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BACKGROUND OBJECTIVE: The purpose of this study was to evaluate prosthetic outcome in patients with severe congenital femoral deficiency and the potential benefits of surgical intervention on prosthetic fitting and gait. METHODS: A retrospective review identified 26 active case records with a proximal femoral focal deficiency using a prosthesis. Validated outcome measures evaluated comfort, function, and prosthetic use and quality-of-life assessment. Outcome compared age groups and surgical intervention. Gait analysis performed in 7 patients further evaluated hip and knee function. RESULTS: Eleven male patients and 15 female patients, including 13 children (mean age 10 years, range 5-16) and 13 adults (mean age 36 years, range 23-63) were evaluated. Better prosthetic function and PedsQL scores were recorded in the pediatric group. There was a trend for better scores after surgery. Gait analysis demonstrated reduced hip extension compensated by knee flexion in 3 patients, 2 patients had hip extension with near normal kinematics, 1 untreated patient walked with an unsteady gait, and the remaining walked well using an ischial-bearing prosthesis with pelvic compensatory movements. CONCLUSION: The management strategy in severe proximal femoral focal deficiency remains a major challenge. Hip reconstruction seems to improve functional scores. Overall, the scores seem to decline into adulthood but not significantly. Gait analysis before further surgical intervention is recommended because compensatory knee flexion may improve step length in terminal stance. Limited numbers, with short follow-up, prevents clear guidance on the benefit of surgery.

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INTRODUCTION: Miserable malalignment syndrome is a complex torsional lower limb deformity with limited consensus on surgical treatment. We present the outcome of de-rotation of the tibia alone using an external fixator. METHODS: Fifteen patients (22 segments) were operated on between 2012 and 2020; 13 presented with anterior knee pain, and two presented with out-toeing. Gait analysis was done in nine patients, and CT scan rotational profile, including tibial tubercle-trochlear groove distance, femoral version, and tibial torsion, were calculated. Kujala knee pain score and visual analogue pain score (VAS) were recorded. All underwent infra-tubercular osteotomy of the tibia and midshaft osteotmy of the fibula and application of a hexapod circular frame to gradually internally rotate the tibia until the foot aligned with the patella. RESULTS: There was no preoperative clinical or radiographic evidence for patellar instability, femoral anteversion 30° (21°-54°), and external tibial torsion 50° (37-70). The mean age at surgery was 21 years (12-37) with a mean follow-up of 20 months (9-83). All osteotomies healed, and the frames were removed at a mean of 111 days (80-168). The mean VAS score improved from 8(5-9) to 1(0-4) postoperatively (P < 0.001). The mean Kujala knee pain score increased from 53 (30-75) to 92 (54-100) postoperatively (P < 0.001). The mean preoperative foot progression angle (FPA) was 37° (20°-50°), with 13 postoperatively walking with neutral FPA. One patient walked with symmetrical + 10° and the other with - 5° FPA. All patients reported relief of knee pain and were satisfied with the alignment. CONCLUSION: Gradual correction of severe external tibia torsion with a hexapod external fixator and an infra-tubercle tibial osteotomy could provide an optimum method to eliminate knee pain and improve limb alignment in miserable malalignment syndrome.

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Background Fractures of the distal radius are a common injury. The British Orthopaedic Association (BOA) and The British Society for Surgery of the Hand (BSSH) have released new guidelines outlining the management of these fractures, specifically identifying "thresholds for intervention," based on radiological parameters for management with open reduction and internal fixation (ORIF). Questions/Purposes Have our distal radius fractures (DRFs), previously managed with ORIF, met the new guidelines' thresholds for intervention, based on radiological parameters? Patients and Methods A retrospective assessment of DRFs treated with ORIF was performed between January 2017 and August 2018. Patients were categorized into three cohorts based on their age. The five radiological parameters of ulnar variance, dorsal tilt, radial inclination, radial height, and intra-articular step were measured on the initial plain radiograph, "pre-manipulation film," postplaster application radiograph, and "post-manipulation film." These were compared with the "thresholds for intervention" outlined in the BOA/BSSH guidelines. Results A total of 94 patients underwent an ORIF with a mean age of 56 years (range 17-86 years). As many as 75.74% of patients on the "pre-manipulation film" met the "threshold for intervention" on at least one radiological parameter, while 53.57% of patients on the "post-manipulation" met at least one "threshold for intervention." Dorsal tilt was the parameter that most often met the threshold in both films at 53.37% and 40.11%, respectively. Conclusion Within our trust, there is a tendency to over manage the distal radius fracture with ORIF, potentially resulting in unnecessary operations. Education surrounding the new guidelines will better serve our decision-making. Level of Evidence This is a level III study.

