Patients With Anterior Cruciate Ligament Rupture and Ipsilateral Segond Fractures Have High Rates of Concurrent Knee Pathology.

Purpose: The purpose of this study was to determine the rates of concomitant knee pathology in patients with ACL injuries and Segond fractures. Methods: A retrospective study is undertaken with patients identified via query of CPT codes for ACL reconstruction from 2014 to 2020. All patients with preoperative radiographs were reviewed for the presence of Segond fractures. Operative reports were analyzed for the presence of concurrent pathology, including meniscus, cartilage, and other ligamentous injuries at the time of arthroscopic ACL reconstruction. Results: A total of 1,058 patients were included in the study. Segond fractures were identified in 50 (4.7%) patients. Ipsilateral concomitant knee pathology was identified in 84% of Segond patients. Thirty-eight (76%) patients had meniscal pathology with a total 49 meniscal injuries, of which 43 were treated operatively. Multiligamentous injuries were present in 16 patients (32%), with 8 patients undergoing further ligament repair/reconstruction at the time of surgery. Chondral injuries were identified in 13 patients (26%). Conclusions: A high prevalence of concomitant meniscal, chondral, and ligamentous injuries was found in patients with Segond fractures. These additional injuries may require further operative management and may place patients at increased risk for future instability or degenerative changes. Patients with Segond fractures should be counseled preoperatively on the nature of their injuries and risk of associated pathologies. Level of Evidence: Level IV, prognostic case series.

Pyoderma Gangrenosum Affecting the Dorsal Hand: A Case Report.

Pyoderma gangrenosum is an uncommon neutrophilic dermatosis characterized by an ulcerative lesion with a violaceous border. Most frequently, these lesions present in the lower extremity and are associated with underlying immune-mediated comorbidities. Infrequently, these lesions may present in the upper extremity, which presents difficult challenges for upper extremity surgeons as the lesions are frequently misdiagnosed as an infectious process. This often leads to inappropriate surgical debridement and antibiotic administration. Local trauma to the lesion can lead to a process of pathergy and worsening of the lesion. Here, we report on a case of cutaneous pyoderma gangrenosum affecting the dorsal hand, originally misidentified as an atypical infection with subsequent unsuccessful surgical debridement. After involvement of a multidisciplinary team, appropriate diagnosis was made, and treatment with local immunosuppressive agents achieved resolution of the lesion.

Two-Tiered Resection of Cam Lesions in Hip Femoroacetabular Impingement: Optimizing Femoral Head Sphericity.

Hip arthroscopy is one of the most rapidly growing fields in orthopaedic surgery. One of the most frequent pathologies treated with hip arthroscopy remains femoroacetabular impingement, which is addressed by labral repair and femoral osteoplasty. The most commonly cited reason for failure of arthroscopic treatment of femoroacetabular impingement is under-resection of the cam lesion. Surgeons frequently use evaluations of preoperative images, intraoperative fluoroscopy, and dynamic range of motion to ensure adequate resection. In this article, we describe a reproducible and standardized technique to assist in appropriate resection. This is achieved by a 2-tiered resection technique: Tier 1 aims to set the depth of resection and restore the head-neck offset. Tier 2 then matches the depth of the resection set by tier 1 and allows for retention of appropriate transition of the proximal convexity to the distal concavity seen in more ideally shaped femoral heads. With this technique, we offer a tool to avoid under-resection in the area of maximal conflict while simultaneously minimizing the risk of proximal over-resection and thus compromising the fluid seal dynamics of the joint in deeper flexion angles.

Ipsilateral ACL injured patients with Segond fractures demonstrate increased posterior tibial slope.

