred sixty-one patients who had PVP procedures with low-viscosity cement (ultra-early injection: 145, normal injection: 135) were included from July 2010 to July 2016 in our hospital. Visual Analog Scale (VAS), Oswestry Disability Index (ODI), Cobb angle, cement leakage, and adjacent vertebral fractures were evaluated. The follow-up period was over 12 months. RESULTS: VAS 3.0 d after surgery was significantly reduced in the ultra-early injection group compared to that in the control group (P = 0.00), but no difference was found at the final follow-up (P = 0.53). Similar results were found for ODI. The Cobb angle in both groups was recovered after PVP (P < 0.05); however, in the control group, the Cobb angle at the final follow-up was significantly increased compared with that 3.0 d after surgery (P = 0.00). There was a significant difference in the Cobb angle between the two groups at the final follow-up (P = 0.00). Regarding cement leakage, there were no significant differences in terms of mild (P = 0.58), moderate (P = 0.68), or severe leakage (P = 0.52). Seven patients in the control group had adjacent vertebral fractures, but only one patient in the ultra-early injection group experienced adjacent fractures (P = 0.03). CONCLUSIONS: Ultra-early injection of low-viscosity cement during PVP procedures in the treatment of OVCFs not only quickly and significantly relieves pain, reduces the incidence of adjacent vertebral fractures, and prevents progressive kyphotic deformity, but also does not increase the risk of cement leakage when compared with that of the traditional injection procedure.