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BACKGROUND: Virtual fracture clinics (VFCs) have become widely adopted, aiming to improve efficiency, standardise patient care and reduce clinic appointments for injuries that can be managed conservatively. A variety of means exist to manage VFC referrals and assessment, including paper-based and digital methods. This study assesses VFC referral quality and outcomes before and after implementation of a digital VFC referral and management system. METHODS: A retrospective analysis was conducted of all VFC referrals and assessments from July 2017-March 2020 in a large UK district general hospital. All referrals and assessments were analysed for quality and completeness of referral information, grade of assessor, outcome of assessment, referral-to-assessment time, and assessment-to-surgery time (for those requiring operative management). RESULTS: 3038 paper and 9,228 digital referrals were analysed by 2 separate reviewers. Quality and completeness of referral information showed significant improvement in 11 predetermined key data points with the digital referral system (p < 0.001). Date and mechanism of injury were the most commonly missing data criteria (67.5% and 68.2%, respectively) with paper referrals. Significant improvements were noted in the proportion of Consultant delivered VFC assessments (84.2% vs 71.0%; p < 0.001), VFC discharge rate (20.8% vs 13.1%; p < 0.001) and patients recalled for urgent review (6.2% vs 0.8%; p < 0.001) with digital referrals. Mean referral-to-assessment (31.2 vs 49.9 h; p < 0.001) and assessment-to-surgery (9.2 vs 13.0 days; p = 0.01) times also reduced significantly with referral digitisation. CONCLUSION: Improvements in virtual referral quality and completeness directly lead to facilitation of more thorough, detailed and appropriate virtual assessments; improving timely decision-making, reducing unnecessary appointments, and permitting better prioritisation of workload and earlier surgery for patients requiring operative treatment. Purpose-built digital solutions are an excellent means of achieving these aims.

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Arthritis of the hip is a degenerative disease characterised by pain and inflammation. It is common and most often affects middle-aged to older adults, with the definitive management being total hip replacement. Advances in the surgical techniques has brought about the popularity of hip preservation surgery in patients with pre-arthritic hip abnormalities, with a goal to prevent progression to early arthritis and subsequently prolong the need for arthroplasty. There is a large body of evidence correlating femoroacetabular impingement (FAI) and the progression of osteoarthritis. Hip arthroscopy is a successful technique in the management of FAI and labral damage. There is, however, less evidence behind its use in those patients with established arthritic changes. After review of such evidence, we believe hip arthroscopy, and other hip preservation procedures, have a key role, and should be considered in the management of early hip arthritis. However, there is no role for such procedures in end-stage arthritis.