BACKGROUND: The anterolateral complex has been demonstrated to assist with rotational stability and prevention of anterior tibial translation during the pivot shift. In this study the Segond fracture is used as a surrogate for an anterolateral complex injury to determine if there is an association between Segond fracture and increased posterior tibial slope. METHODS: Patients' charts and radiographs were analyzed retrospectively for the presence of Segond fractures on injury radiographs. These patients, the Segond cohort, were then age and gender matched to a control cohort. Demographic as well MRI measurements of medial and lateral posterior tibial slope and lateral-to-medial slope asymmetry were collected for each cohort. Secondary outcome of anterior cruciate ligament reconstruction failure data was also collected. RESULTS: The Segond group demonstrated a statistically significantly greater lateral posterior tibial slope (8.42° versus 6.55°, P = 0.003) as well as medial posterior tibial slope (6.57° versus 5.34° degrees, P = 0.045). There was no significant differences between lateral-to-medial asymmetry (2.18°versus 1.83°, P = 0.246). CONCLUSION: Patients with Segond fractures at the time of anterior cruciate ligament injury have increased medial and lateral posterior tibial slope. This may relate to increased rotational and translational instability associated with anterolateral complex injuries. Surgeons treating these patient may use this information to counsel their patients on the risks of associated pathology at the time of arthroscopy such as lateral meniscal posterior root tears.

The Use of Tumor Prostheses for Primary or Revision Reverse Total Shoulder Arthroplasty With Proximal Humeral Bone Loss.

Background: During shoulder arthroplasty with substantial bone and soft tissue loss, reverse shoulder arthroplasty (RSA) with a tumor prosthesis may restore function, reduce pain, and improve implant fixation. Methods: Thirteen adult patients undergoing RSA using a tumor prosthesis system were retrospectively reviewed. Preoperative visual analog score (VAS), single assessment numeric evaluation (SANE), American Shoulder and Elbow Surgeons (ASES) score, simple shoulder test (SST), and forward flexion were compared to latest follow up. Postoperative radiographs and complications were recorded. Results: Mean age at surgery was 68.4 years. Eight patients had undergone at least 1 prior operation on the indicated shoulder. Six patients required wide excision of proximal humerus tumor. At mean of 34 months postoperatively, significant improvements were noted in VAS (P = .03) and ASES score (P = .04). Active forward elevation was 81.1 degrees. For all patients, postoperative radiographs demonstrated satisfactory alignment. Complications occurred in 38% of patients, with 31% requiring reoperation. Conclusion: In cases of failed shoulder arthroplasty with excessive bone and soft tissue loss or substantial tumor burden, RSA with a tumor prosthesis can reduce pain levels and improve functional outcomes. However, forward elevation remains limited, and postoperative complications are a concern.

Is pin-plate overlap in tibial plateau fractures associated with increased infection rates?

INTRODUCTION: Despite advances in the treatment of high energy proximal tibia fractures, including the utilization of staged management with external fixation, the infection rate remains high. Overlap between external fixator pin sites and definitive internal fixation has been proposed as a risk factor for infection. METHODS: This retrospective study reviews 244 patients with staged knee-spanning external fixation followed by delayed definitive internal fixation at two separate level one trauma centers. Presence of pin-plate overlap as well as several other known risk factors for infection were recorded and measured to include open fractures, compartment syndrome, operative time and number of incisions. Development of deep infection was the primary outcome. Both univariate and multivariate statistics were applied to determine differences in rates of infection. RESULTS: 65 (26.6%) patients had presence of pin-plate overlap while 179 (73.4%) patients had no overlap. There were no differences between overlapping and non-overlapping groups with respect to other infectious risk factors. Deep infection occurred in 34 (13.9%) total patients, 18 (27.7%) were in patients with pin-plate overlap and 16 (8.9%) in those without overlap. (P = 0.003; RR 3.01, 95% CI 1.51-4.76). DISCUSSION: This large, multicenter study demonstrated a statistically significant association between pin-plate overlap and the development of deep infection in tibial plateau fractures. On multivariate analysis, pin-plate overlap was identified as an independent risk factor for infection. When treating these complex injuries, surgeons should consider the definitive fixation construct when placing external fixation pins.

Immunologic Contributions Following Rotator Cuff Injury and Development of Cuff Tear Arthropathy.

