Discovery of a novel esterase subfamily sharing an identified arm sequence (ArmEst) by gene-specific metagenomic PCR.

A gene-specific, metagenomic PCR method has led to the discovery of a novel esterase subfamily consisting of five homologous members. Sequence analysis of this esterase subfamily, named the ArmEst subfamily, revealed a unique conserved pattern with a significant variable interior sequence flanked by two symmetric and identical long arm sequences. The two homologous long arm sequences had 100 % sequence identity and symmetry at both ends between the five members of this esterase class, but only 17-58 % identity was shared for the internal sequence. The biochemical properties of two of the ArmEst esterases definitively demonstrated that they are true active esterases rather than pseudogenes. This is the first report presenting an esterase subfamily containing a unique arm sequence, indicating a rare homologous recombination occurring in the coding area of a functional gene to generate their functional diversity.

[Effect of platelet-rich plasma injection on early healing of Achilles tendon rupture in rats].

OBJECTIVE: Platelet-rich plasma (PRP) can promote wound healing. To observe the effect of PRP injection on the early healing of rat's Achilles tendon rupture so as to provide the experimental basis for clinical practice. METHODS: Forty-six Sprague Dawley rats were included in this experiment, female or male and weighing 190-240 g. PRP and platelet-poor plasma (PPP) were prepared from the heart arterial blood of 10 rats; other 36 rats were made the models of Achilles tendon rupture, and were randomly divided into 3 groups (control group, PPP group, and PRP group), 12 rats for each group. In PPP and PRP groups, PPP and PRP of 100 microL were injected around the tendons once a week, respectively; in the control group, nothing was injected. The tendon tissue sample was harvested at 1, 2, 3, and 4 weeks after operation for morphology, histology, and immunohistochemistry observations. The content of collagen type I fibers also was measured. Specimens of each group were obtained for biomechanical test at 4 weeks. RESULTS: All the animals survived till the end of the experiment. Tendon edema gradually decreased and sliding improved with time. The tendon adhesion increased steadily from 1 week to 3 weeks postoperatively, and it was relieved at 4 weeks in 3 groups. There was no significant difference in the grading of tendon adhesion among 3 groups at 1 week and at 4 weeks (P > 0.05), respectively. The inflammatory cell infiltration, angiogenesis, and collagen fibers were more in PRP group than in PPP group and control group at 1 week; with time, inflammatory cell infiltration and angiogenesis gradually decreased. Positive staining of collagen type I fibers was observed at 1-4 weeks postoperatively in 3 groups. The positive density of collagen type I fibers in group PRP was significantly higher than that in control group and PPP group at 1, 2, and 3 weeks (P < 0.05), but no significant difference was found among 3 groups at 4 weeks (P > 0.05). The biomechanical tests showed that there was no significant difference in the maximal gliding excursion among 3 groups at 4 weeks postoperatively (P > 0.05); the elasticity modulus and the ultimate tensile strength of PRP group were significantly higher than those of control group and PPP group at 4 weeks (P < 0.05). CONCLUSION: PRP injection can improve the healing of Achilles tendon in early repair of rat's Achilles tendon rupture.

Retrieval of glycoside hydrolase family 9 cellulase genes from environmental DNA by metagenomic gene specific multi-primer PCR.

A new method, termed metagenomic gene specific multi-primer PCR (MGSM-PCR), is presented that uses multiple gene specific primers derived from an isolated gene from a constructed metagenomic library rather than degenerate primers designed based on a known enzyme family. The utility of MGSM-PCR was shown by applying it to search for homologues of the glycoside hydrolase family 9 cellulase in metagenomic DNA. The success of the multiplex PCR was verified by visualizing products on an agarose gel following gel electrophoresis. A total of 127 homologous genes were amplified with combinatorial multi-primer reactions from 34 soil DNA samples. Multiple alignments revealed extensive sequence diversity among these captured sequences with sequence identity varying from 26 to 99.7%. These results indicated that significantly diverse homologous genes were indeed readily accessible when using multiple metagenomic gene specific primers.

Overexpression and characterization of a new organic solvent-tolerant esterase derived from soil metagenomic DNA.

In this study, an esterase, designated EstC23, was isolated from a soil metagenomic library. The protein was amenable to overexpression in Escherichia coli under control of the T7 promoter, resulting in expression of the active, soluble protein that constituted 30% of the total cell protein content. This enzyme showed optimal activity at 40 °C and retained about 50% maximal activity at 5-10 °C. EstC23 showed remarkable stability in up to 50% (v/v) benzene and alkanes (high logP solvents). When incubated for 7 days in the presence of 50% benzene or alkanes, the enzyme maintained its 2-3 fold elevated activity. The purified enzyme also cleaved sterically hindered esters of tertiary alcohols. These results indicate that EstC23 has potential for use in industrial processes.

Internal versus external fixation for unstable distal radius fractures: an up-to-date meta-analysis.

PURPOSE: Our aim was to compare the effect of internal vs external fixation for unstable distal radius fractures regarding postoperative complications, clinical results and radiological outcomes. METHODS: We selected PubMed; Cochrane Library; EMBASE; BIOSIS; Ovid and the relevant English orthopaedic journals and pooled data from ten eligible randomised controlled trials containing 738 patients to conduct a subgroup analysis according to different periods of follow-up. Our aim was to summarise the best available evidence. RESULTS: Results showed that compared with external fixation, internal fixation led to significantly fewer total surgical complications [95% confidence interval (CI) 0.39-0.81, P = 0.002] and reduced the incidence of pin-track infections (95% CI 0.08-0.46, P = 0.0002) after a one year follow-up. For clinical results, grip strength (95% CI 1.59-8.25, P = 0.004), supination (95% CI 13.99-48.83, P = 0.0004) and pronation (95% CI 5.61-26.09, P = 0.002) were superior in the internal fixation group six weeks postoperatively, and the same results were obtained three months postoperatively for grip strength (95% CI 3.21-13.47, P = 0.001) and supination (95% CI 3.61-16.01, P = 0.002). Meanwhile, the Disabilities of the Arm, Shoulder and Hand (DASH) score was superior in the internal fixation group at three months (95% CI -20.62 to -2.07, P = 0.02) and after one year (95% CI -14.37 to -2.32, P = 0.007) follow-up. CONCLUSIONS: We suggest that the final results are significant and there is some evidence supporting the use of open reduction and internal fixation.

Prospecting metagenomic enzyme subfamily genes for DNA family shuffling by a novel PCR-based approach.

DNA family shuffling is a powerful method for enzyme engineering, which utilizes recombination of naturally occurring functional diversity to accelerate laboratory-directed evolution. However, the use of this technique has been hindered by the scarcity of family genes with the required level of sequence identity in the genome database. We describe here a strategy for collecting metagenomic homologous genes for DNA shuffling from environmental samples by truncated metagenomic gene-specific PCR (TMGS-PCR). Using identified metagenomic gene-specific primers, twenty-three 921-bp truncated lipase gene fragments, which shared 64-99% identity with each other and formed a distinct subfamily of lipases, were retrieved from 60 metagenomic samples. These lipase genes were shuffled, and selected active clones were characterized. The chimeric clones show extensive functional and genetic diversity, as demonstrated by functional characterization and sequence analysis. Our results indicate that homologous sequences of genes captured by TMGS-PCR can be used as suitable genetic material for DNA family shuffling with broad applications in enzyme engineering.