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BACKGROUND: The prevalence of self-reported shoulder pain in the UK has been estimated at 16%. This has been linked with significant sleep disturbance. It is possible that this relationship is bidirectional, with both symptoms capable of causing the other. Within the field of sleep monitoring, there is a requirement for a mobile and unobtrusive device capable of monitoring sleep posture and quality. This study investigates the feasibility of a wearable sleep system (WSS) in accurately detecting sleeping posture and physical activity. METHODS: Sixteen healthy subjects were recruited and fitted with three wearable inertial sensors on the trunk and forearms. Ten participants were entered into a 'Posture' protocol; assuming a series of common sleeping postures in a simulated bedroom. Five participants completed an 'Activity' protocol, in which a triphasic simulated sleep was performed including awake, sleep and REM phases. A combined sleep posture and activity protocol was then conducted as a 'Proof of Concept' model. Data were used to train a posture detection algorithm, and added to activity to predict sleep phase. Classification accuracy of the WSS was measured during the simulations. RESULTS: The WSS was found to have an overall accuracy of 99.5% in detection of four major postures, and 92.5% in the detection of eight minor postures. Prediction of sleep phase using activity measurements was accurate in 97.3% of the simulations. The ability of the system to accurately detect both posture and activity enabled the design of a conceptual layout for a user-friendly tablet application. CONCLUSIONS: The study presents a pervasive wearable sensor platform, which can accurately detect both sleeping posture and activity in non-specialised environments. The extent and accuracy of sleep metrics available advances the current state-of-the-art technology. This has potential diagnostic implications in musculoskeletal pathology and with the addition of alerts may provide therapeutic value in a range of areas including the prevention of pressure sores.

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Fractures of the coracoid process are uncommon and when they do occur, are often mistaken for injuries to the acromi oclavicular joint. We report a case of a 15-year-old boy who sustained a Salter-Harris Type 1 fracture through his coracoid process alongside strain of the acromioclavicular and coracoclavicular ligaments. Additional imaging, specifically MRI, was critical in both correctly identifying this injury as a coracoid process fracture and also in determining that conservative management was the best course of action. Optimum management of such injuries remains controversial, specifically with regards to skeletally immature patients. In our case, the injury was identified clearly on MRI and managed conservatively, with the patient making a full recovery and a return to contact rugby after 3 months.

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African trypanosomes are mammalian pathogens that must regularly change their protein coat to survive in the host bloodstream. Chronic trypanosome infections are potentiated by their ability to access a deep genomic repertoire of Variant Surface Glycoprotein (VSG) genes and switch from the expression of one VSG to another. Switching VSG expression is largely based in DNA recombination events that result in chromosome translocations between an acceptor site, which houses the actively transcribed VSG, and a donor gene, drawn from an archive of more than 2,000 silent VSGs. One element implicated in these duplicative gene conversion events is a DNA repeat of approximately 70 bp that is found in long regions within each BES and short iterations proximal to VSGs within the silent archive. Early observations showing that 70-bp repeats can be recombination boundaries during VSG switching led to the prediction that VSG-proximal 70-bp repeats provide recombinatorial homology. Yet, this long held assumption had not been tested and no specific function for the conserved 70-bp repeats had been demonstrated. In the present study, the 70-bp repeats were genetically manipulated under conditions that induce gene conversion. In this manner, we demonstrated that 70-bp repeats promote access to archival VSGs. Synthetic repeat DNA sequences were then employed to identify the length, sequence, and directionality of repeat regions required for this activity. In addition, manipulation of the 70-bp repeats allowed us to observe a link between VSG switching and the cell cycle that had not been appreciated. Together these data provide definitive support for the long-standing hypothesis that 70-bp repeats provide recombinatorial homology during switching. Yet, the fact that silent archival VSGs are selected under these conditions suggests the 70-bp repeats also direct DNA pairing and recombination machinery away from the closest homologs (silent BESs) and toward the rest of the archive.

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Trypanosoma brucei, a causative agent of African Sleeping Sickness, constantly changes its dense variant surface glycoprotein (VSG) coat to avoid elimination by the immune system of its mammalian host, using an extensive repertoire of dedicated genes. However, the dynamics of VSG expression in T. brucei during an infection are poorly understood. We have developed a method, based on de novo assembly of VSGs, for quantitatively examining the diversity of expressed VSGs in any population of trypanosomes and monitored VSG population dynamics in vivo. Our experiments revealed unexpected diversity within parasite populations and a mechanism for diversifying the genome-encoded VSG repertoire. The interaction between T. brucei and its host is substantially more dynamic and nuanced than previously expected.