¼: Rotator cuff tear arthropathy (RCTA) describes a pattern of glenohumeral degenerative changes following chronic rotator cuff tears that is characterized by superior humeral head migration, erosion of the greater tuberosity of the humeral head, contouring of the coracoacromial arch to create a socket for the humeral head, and eventual glenohumeral arthritis. ¼: Acute and chronic inflammatory changes following rotator cuff tears are thought to contribute to cartilage damage, muscle fibrosis, and fatty infiltration in the glenohumeral joint. ¼: In vitro animal studies targeting various inflammatory modulators, including macrophages, insulin-like growth factor-I, and transforming growth factor-beta pathways, provide promising therapeutic targets to improve healing after rotator cuff tears. ¼: The role of platelet-rich plasma in the treatment and prevention of RCTA has been investigated, with conflicting results.

Identification of Root Rot Resistance QTLs in Pea Using Fusarium solani f. sp. pisi-Responsive Differentially Expressed Genes.

Pisum sativum (pea) yields in the United States have declined significantly over the last decades, predominantly due to susceptibility to root rot diseases. One of the main causal agents of root rot is the fungus Fusarium solani f. sp. pisi (Fsp), leading to yield losses ranging from 15 to 60%. Determining and subsequently incorporating the genetic basis for resistance in new cultivars offers one of the best solutions to control this pathogen; however, no green-seeded pea cultivars with complete resistance to Fsp have been identified. To date, only partial levels of resistance to Fsp has been identified among pea genotypes. SNPs mined from Fsp-responsive differentially expressed genes (DEGs) identified in a preceding study were utilized to identify QTLs associated with Fsp resistance using composite interval mapping in two recombinant inbred line (RIL) populations segregating for partial root rot resistance. A total of 769 DEGs with single nucleotide polymorphisms (SNPs) were identified, and the putative SNPs were evaluated for being polymorphic across four partially resistant and four susceptible P. sativum genotypes. The SNPs with validated polymorphisms were used to screen two RIL populations using two phenotypic criteria: root disease severity and plant height. One QTL, WB.Fsp-Ps 5.1 that mapped to chromosome 5 explained 14.8% of the variance with a confidence interval of 10.4 cM. The other four QTLs located on chromosomes 2, 3, and 5, explained 5.3-8.1% of the variance. The use of SNPs derived from Fsp-responsive DEGs for QTL mapping proved to be an efficient way to identify molecular markers associated with Fsp resistance in pea. These QTLs are potential candidates for marker-assisted selection and gene pyramiding to obtain high levels of partial resistance in pea cultivars to combat root rot caused by Fsp.

Systematic Isolation of Key Parameters for Estimating Skeletal Maturity on AP Hip Radiographs.

BACKGROUND: The ability to estimate skeletal maturity using a hip radiograph does not yet exist, but may have utility in the treatment of scoliosis, slipped capital femoral epiphysis, and lower limb deformity. We sought to develop a fast, accurate, and reproducible method. METHODS: Fourteen hip radiologic parameters were evaluated on serial anteroposterior hip radiographs from 3 years before to 2 years after the skeletal age associated with 90% of final height, a validated skeletal maturity definition which correlates with the timing of peak height velocity. The Greulich and Pyle (GP) left hand bone age was obtained for comparison. Stepwise linear regression and generalized estimating equation analyses were used to isolate key hip and demographic parameters, creating the "optimized Oxford" skeletal maturity system. The accuracy of the optimized Oxford system in predicting years from 90% of final height was evaluated and compared with systems of demographics only, the modified Oxford, demographics+modified Oxford, and demographics+GP. RESULTS: A total of 284 hip radiographs from 41 girls (range: 7 to 15 y) and 38 boys (range: 9 to 17 y) were included. Following multivariate analyses, 5 of the original 14 hip radiographic parameters remained significant. The predictions made by the optimized Oxford model had greater accuracy and fewer outlier predictions (predictions >1 y off from actual years from 90% of final height) than the demographics only and modified Oxford only models (P<0.05 for all). The optimized Oxford model had greater prediction accuracy than the demographics+modified Oxford model, but similar rates of outlier predictions (P=0.903). No differences in mean prediction accuracy or rate of outlier predictions were observed between the optimized Oxford and the demographics+GP model (P>0.05). CONCLUSION: High precision in skeletal maturity estimation can be achieved by using chronological age, sex, and 5 hip radiographic parameters. CLINICAL RELEVANCE: We have developed a skeletal maturity system that utilizes anteroposterior hip radiographs and performs as accurately as GP.

