Studies of the dental caries pathogen Streptococcus mutans have benefitted tremendously from its sophisticated genetic system. As part of our own efforts to further improve upon the S. mutans genetic toolbox, we previously reported the development of the first cloning-independent markerless mutagenesis (CIMM) system for S. mutans and illustrated how this approach could be adapted for use in many other organisms. The CIMM approach only requires overlap extension PCR (OE-PCR) protocols to assemble counterselectable allelic replacement mutagenesis constructs, and thus greatly increased the speed and efficiency with which markerless mutations could be introduced into S. mutans. Despite its utility, the system is still subject to a couple limitations. Firstly, CIMM requires negative selection with the conditionally toxic phenylalanine analog p-chlorophenylalanine (4-CP), which is efficient, but never perfect. Typically, 4-CP negative selection results in a small percentage of naturally resistant background colonies. Secondly, CIMM requires two transformation steps to create markerless mutants. This can be inherently problematic if the transformability of the strain is negatively impacted after the first transformation step, which is used to insert the counterselection cassette at the mutation site on the chromosome. In the current study, we develop a next-generation counterselection cassette that eliminates 4-CP background resistance and combine this with a new direct repeat-mediated cloning-independent markerless mutagenesis (DR-CIMM) system to specifically address the limitations of the prior approach. DR-CIMM is even faster and more efficient than CIMM for the creation of all types of deletions, insertions, and point mutations and is similarly adaptable for use in a wide range of genetically tractable bacteria.
Temporomandibular joint (TMJ) ankylosis is a refractory disease that is difficult to predictably treat. This study evaluated the prognosis of using standard alloplastic TMJ prostheses for the treatment of TMJ ankylosis in Chinese patients with severe mandibular deficiency. Patients treated from 2013 to 2015 were reviewed. The computer-aided design and manufacture (CAD/CAM) technique was used to guide bony mass removal and locate the TMJ prosthesis (Biomet, USA). Eleven patients were included in this study. All prostheses were successfully installed and stabilized intraoperatively. In 4 patients with severe mandibular deficiency, their mandibular ramus was elongated by the TMJ prosthesis and 2 patients were combined with Le Fort I osteotomy guided by digital templates. Their mean chin advancement was 10.19 mm. Their SNB and ramus heights were also significantly improved after operation (P < 0.05). There was no prosthesis loosening, breakage, or infection leading to removal after a mean follow-up period of 22 months (range, 12-31mos.). Mouth opening was significantly improved from 5.5 mm preoperatively to 31.5 mm postoperatively. TMJ reconstruction with standard alloplastic prosthesis is a reliable treatment for ankylosis, especially in recurrent cases. By CAD/CAM technique, it can correct jaw deformities simultaneously and produce stable results.
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