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Trypanosoma brucei evades the adaptive immune response through the expression of antigenically distinct Variant Surface Glycoprotein (VSG) coats. To understand the progression and mechanisms of VSG switching, and to identify the VSGs expressed in populations of trypanosomes, it is desirable to predetermine the available repertoire of VSG genes (the 'VSGnome'). To date, the catalog of VSG genes present in any strain is far from complete and the majority of current information regarding VSGs is derived from the TREU927 strain that is not commonly used as an experimental model. We have assembled, annotated and analyzed 2563 distinct and previously unsequenced genes encoding complete and partial VSGs of the widely used Lister 427 strain of T. brucei. Around 80% of the VSGnome consists of incomplete genes or pseudogenes. Read-depth analysis demonstrated that most VSGs exist as single copies, but 360 exist as two or more indistinguishable copies. The assembled regions include five functional metacyclic VSG expression sites. One third of minichromosome sub-telomeres contain a VSG (64-67 VSGs on ∼96 minichromosomes), of which 85% appear to be functionally competent. The minichromosomal repertoire is very dynamic, differing among clones of the same strain. Few VSGs are unique along their entire length: frequent recombination events are likely to have shaped (and to continue to shape) the repertoire. In spite of their low sequence conservation and short window of expression, VSGs show evidence of purifying selection, with ∼40% of non-synonymous mutations being removed from the population. VSGs show a strong codon-usage bias that is distinct from that of any other group of trypanosome genes. VSG sequences are generally very divergent between Lister 427 and TREU927 strains of T. brucei, but those that are highly similar are not found in 'protected' genomic environments, but may reflect genetic exchange among populations.

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We describe two gene-knockout (KO) strategies in Trypanosoma brucei using Cre recombinase and loxP sites. Due to the limited number of selection markers for T. brucei, it has been difficult to generate a mutant with two genes knocked out and impractical to simultaneously knockout more than two genes, deterring detailed studies of important cellular mechanisms. The first KO strategy described can overcome the marker problem by allowing continuous re-use of drug-resistance markers. The same KO vector can be used to make a conditional KO system, when a gene of interest is essential for cell viability. As a gene of interest is removed from its original chromosomal locus by the induction of Cre recombinase, deletion is complete and instantaneous. This makes it easier to identify primary effects rather than having secondary effects obscuring phenotypic assessment, as is often the case with RNAi silencing.

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Trypanosoma brucei variant surface glycoprotein (VSG) expression is a classic example of allelic exclusion. While the genome of T. brucei contains >2,000 VSG genes and VSG pseudogenes, only one allele is expressed at the surface of each infectious trypanosome and the others are repressed. Along with recombinatorial VSG switching, allelic exclusion provides a major host evasion mechanism for trypanosomes, a phenomenon known as antigenic variation. To extend our understanding of how trypanosomes escape host immunity by differential expression of VSGs, we attempted to identify genes that contribute to VSG silencing, by performing a loss-of-silencing screen in T. brucei using a transposon-mediated random insertional mutagenesis. One identified gene, which we initially named LOS1, encodes a T. brucei MCM-Binding Protein (TbMCM-BP). Here we show that TbMCM-BP is essential for viability of infectious bloodstream-form (BF) trypanosome and is required for proper cell-cycle progression. Tandem affinity purification of TbMCM-BP followed by mass spectrometry identified four subunits (MCM4-MCM7) of the T. brucei MCM complex, a replicative helicase, and MCM8, a subunit that is uniquely co-purified with TbMCM-BP. TbMCM-BP is required not only for repression of subtelomeric VSGs but also for silencing of life-cycle specific, insect-stage genes, procyclin and procyclin-associated genes (PAGs), that are normally repressed in BF trypanosomes and are transcribed by RNA polymerase I. Our study uncovers a functional link between chromosome maintenance and RNA pol I-mediated gene silencing in T. brucei.