The Current Status of Concussion Assessment Scales: A Critical Analysis Review.

¼: Concussion is a complex pathophysiologic process that affects the brain; it is induced by biomechanical forces, with alteration in mental status with or without loss of consciousness. ¼: Concussion assessment tools may be broadly categorized into (1) screening tests such as the SAC (Standardized Assessment of Concussion), the BESS (Balance Error Scoring System), and the King-Devick (KD) test; (2) confirmatory tests including the SCAT (Sport Concussion Assessment Tool), the ImPACT (Immediate Post-Concussion Assessment and Cognitive Testing), and the VOMS (Vestibular Oculomotor Screening); and (3) objective examinations such as brain network activation (BNA) analysis, imaging studies, and physiologic markers. ¼: The KD, child SCAT3 (cSCAT3), child ImPACT (cImPACT), and VOMS tests may be used to evaluate for concussion in the pediatric athlete. ¼: Future work with BNA, functional magnetic resonance imaging, diffusion tensor imaging, and serum biomarkers may provide more objective assessment of concussion, neurologic injury, and subsequent recovery.

The Natural History of Benign Bone Tumors of the Extremities in Asymptomatic Children: A Longitudinal Radiographic Study.

BACKGROUND: Benign bone tumors are common incidental findings in the pediatric population during radiographic evaluation. Counseling these patients requires reassurance and raises questions about the natural history of these tumors over time. The purpose of this study was to estimate the prevalence and observe the behavior of benign childhood bone tumors in an asymptomatic population. METHODS: A historical, longitudinal radiographic collection of healthy children was reviewed, which included comprehensive left-sided radiographs of the extremities at yearly intervals. In this study, 262 subjects with 25,555 radiographs were screened for benign bone tumors at a median age of 8 years (range, 0 to 18 years). All potential tumors were reviewed by a multidisciplinary panel, which confirmed the radiographic diagnosis of each lesion, the age at which the lesion first appeared, and the age at which it had resolved. Prevalence rates were calculated using the number of distinct subjects available for each radiographic location and age. RESULTS: Thirty-five tumors were identified in 33 subjects, including 19 nonossifying fibromas, 8 enostoses, 6 osteochondromas, and 2 enchondromas. The prevalence rate for all tumors combined increased with age and was 18.9% overall. The overall prevalence rates for specific tumor types were 7.5% for nonossifying fibromas, 5.2% for enostoses, 4.5% for osteochondromas, and 1.8% for enchondromas. Nonossifying fibromas demonstrated a bimodal distribution of prevalence, with a peak at 5 years (10.8%) and another after skeletal maturity (13.3%). The median age at the first appearance for all tumors combined was 9 years (range, 2 to 15 years), but varied by tumor type. Nonossifying fibromas often resolved (7 [37%] of 19), with further resolution possible beyond the last available radiograph. Enostoses, osteochondromas, and enchondromas persisted until the last available radiographs in all subjects. CONCLUSIONS: The prevalence of benign childhood bone tumors of the extremities was 18.9% in a historical asymptomatic population. Longitudinal radiographs allowed observation of the timing of the first appearance and the potential for resolution for each tumor type. These findings provide unique evidence to answer many commonly encountered questions when counseling patients and their families on benign bone tumors. LEVEL OF EVIDENCE: Prognostic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Greater Trochanter Height: A Quantitative Predictor of Skeletal Maturity.

