Nicotiana benthamiana, a Surrogate Host to Study Novel Virulence Mechanisms of Gram-Positive Bacteria, Clavibacter michiganensis, and C. capsici in Plants.

Clavibacter michiganensis is a Gram-positive bacterium that causes bacterial canker and wilting in host plants like tomato. Two major virulence genes encoding a cellulase (celA) and a putative serine protease (pat-1) have been reported. Here we show that Nicotiana benthamiana, a commonly used model plant for studying molecular plant-pathogen interactions, is a surrogate host of C. michiganensis and C. capsici. When a low concentration of two Clavibacter species, C. michiganensis and C. capsici, were infiltrated into N. benthamiana leaves, they caused blister-like lesions closely associated with cell death and the generation of reactive oxygen species and proliferated significantly like a pathogenic bacterium. By contrast, they did not cause any disease symptoms in N. tabacum leaves. The celA and pat-1 mutants of C. michiganensis still caused blister-like lesions and cankers like the wild-type strain. When a high concentration of two Clavibacter species and two mutant strains were infiltrated into N. benthamiana leaves, all of them caused strong and rapid necrosis. However, only C. michiganensis strains, including the celA and pat-1 mutants, caused wilting symptoms when it was injected into stems. When two Clavibacter species and two mutants were infiltrated into N. tabacum leaves at the high concentration, they (except for the pat-1 mutant) caused a strong hypersensitive response. These results indicate that C. michiganensis causes blister-like lesions, canker, and wilting in N. benthamiana, and celA and pat-1 genes are not necessary for the development of these symptoms. Overall, N. benthamiana is a surrogate host of Clavibacter species, and their novel virulence factors are responsible for disease development in this plant.

An Apoplastic Effector Pat-1Cm of the Gram-Positive Bacterium Clavibacter michiganensis Acts as Both a Pathogenicity Factor and an Immunity Elicitor in Plants.

Clavibacter michiganensis, a Gram-positive plant-pathogenic bacterium, utilizes apoplastic effectors for disease development in host plants. Here, we determine the roles of Pat-1Cm (a putative serine protease) in pathogenicity and plant immunity. Pat-1Cm was found to be a genuine secreted protein, and the secreted mature form did not carry the first 33 amino acids predicted to be a signal peptide (SP). The pat-1Cm mutant impaired to cause wilting, but still caused canker symptom in tomato. Moreover, this mutant failed to trigger the hypersensitive response (HR) in a nonhost Nicotiana tabacum. Among orthologs and paralogs of pat-1Cm , only chp-7Cs from Clavibacter sepedonicus, a potato pathogen, successfully complemented pat-1Cm function in pathogenicity in tomato, whereas all failed to complement pat-1Cm function in HR induction in N. tabacum. Based on the structural prediction, Pat-1Cm carried a catalytic triad for putative serine protease, and alanine substitution of any amino acids in the triad abolished both pathogenicity and HR-inducing activities of Pat-1Cm in C. michiganensis. Ectopic expression of pat-1Cm with an SP from tobacco secreted protein triggered HR in N. tabacum, but not in tomato, whereas a catalytic triad mutant failed to induce HR. Inoculation of the pat-1Cm mutant mixed with the mutant of another apoplastic effector CelA (cellulase) caused severe wilting in tomato, indicating that these two apoplastic effectors can functionally cooperate in pathogenicity. Overall, these results indicate that Pat-1Cm is a distinct secreted protein carrying a functional catalytic triad for serine protease and this enzymatic activity might be critical for both pathogenicity and HR-eliciting activities of Pat-1Cm in plants.

Is a Titanium Implant for Total Knee Arthroplasty Better? A Randomized Controlled Study.

