R. K. W. Smith

Genome Sequence of Babesia bovis and Comparative Analysis of Apicomplexan Hemoprotozoa

Babesia bovis is an apicomplexan tick-transmitted pathogen of cattle imposing a global risk and severe constraints to livestock health and economic development. The complete genome sequence was undertaken to facilitate vaccine antigen discovery, and to allow for comparative analysis with the related apicomplexan hemoprotozoa Theileria parva and Plasmodium falciparum. At 8.2 Mbp, the B. bovis genome is similar in size to that of Theileria spp. Structural features of the B. bovis and T. parva genomes are remarkably similar, and extensive synteny is present despite several chromosomal rearrangements. In contrast, B. bovis and P. falciparum, which have similar clinical and pathological features, have major differences in genome size, chromosome number, and gene complement. Chromosomal synteny with P. falciparum is limited to microregions. The B. bovis genome sequence has allowed wide scale analyses of the polymorphic variant erythrocyte surface antigen protein (ves1 gene) family that, similar to the P. falciparum var genes, is postulated to play a role in cytoadhesion, sequestration, and immune evasion. The ∼150 ves1 genes are found in clusters that are distributed throughout each chromosome, with an increased concentration adjacent to a physical gap on chromosome 1 that contains multiple ves1-like sequences. ves1 clusters are frequently linked to a novel family of variant genes termed smorfs that may themselves contribute to immune evasion, may play a role in variant erythrocyte surface antigen protein biology, or both. Initial expression analysis of ves1 and smorf genes indicates coincident transcription of multiple variants. B. bovis displays a limited metabolic potential, with numerous missing pathways, including two pathways previously described for the P. falciparum apicoplast. This reduced metabolic potential is reflected in the B. bovis apicoplast, which appears to have fewer nuclear genes targeted to it than other apicoplast containing organisms. Finally, comparative analyses have identified several novel vaccine candidates including a positional homolog of p67 and SPAG-1, Theileria sporozoite antigens targeted for vaccine development. The genome sequence provides a greater understanding of B. bovis metabolism and potential avenues for drug therapies and vaccine development.

Implantation of bone marrow‐derived mesenchymal stem cells demonstrates improved outcome in horses with overstrain injury of the superficial digital flexor tendon

REASONS FOR PERFORMING STUDY Mesenchymal stem (progenitor; stromal) cell (MSC) therapy has gained popularity for the treatment of equine tendon injuries but without reports of long-term follow-up. OBJECTIVES To evaluate the safety and reinjury rate of racehorses after intralesional MSC injection in a large study of naturally occurring superficial digital flexor tendinopathy and to compare these data with those published for other treatments. METHODS Safety was assessed clinically, ultrasonographically, scintigraphically and histologically in a cohort of treated cases: 141 client-owned treated racehorses followed-up for a minimum of 2 years after return to full work. Reinjury percentages were compared to 2 published studies of other treatments with similar selection criteria and follow-up. The number of race starts, discipline, age, number of MSCs injected and interval between injury and treatment were analysed. RESULTS There were no adverse effects of the treatment with no aberrant tissue on histological examination. The reinjury percentage of all racehorses with follow-up (n = 113) undergoing MSC treatment was 27.4%, with the rate for flat (n = 8) and National Hunt (n = 105) racehorses being 50 and 25.7%, respectively. This was significantly less than published for National Hunt racehorses treated in other ways. No relationship between outcome and age, discipline, number of MSCs injected or injury to implantation interval was found. CONCLUSIONS Whilst recognising the limitations of historical controls, this study has shown that MPC implantation is safe and appears to reduce the reinjury rate after superficial digital flexor tendinopathy, especially in National Hunt racehorses. POTENTIAL RELEVANCE This study has provided evidence for the long-term efficacy of MSC treatment for tendinopathy in racehorses and provides support for translation to human tendon injuries.

THE DISTRIBUTION OF CARTILAGE OLIGOMERIC MATRIX PROTEIN (COMP) IN TENDON AND ITS VARIATION WITH TENDON SITE, AGE AND LOAD

A protein prominent in guanidine hydrochloride extracts of adult bovine and equine digital flexor tendons was confirmed to be Cartilage Oligomeric Matrix Protein (COMP) by non-reducing and reducing SDS-PAGE, reaction with rabbit anti-COMP polyclonal antiserum on Western blots, trypsin digestion followed by HPLC on a C2/C18 column, and identification of COMP mRNA from tendon on Northern blots. Immunohistochemistry and Western blots of extracts showed COMP to be present in all regions of digital flexor tendons. Equine tendon COMP was purified by ion exchange chromatography and gel filtration and used in a heterologous inhibition ELISA to quantify COMP in equine digital flexor tendons at different ages, and in other tendons and ligaments. Mean COMP levels in digital flexor tendon were approximately 2-5mg/g wet weight, but they showed a large variation. Levels were low in neonatal tendon but rose rapidly during growth, with the metacarpal (tensional) superficial digital flexor tendon having the highest levels (approximately 10mg/g wet weight). Levels subsequently declined in this region, while in areas which experience a variable amount of compression, levels increased less but then remained constant. Extensor tendons and collateral ligaments, which experience less loading in vivo, had levels similar to those in neonatal tendon. COMP was identified in scarred skin and granulation tissue but not in normal skin, chronic fibrosis, or a fibrosarcomatous skin growth. A unilateral non-weight-bearing growing animal contained three to six times more COMP in the weight-bearing digital flexor tendons compared to the paralyzed limb, while the extensor tendons had similar amounts in both limbs. With the recent discovery of a COMP gene mutation causing pseudoachondroplasia (Hecht et al., 1995), in which lax tendons and ligaments are a feature, the present data suggest that COMP is synthesized in response to, and is necessary for tendon to resist, load.