BACKGROUND: The purpose of this investigation was to develop a quantitative and reproductible method for estimating skeletal maturity based on measurements of the height of the bony greater trochanter (GT) using timing to 90% of final height as a gold standard. METHODS: Bony GT height was measured using serial anteroposterior pelvic radiographs in 76 healthy pediatric patients obtained from the Bolton-Brush (BB) Study with corresponding Greulich-Pyle (GP) bone ages. Chronologic age at 90% of final height was calculated. GT height was then measured in 300 contemporary patients aged 4 to 18 years, evenly divided based on sex and race. Bony GT height was compared between BB and contemporary patients, while linear mixed-effects models were used to examine for potential predictors of years to 90% final height using patient sex, GP bone age and bony GT height measurements. RESULTS: Bony GT height was measured in 303 radiographs from the BB Collection (n=37 males; n=39 females) with corresponding GP bone ages, chronological ages, and heights to represent skeletal maturity. Mean age at 90% final height was 13.3±0.6 years for males and 11.4±0.8 years for females. When controlling for patient age and sex, multiple regression analysis revealed that contemporary patients possessed significantly greater bony GT height (mean difference: 1.15 mm; P=0.001) when compared with BB patients. Multivariate analysis showed that combining bony GT height, GP bone age, and sex significantly predicted years to 90% total growth (P<0.001) and explained ∼85% (95% confidence interval for R2: 82%-87%) of the total variance in years using 90% of final height, with sex, GP bone age, and GT height all significant contributors. CONCLUSIONS: Including bony GT height provides more accurate prediction of 90% final height when combined with GP bone age and sex. GT height offers an efficient and accurate parameter that may be utilized in pediatric orthopedic conditions requiring a quantitative estimate of bone age in children with prior pelvis or hip imaging. LEVEL OF EVIDENCE: Level II-diagnostic study.

Identification of Fusarium solani f. sp. pisi (Fsp) Responsive Genes in Pisum sativum.

Pisum sativum (pea) is rapidly emerging as an inexpensive and significant contributor to the plant-derived protein market. Due to its nitrogen-fixation capability, short life cycle, and low water usage, pea is a useful cover-and-break crop that requires minimal external inputs. It is critical for sustainable agriculture and indispensable for future food security. Root rot in pea, caused by the fungal pathogen Fusarium solani f. sp. pisi (Fsp), can result in a 15-60% reduction in yield. It is urgent to understand the molecular basis of Fsp interaction in pea to develop root rot tolerant cultivars. A complementary genetics and gene expression approach was undertaken in this study to identify Fsp-responsive genes in four tolerant and four susceptible pea genotypes. Time course RNAseq was performed on both sets of genotypes after the Fsp challenge. Analysis of the transcriptome data resulted in the identification of 42,905 differentially expressed contigs (DECs). Interestingly, the vast majority of DECs were overexpressed in the susceptible genotypes at all sampling time points, rather than in the tolerant genotypes. Gene expression and GO enrichment analyses revealed genes coding for receptor-mediated endocytosis, sugar transporters, salicylic acid synthesis, and signaling, and cell death were overexpressed in the susceptible genotypes. In the tolerant genotypes, genes involved in exocytosis, and secretion by cell, the anthocyanin synthesis pathway, as well as the DRR230 gene, a pathogenesis-related (PR) gene, were overexpressed. The complementary genetic and RNAseq approach has yielded a set of potential genes that could be targeted for improved tolerance against root rot in P. sativum. Fsp challenge produced a futile transcriptomic response in the susceptible genotypes. This type of response is hypothesized to be related to the speed at which the pathogen infestation advances in the susceptible genotypes and the preexisting level of disease-preparedness in the tolerant genotypes.

Plastid transit peptides-where do they come from and where do they all belong? Multi-genome and pan-genomic assessment of chloroplast transit peptide evolution.