BACKGROUND: Lighter weight and lower modulus are potential advantages of titanium (Ti) implants over cobalt chrome (CoCr) implants in total knee arthroplasty (TKA). This study was conducted to determine whether Ti implants in TKA resulted in better clinical outcomes and radiologic results. METHODS: One hundred and eight patients (216 knees) with knee arthritis warranting bilateral primary TKA were randomly allocated to undergo Ti rotating-platform TKA in one knee and CoCr rotating-platform TKA in the contralateral knee. The mean follow-up period was 5.3 years (range, 1-7 years). The weight of Ti implants was one-third lighter than that of CoCr implants (133.9 g vs 390.1 g, P < .01). Clinical outcomes were evaluated using clinical scores, patient preferences (lightness, comfort, naturalness, and satisfaction), gait analysis (kinetic and kinematic data), range of motion, and degree of pain. Radiologic results were evaluated based on the radiolucent line (RLL), degree of medial tibial bone loss, and loosening as seen on X-ray. RESULTS: No significant differences were observed in clinical scores or patient preference. Regarding implant weight, approximately 70% of patients did not perceive the Ti implant as lighter. No significant differences were observed in gait analysis, range of motion, or degree of pain. The RLL was seen in 9% of the Ti implant group and 19% of the CoCr implant group. CONCLUSION: The lighter Ti implant did not show any clinical benefit over CoCr implants. The lightness of the Ti implant is not sufficient to matter or be noticeable. However, the Ti implant showed lower rate of RLL than the CoCr implant. LEVEL OF EVIDENCE: level I, randomized controlled trial.

Comparative Genome Analyses of Clavibacter michiganensis Type Strain LMG7333T Reveal Distinct Gene Contents in Plasmids From Other Clavibacter Species.

Clavibacter michiganensis, a Gram-positive, plant-pathogenic bacterium belonging to Actinobacteria, is a causal agent of bacterial canker in tomatoes. Although LMG7333T is the type strain of C. michiganensis, it has not been used in many studies, probably because of a lack of the complete genome sequence being available. Therefore, in this study, the complete genome sequence of this type strain was obtained, and comparative genome analysis was conducted with the genome sequences of two other C. michiganensis strains and type strains of Clavibacter species, of which their complete genome sequences are available. C. michiganensis LMG7333T carries one chromosome and two plasmids, pCM1 and pCM2, like two other C. michiganensis strains. All three chromosomal DNA sequences were almost identical. However, the DNA sequences of two plasmids of LMG7333T are similar to those of UF1, but different from those of NCPPB382, indicating that both plasmids carry distinct gene content among C. michiganensis strains. Moreover, 216 protein-coding sequences (CDSs) were only present in the LMG7333T genome compared with type strains of other Clavibacter species. Among these 216 CDSs, approximately 83% were in the chromosome, whereas others were in both plasmids (more than 6% in pCM1 and 11% in pCM2). However, the ratio of unique CDSs of the total CDSs in both plasmids were approximately 38% in pCM1 and 30% in pCM2, indicating that the high gene content percentage in both plasmids of C. michiganensis are different from those of other Clavibacter species, and plasmid DNAs might be derived from different origins. A virulence assay with C. michiganensis LMG7333T using three different inoculation methods, root-dipping, leaf-clipping, and stem injection, resulted in typical disease symptoms, including wilting and canker in tomato. Altogether, our results indicate that two plasmids of C. michiganensis carry distinct gene content, and the genome information of the type strain LMG7333T will help to understand the genetic diversity of the two plasmids of Clavibacter species, including C. michiganensis.

Sex differences in knee joint loading: Cross-sectional study in geriatric population.