Mutations of the intronic IgH enhancer and its flanking sequences differentially affect accessibility of the JH locus.

To investigate the role of intronic immunoglobulin heavy chain (IgH) enhancer (E mu) in generating accessibility of the JH locus for VDJ recombination, we generated ES cells in which E mu or its flanking sequences were mutated by replacement with or insertion of an expressed neor gene. Heterozygous mutant ES cells were used to generate chimeric mice from which pre‐B cell lines were derived by transformation of bone marrow cells with Abelson murine leukemia virus (A‐MuLV). Comparison of the rearrangement status of the normal and mutated alleles in individual pre‐B cell lines allowed us to assay for cis‐acting effects of the mutations. Replacement of a 700 bp region immediately downstream from the core E mu [which includes part of the 3′ matrix associated region (MAR) and the I mu exon] had no obvious effect on rearrangement of the targeted allele, indicating that insertion of a transcribed neor gene into the JH‐C mu intron does not affect JH accessibility. In contrast, replacement of an overlapping 1 kb DNA fragment that contains the E mu resulted in a dramatic cis‐acting inhibition of rearrangement, demethylation and germline transcription of the associated JH locus. Surprisingly, insertion of the neor gene into the 5′ MAR sequence approximately 100 bp upstream of the core E mu also dramatically decreased recombination of the linked JH locus; but, in many lines, did not prevent demethylation of this locus. We conclude that integrity of the E mu and upstream flanking sequences is required for efficient rearrangement of the JH locus and that demethylation of this locus, per se, does not necessarily make it a good substrate for VDJ recombination.

Superficial digital flexor tendonitis in the horse

The superficial digital flexor tendon (SDFT) is an elastic structure that during maximal exercise appears to operate close to its functional limits. The biomechanical and biochemical responses to exercise, injury, and healing are still poorly understood but ongoing research is providing valuable new information which is addressed in this review. It appears that the SDFT matures early, after which time it has limited ability to adapt to stress and undergoes progressive degeneration. Focal hypocellularity, collagen fibril degeneration, selective fibril loading and alterations in the noncollagenous matrix occur primarily within the central core region of the midmetacarpal segment. Current treatment strategies have had equivocal results in returning animals to optimal athletic activity. To date it would seem that progressive rehabilitation programmes coupled with regular ultrasonographic evaluations are a cost-effective and comparable strategy when compared to surgical treatment methods. Recent interest in pharmacological modulation of intrinsic healing of collagenous structures has led to the investigation of various growth factors as potential therapeutic aids in the healing of tendon injuries. However, one of the major goals in tendon research, and one which holds the most optimism for success in the immediate future, is the prevention of tendon injuries.

Mesenchymal stem cell therapy for equine tendinopathy

Optimal management of tendon overuse injuries in equine and human athletes should avoid the formation of excessive scar tissue, regenerate normal tendon matrix, and reduce re-injury rates. We hypothesized that the implantation of marrow-derived stromal stem cells (BM-MSCs), in far greater numbers than are present normally within tendon tissue, would synthesize a matrix more closely resembling tendon matrix than scar tissue, and hence increase the capacity to return to performance successfully. This article reviews the technique used clinically in the horse and the current outcome data for horses treated by the autologous implantation of BM-MSCs into moderate to severe acute superficial digital flexor tendon (SDFT) injuries. Bone marrow was aspirated from the sternum under standing sedation. The nucleated adherent cell population (containing the BM-MSCs) were isolated and expanded so that, after approximately three weeks, the cells were re-suspended in the supernatant from the bone marrow and implanted into injured SDFT under ultrasonographic guidance. The horses then entered a 48-week rehabilitation period consisting of an ascending exercise regime. By September 2006, 168 racehorses had undergone this regimen. For horses which had returned to full work, 18% had re-injured, which compared favourably to previous studies on conventional management (56% re-injury rate). No adverse effects were noted other than needle tracts visible ultrasonographically. Autologous implantation of mesenchymal stem cells into tendon injuries may therefore improve clinical outcome although definitive proof of efficacy, which is still lacking, will require randomized controlled trials.