Subcellular relocalization of proteins determines an organism's metabolic repertoire and thereby its survival in unique evolutionary niches. In plants, the plastid and its various morphotypes import a large and varied number of nuclear-encoded proteins to orchestrate vital biochemical reactions in a spatiotemporal context. Recent comparative genomics analysis and high-throughput shotgun proteomics data indicate that there are a large number of plastid-targeted proteins that are either semi-conserved or non-conserved across different lineages. This implies that homologs are differentially targeted across different species, which is feasible only if proteins have gained or lost plastid targeting peptides during evolution. In this study, a broad, multi-genome analysis of 15 phylogenetically diverse genera and in-depth analyses of pangenomes from Arabidopsis and Brachypodium were performed to address the question of how proteins acquire or lose plastid targeting peptides. The analysis revealed that random insertions or deletions were the dominant mechanism by which novel transit peptides are gained by proteins. While gene duplication was not a strict requirement for the acquisition of novel subcellular targeting, 40% of novel plastid-targeted genes were found to be most closely related to a sequence within the same genome, and of these, 30.5% resulted from alternative transcription or translation initiation sites. Interestingly, analysis of the distribution of amino acids in the transit peptides of known and predicted chloroplast-targeted proteins revealed monocot and eudicot-specific preferences in residue distribution.

Structural and biochemical analysis of phosphoethanolamine methyltransferase from the pine wilt nematode Bursaphelenchus xylophilus.

In free-living and parasitic nematodes, the methylation of phosphoethanolamine to phosphocholine provides a key metabolite to sustain phospholipid biosynthesis for growth and development. Because the phosphoethanolamine methyltransferases (PMT) of nematodes are essential for normal growth and development, these enzymes are potential targets of inhibitor design. The pine wilt nematode (Bursaphelenchus xylophilus) causes extensive damage to trees used for lumber and paper in Asia. As a first step toward testing BxPMT1 as a potential nematicide target, we determined the 2.05 Å resolution x-ray crystal structure of the enzyme as a dead-end complex with phosphoethanolamine and S-adenosylhomocysteine. The three-dimensional structure of BxPMT1 served as a template for site-directed mutagenesis to probe the contribution of active site residues to catalysis and phosphoethanolamine binding using steady-state kinetic analysis. Biochemical analysis of the mutants identifies key residues on the ß1d-α6 loop (W123F, M126I, and Y127F) and ß1e-α7 loop (S155A, S160A, H170A, T178V, and Y180F) that form the phosphobase binding site and suggest that Tyr127 facilitates the methylation reaction in BxPMT1.

Poverty, a risk factor overlooked: a cross-sectional cohort study comparing poverty rate and cardiovascular disease outcomes in the state of Florida.

The purpose of this study is to examine the relationship between poverty rate and heart disease in our state. A cross-sectional data analysis was performed using figures provided by the Center for Disease Control's Interactive Atlas of Heart Disease and Stroke Tables. Spearman's correlations and simple regressions were used to determine if there was a relationship between poverty and cardiovascular hospitalization rate and cardiovascular death rate. There was a positive monotonic correlation between poverty rate and cardiovascular hospitalization rate (Rho=0.384, P=0.001). There was a positive monotonic correlation between poverty rate and cardiovascular death rate (Rho=0.646, P<0.0001). County poverty rate had a statistically significant positive relationship with cardiovascular hospitalization and cardiovascular mortality in the state of Florida.

Rapid Onset of B12 Deficiency in the Setting of Worsening Multiple Myeloma: Correlations between B12 Deficiency and Multiple Myeloma.

A 67-year-old female with a relapse of multiple myeloma after being in remission for approximately 2 years following autologous stem cell transplant presented with worsening pancytopenia, over a three-month period. There were an increase in her monoclonal spike at 3.13 g/dL on serum protein electrophoresis, low serum B12 levels, and positive intrinsic factor antibodies. Three months before, she had normal B12 levels and a significantly lower monoclonal spike of 1.07 g/dL. She was diagnosed with B12 deficiency with pernicious anaemia in the setting of her worsening myeloma. Multiple myeloma (MM) has been linked with B12 deficiency and pernicious anaemia. Several mechanisms have been described regarding the pathogenesis of B12 deficiency in such patients. Increased tumour activity can further perpetuate the development of B12 deficiency in such patients. With regard to our case, the increase in tumour activity and onset of pernicious anaemia could have contributed to the rapid development of B12 deficiency. In contrast to this, rapid development of B12 deficiency could also signify relapse or worsening of the myeloma as seen in our case. Physicians ought to consider B12 deficiency in patients with worsening pancytopenia and myeloma.