This study investigated sex differences in knee biomechanics and investigated determinants for difference in a geriatric population. Age-matched healthy volunteers (42 males and 42 females, average age 65 years) without knee OA were included in the study. Subjects underwent physical examination on their knee and standing full-limb radiography for anthropometric measurements. Linear, kinetic, and kinematic parameters were compared using a three-dimensional, 12-camera motion capture system. Gait parameters were evaluated and determinants for sex difference were evaluated with multiple regression analysis. Females had a higher peak knee adduction moment (KAM) during gait (p = 0.004). Females had relatively wider pelvis and narrower step width (both p < 0.001). However, coronal knee alignment was not significantly different between the sexes. Multiple regression analysis revealed that coronal alignment (b = 0.014, p < 0.001), step width (b = -0.010, p = 0.011), and pelvic width/height ratio (b = 1.703, p = 0.046) were significant determinants of peak KAM. Because coronal alignment was not different between the sexes, narrow step width and high pelvic width/height ratio of female were the main contributors to higher peak KAM in females. Sex differences in knee biomechanics were present in the geriatric population. Increased mechanical loading on the female knee, which was associated with narrow step width and wide pelvis, may play an important role in future development and progression of OA. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1283-1289, 2017.

Intramedullary Nailing for Pathological Fractures of the Proximal Humerus.

BACKGROUND: Endoprosthetic reconstruction is widely applied for pathological fractures of the proximal humerus; however, functional impairment is usually unsatisfactory. The aims of the current study are to evaluate the reliability of interlocking intramedullary (IM) nailing with cement augmentation as a fixation method in proximal humeral lesions and to assess functional outcomes. METHODS: We reviewed 32 patients with pathological fractures of the proximal humerus who underwent interlocking IM nailing and cement augmentation. Functional scores and pain relief were assessed as outcomes. RESULTS: The mean follow-up period was 14.2 months. The mean Musculoskeletal Tumor Society functional score and Karnofsky performance status scale score were 27.7 and 75.6, respectively. Improvement of pain assessed using the visual analogue scale was 6.2 on average. Thirty-one patients (97%) experienced no pain after surgery. The mean ranges of forward flexion and abduction were 115° and 112.6°, respectively. All patients achieved stability and had no local recurrence without failure of fixation until the last follow-up. CONCLUSIONS: Proximal interlocking IM nailing with cement augmentation appears to be a reliable treatment option for pathological or impending fractures of the proximal humerus in selected patients with metastatic tumors, even with extensive bone destruction.

Multiple channeling improves the structural integrity of rotator cuff repair.

BACKGROUND: Multiple channeling is a straightforward additional procedure for rotator cuff repair that creates multiple channels in the greater tuberosity, through which bone marrow of the proximal humerus communicates with the repair site. PURPOSE: To investigate the effect of multiple channeling on clinical and structural outcomes of arthroscopic rotator cuff repair. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: A total of 124 patients with a full-thickness rotator cuff tear were included in the study. Fifty-seven patients underwent arthroscopic rotator cuff repair with multiple channeling (the multiple channeling group) and 67 without it (the conventional group). Analysis of surface markers was performed to characterize the cells recruited by multiple channeling using flow cytometry. Clinical outcomes were evaluated preoperatively and at a minimum of 2 years after surgery (average, 36.79 ± 13.69 months) with respect to pain, range of motion, muscle strength, overall satisfaction, and commonly used functional scores. At a minimum of 9 months after surgery, structural integrity was assessed by magnetic resonance imaging or computed tomography arthrography. RESULTS: Mesenchymal stem cells (MSCs) positive for CD73, CD90, and CD105 and negative for CD45 could be isolated and cultured from bone marrow mononuclear cells of the proximal humerus. Clinical outcomes, including pain, range of motion, strength, overall satisfaction, and functional scores, showed no statistical difference between the 2 groups (all P > .05). The retear rate of the multiple channeling group (22.2%) was significantly lower than that of the conventional group (45.2%) (P = .023). CONCLUSION: The findings of the study showed that multiple channeling significantly decreased the retear rate after arthroscopic rotator cuff repair, probably via the recruitment of endogenous MSCs from the proximal humerus. Although the results did not show significant differences in the clinical outcomes between the 2 groups, better clinical outcomes might be anticipated in the multiple channeling group via improved structural integrity in a long-term follow-up.