Complete reannotation of the Arabidopsis genome: methods, tools, protocols and the final release

BackgroundSince the initial publication of its complete genome sequence, Arabidopsis thaliana has become more important than ever as a model for plant research. However, the initial genome annotation was submitted by multiple centers using inconsistent methods, making the data difficult to use for many applications.ResultsOver the course of three years, TIGR has completed its effort to standardize the structural and functional annotation of the Arabidopsis genome. Using both manual and automated methods, Arabidopsis gene structures were refined and gene products were renamed and assigned to Gene Ontology categories. We present an overview of the methods employed, tools developed, and protocols followed, summarizing the contents of each data release with special emphasis on our final annotation release (version 5).ConclusionOver the entire period, several thousand new genes and pseudogenes were added to the annotation. Approximately one third of the originally annotated gene models were significantly refined yielding improved gene structure annotations, and every protein-coding gene was manually inspected and classified using Gene Ontology terms.

Annotation of the Arabidopsis Genome

The Arabidopsis Genome Sequencing Project was officially completed in late 2000, leading to the publication of a landmark paper describing, in broad outline, many salient features of the Arabidopsis genome ([Arabidopsis Genome Initiative [AGI], 2000][1]). However, the genome annotation, generated by

Prevalence of superficial digital flexor tendonitis and suspensory desmitis in Japanese Thoroughbred flat racehorses in 1999

REASONS FOR PERFORMING STUDY Overstrain injuries to the superficial digital flexor tendon (SDFT) and suspensory ligament (SI) are among the most common musculoskeletal injuries which contribute to the considerable wastage of racing Thoroughbreds. Many epidemiological studies have demonstrated the prevalence of and risk factors for tendon injury when racing but have not included those injuries sustained during training. However, since tendon injury during training is seen commonly in clinical practice, it is appropriate to determine the overall prevalence of tendon injury sustained during both training and racing. OBJECTIVE To determine the prevalence of overstrain injury to the SDFT and SL during training and racing among Thoroughbred flat racehorses in Japan in 1999. METHODS A retrospective study was performed using a sample population of 10,262 Thoroughbred racehorses. The medical information database of Thoroughbred racehorses registered by the Japan Racing Association (JRA) in 1999 was analysed for SDFT and SL overstrain injury diagnosed by a veterinarian employed by JRA during training and racing. Jump racehorses were excluded from this study. RESULTS The prevalence of forelimb SDFT tendonitis and SL desmitis was 11.1% (1130 cases) and 3.61% (370 cases) of the population, respectively. In the hindlimb, there were 0.06% (6 cases) and 0.14% (14 cases), respectively. Risks of SDF tendonitis in the forelimb in 3-year-olds or older horses were significantly higher than in 2-year-olds. In contrast, the risk of SL desmitis in the forelimb at age 3 and 4 years was 2.23 and 2.11 times higher, respectively, than in 2-year-olds, but this increased to 5.07 times in those age > or = 5 years. Entire males were at greater risk in comparison to females and geldings. CONCLUSIONS The results suggest that the prevalence of SDF tendonitis and SL desmitis in the forelimb was associated with the horses age and sex. The prevalence of SL desmitis increased further with age compared with SDF tendonitis, possibly reflecting a more rapid accumulation of degeneration in this structure. POTENTIAL RELEVANCE The age-related risk demonstrated in this study provides further support that overstrain injuries are associated with accumulated degeneration. These data provide a valuable resource for further research into the aetiology of tendon injury in the racehorse.

Variants within the MMP3 gene are associated with Achilles tendinopathy: possible interaction with the COL5A1 gene

Objectives: Sequence variation within the COL5A1 and TNC genes are known to associate with Achilles tendinopathy. The primary aim of this case-control genetic association study was to investigate whether variants within the matrix metalloproteinase 3 (MMP3) gene also contributed to both Achilles tendinopathy and Achilles tendon rupture in a Caucasian population. A secondary aim was to establish whether variants within the MMP3 gene interacted with the COL5A1 rs12722 variant to raise risk of these pathologies. Methods: 114 subjects with symptoms of Achilles tendon pathology and 98 healthy controls were genotyped for MMP3 variants rs679620, rs591058 and rs650108. Results: As single markers, significant associations were found between the GG genotype of rs679620 (OR = 2.5, 95% CI 1.2 to 4.90, p = 0.010), the CC genotype of rs591058 (OR = 2.3, 95% CI 1.1 to 4.50, p = 0.023) and the AA genotype of rs650108 (OR = 4.9, 95% CI 1.0 to 24.1, p = 0.043) and risk of Achilles tendinopathy. The ATG haplotype (rs679620, rs591058, and rs650108) was under-represented in the tendinopathy group when compared to the control group (41% vs 53%, p = 0.038). Finally, the G allele of rs679620 and the T allele of COL5A1 rs12722 significantly interacted to raise risk of AT (p = 0.006). No associations were found between any of the MMP3 markers and Achilles tendon rupture. Conclusion: Variants within the MMP3 gene are associated with Achilles tendinopathy. Furthermore, the MMP3 gene variant rs679620 and the COL5A1 marker rs12722 interact to modify the risk of tendinopathy. These data further support a genetic contribution to a common sports related injury.