Necrotizing Pseudomonas aeruginosa Community-Acquired Pneumonia: A Case Report and Review of the Literature.

Lung cavities are not typically associated with community-acquired pneumonia (CAP). CAP due to P. aeruginosa is rare and even less commonly causes necrotizing pneumonia. We report a case of P. aeruginosa CAP that progressed to necrotizing pneumonia and was eventually fatal. Procalcitonin (PCT) has been well investigated in guiding antibiotic therapy (especially CAP) in adults. In this case, PCT at presentation and sequentially was negative. We discuss this caveat and present hypotheses as to the sensitivity and specificity of PCT and C-reactive protein (CRP) in these patients. To better characterize P. aeruginosa CAP, we undertook a review of cases indexed in PubMed from 2001 to 2016 (n = 9). The data reveal that risk factors for P. aeruginosa CAP include smoking, alcohol use, obstructive lung disease, sinusitis, and hot tub use. The route of infection for P. aeruginosa CAP remains unknown. One of the most interesting findings on reviewing cases was that P. aeruginosa CAP involves the right upper lobe in the vast majority. We suggest that when physicians in the community see patients with distinctly upper lobe necrotizing or cavitary pneumonia, they should consider P. aeruginosa in their differential diagnosis. Further studies are needed to clarify route of infection, role of PCT and CRP, and optimal therapy including drug and duration.

Sequence analysis and functional characterization of the dialkylglycine decarboxylase gene DGD1 from Mycosphaerella graminicola.

Dialkylglycine decarboxylase is a pyridoxal phosphate-dependent enzyme in the aminotransferases class III group of enzymes. The enzyme is unique in terms of catalyzing both decarboxylation and transamination. Although the enzymatic activity is present in some bacteria and fungi, the biological role is unclear. We identified and disrupted the dialkylglycine decarboxylase-encoding gene DGD1 in the wheat blotch fungus Mycosphaerella graminicola by transposon-arrayed gene knockout. The DGD1 gene is highly similar to dialkylglycine decarboxylase from the soil bacterium Burkholderia cepacia. Phylogenetic analysis of various class III aminotransferases showed that dialkylglycine decarboxylases from bacteria and fungi are found in a distinct cluster. Functional analysis revealed that dgd1 disruption mutants display wild-type morphology and pathogenicity to wheat. The dgd1 mutants cannot utilize 2-methylalanine as a sole nitrogen source, as assessed by large-scale nutritional utilization analysis. This is the first description of a mutant phenotype of the fungal dialkylglycine decarboxylase gene.

Efficient gene identification and targeted gene disruption in the wheat blotch fungus Mycosphaerella graminicola using TAGKO.

TAGKO ( transposon- arrayed gene knock out) is a highly efficient method for gene discovery and gene function assignment in the rice blast fungus Magnaporthe grisea. Here, we report the application of genome-wide TAGKO to the wheat blotch fungus Mycosphaerella graminicola, including the successful development of electroporation-based transformation for this fungus. A M. graminicola genomic cosmid library was constructed and a pool of 250 cosmid clones was mutagenized by in vitro transposition. Sequence analysis identified 5,110 unique insertion events in the M. graminicola genome. Eleven transposon-tagged cosmid clones (TAGKO clones) were chosen and transformed into the wild-type strain by electroporation. Ten TAGKO clones out of 11 produced gene-specific mutants at a targeting frequency of 15-28%, significantly higher than that of conventional gene-disruption constructs. The remaining clone failed to produce viable mutants, thereby providing indirect evidence for the identification of an essential gene.