Application of suture anchors for a clinically relevant rat model of rotator cuff tear.

Current rat model of rotator cuff (RC) tear could not mimic the suture anchor (SA) repair technique in the clinical practice. We designed a novel SA for RC repair of rats to establish a clinically relevant animal model. Small suture anchors that fit the rat shoulder were assembled. 60 rats were assigned to the transosseous (TO) repair group or SA repair group (n = 30/group). Micro-computed tomography (Micro-CT) scanning, biomechanical test and histological analysis were implemented at 2, 4, and 8-week post-repair. The failure load and stiffness in the SA group were significantly higher than those of TO group at 4-week post-repair. Micro-computed tomography analysis showed the bone mineral density and trabecular thickness of the SA group were significantly lower than those of TO group. The SA group showed a better insertion continuity at 4-week post-repair compared to TO group. No significant difference in gait parameters was found between groups. Therefore, SA repair is applicable for the rat model of RC tears. The SA repair achieved superior RC tendon healing, but more extensive initial bone damage compared to TO repair, while the shoulder function was comparable. This model could replicate the current repair technique in the clinical situation and be considered for future preclinical studies on healing enhancement for RC tears. Statement of Clinical Significance: With high clinical relevance, this model may facilitate the translation from an animal study into clinical trials.

Two new Sinorhizobium meliloti LysR-type transcriptional regulators required for nodulation.

The establishment of an effective nitrogen-fixing symbiosis between Sinorhizobium meliloti and its legume host alfalfa (Medicago sativa) depends on the timely expression of nodulation genes that are controlled by LysR-type regulators. Ninety putative genes coding for LysR-type transcriptional regulators were identified in the recently sequenced S. meliloti genome. All 90 putative lysR genes were mutagenized using plasmid insertions as a first step toward determining their roles in symbiosis. Two new LysR-type symbiosis regulator genes, lsrA and lsrB, were identified in the screening. Both the lsrA and lsrB genes are expressed in free-living S. meliloti cells, but they are not required for cell growth. An lsrA1 mutant was defective in symbiosis and elicited only white nodules that exhibited no nitrogenase activity. Cells of the lsrA1 mutant were recovered from the white nodules, suggesting that the lsrA1 mutant was blocked early in nodulation. An lsrB1 mutant was deficient in symbiosis and elicited a mixture of pink and white nodules on alfalfa plants. These plants exhibited lower overall nitrogenase activity than plants inoculated with the wild-type strain, which is consistent with the fact that most of the alfalfa plants inoculated with the lsrB1 mutant were short and yellow. Cells of the lsrB1 mutant were recovered from both pink and white nodules, suggesting that lsrB1 mutants could be blocked at multiple points during nodulation. The identification of two new LysR-type symbiosis transcriptional regulators provides two new avenues for understanding the complex S. meliloti-alfalfa interactions which occur during symbiosis.

Role of oxyR from Sinorhizobium meliloti in regulating the expression of catalases.

The process of symbiotic nitrogen fixation results in the generation of reactive oxygen species such as the superoxide anion (O2-) and hydrogen peroxide (H2O2). The response of rhizobia to these toxic oxygen species is an important factor in nodulation and nitrogen fixation. In Sinorhizobium meliloti, one oxyR homologue and three catalase genes, katA, katB, and katC were detected by sequence analysis. This oxyR gene is located next to and divergently from katA on the chromosome. To investigate the possible roles of oxyR in regulating the expression of catalases at the transcriptional level in S. meliloti, an insertion mutant of this gene was constructed. The mutant was more sensitive and less adaptive to H2O2 than the wild type strain, and total catalase/peroxidase activity was reduced approximately fourfold with the OxyR mutation relative to controls. The activities of KatA and KatB and the expression of katA::lacZ and katB::lacZ promoter fusions were increased in the mutant strain compared with the parental strain grown in the absence of H2O2, indicating that katA and katB are repressed by OxyR. However, when exposed to H2O2, katA expression was also increased in both S. meliloti and Escherichia coli. When exposed to H2O2, OxyR is converted from a reduced to an oxidized form in E. coli. We concluded that the reduced form of OxyR functions as a repressor of katA and katB expression. Thus, in the presence of H2O2, reduced OxyR is converted to the oxidized form of OxyR that then results in increased katA expression. We further showed that oxyR expression is autoregulated via negative feedback.

Sinorhizobium meliloti ExoR and ExoS proteins regulate both succinoglycan and flagellum production.

The production of the Sinorhizobium meliloti exopolysaccharide, succinoglycan, is required for the formation of infection threads inside root hairs, a critical step during the nodulation of alfalfa (Medicago sativa) by S. meliloti. Two bacterial mutations, exoR95::Tn5 and exoS96::Tn5, resulted in the overproduction of succinoglycan and a reduction in symbiosis. Systematic analyses of the symbiotic phenotypes of the two mutants demonstrated their reduced efficiency of root hair colonization. In addition, both the exoR95 and exoS96 mutations caused a marked reduction in the biosynthesis of flagella and consequent loss of ability of the cells to swarm and swim. Succinoglycan overproduction did not appear to be the cause of the suppression of flagellum biosynthesis. Further analysis indicated that both the exoR95 and exoS96 mutations affected the expression of the flagellum biosynthesis genes. These findings suggest that both the ExoR protein and the ExoS/ChvI two-component regulatory system are involved in the regulation of both succinoglycan and flagellum biosynthesis. These findings provide new avenues of understanding of the physiological changes S. meliloti cells go through during the early stages of symbiosis and of the signal transduction pathways that mediate such changes.

The key Sinorhizobium meliloti succinoglycan biosynthesis gene exoY is expressed from two promoters.

Bacterial exopolysaccharide, succinoglycan, plays an important role in eliciting infection thread formation, which is a key step in the establishment of Sinorhizobium meliloti-alfalfa (Medicago sativa) nitrogen fixing symbiosis. To understand the regulatory mechanisms that control production of succinoglycan, the expression of the key succinoglycan biosynthesis gene, exoY, was analyzed by constructing a set of nested deletions of the exoY promoter region. Two exoY promoters were identified based on the promoter activities and confirmed by direct detection of the transcripts. The expression from both promoters was induced in the exoR95 and exoS96 mutant backgrounds suggesting that both promoters are regulated by the ExoR protein and the ExoS/ChvI two-component signal transduction system. The identification of the exoY promoters provides additional avenue for further analysis of the role of succinoglycan in S. meliloti-alfalfa symbiosis.