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Binding of the Origin Recognition Complex (ORC) to replication origins is essential for initiation of DNA replication, but ORC has non-essential functions outside of DNA replication, including in heterochromatic gene silencing and telomere maintenance. Trypanosoma brucei, a protozoan parasite that causes human African trypanosomiasis, uses antigenic variation as a major virulence mechanism to evade the host's immune attack by expressing its major surface antigen, the Variant Surface Glycoprotein (VSG), in a monoallelic manner. An Orc1/Cdc6 homologue has been identified in T. brucei, but its role in DNA replication has not been directly confirmed and its potential involvement in VSG repression or switching has not been thoroughly investigated. In this study, we show that TbOrc1 is essential for nuclear DNA replication in mammalian-infectious bloodstream and tsetse procyclic forms (BF and PF). Depletion of TbOrc1 resulted in derepression of telomere-linked silent VSGs in both BF and PF, and increased VSG switching particularly through the in situ transcriptional switching mechanism. TbOrc1 associates with telomere repeats but appears to do so independently of two known T. brucei telomere proteins, TbRAP1 and TbTRF. We conclude that TbOrc1 has conserved functions in DNA replication and is also required to control telomere-linked VSG expression and VSG switching.

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Unraveling the intricate interactions between Trypanosoma brucei, the protozoan parasite causing African trypanosomiasis, and the tsetse (Glossina) vector remains a challenge. Metacyclic trypanosomes, which inhabit the tsetse salivary glands, transmit the disease and are produced through a complex differentiation and unknown program. By overexpressing a single RNA-binding protein, TbRBP6, in cultured noninfectious trypanosomes, we recapitulated the developmental stages that have been observed in tsetse, including the generation of infective metacyclic forms expressing the variant surface glycoprotein. Thus, events leading to acquisition of infectivity in the insect vector are now accessible to laboratory investigation, providing an opening for new intervention strategies.

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Trypanosoma brucei is a master of antigenic variation and immune response evasion. Utilizing a genomic repertoire of more than 1000 Variant Surface Glycoprotein-encoding genes (VSGs), T. brucei can change its protein coat by "switching" from the expression of one VSG to another. Each active VSG is monoallelically expressed from only one of approximately 15 subtelomeric sites. Switching VSG expression occurs by three predominant mechanisms, arguably the most significant of which is the non-reciprocal exchange of VSG containing DNA by duplicative gene conversion (GC). How T. brucei orchestrates its complex switching mechanisms remains to be elucidated. Recent work has demonstrated that an exogenous DNA break in the active site could initiate a GC based switch, yet the source of the switch-initiating DNA lesion under natural conditions is still unknown. Here we investigated the hypothesis that telomere length directly affects VSG switching. We demonstrate that telomerase deficient strains with short telomeres switch more frequently than genetically identical strains with long telomeres and that, when the telomere is short, switching preferentially occurs by GC. Our data supports the hypothesis that a short telomere at the active VSG expression site results in an increase in subtelomeric DNA breaks, which can initiate GC based switching. In addition to their significance for T. brucei and telomere biology, the findings presented here have implications for the many diverse pathogens that organize their antigenic genes in subtelomeric regions.

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RNA-binding proteins that target mRNA coding regions are emerging as regulators of post-transcriptional processes in eukaryotes. Here we describe a newly identified RNA-binding protein, RBP42, which targets the coding region of mRNAs in the insect form of the African trypanosome, Trypanosoma brucei. RBP42 is an essential protein and associates with polysome-bound mRNAs in the cytoplasm. A global survey of RBP42-bound mRNAs was performed by applying HITS-CLIP technology, which captures protein-RNA interactions in vivo using UV light. Specific RBP42-mRNA interactions, as well as mRNA interactions with a known RNA-binding protein, were purified using specific antibodies. Target RNA sequences were identified and quantified using high-throughput RNA sequencing. Analysis revealed that RBP42 bound mainly within the coding region of mRNAs that encode proteins involved in cellular energy metabolism. Although the mechanism of RBP42's function is unclear at present, we speculate that RBP42 plays a critical role in modulating T. brucei energy metabolism.

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Histone variants are non-allelic protein isoforms that play key roles in diversifying chromatin structure. The known number of such variants has greatly increased in recent years, but the lack of naming conventions for them has led to a variety of naming styles, multiple synonyms and misleading homographs that obscure variant relationships and complicate database searches. We propose here a unified nomenclature for variants of all five classes of histones that uses consistent but flexible naming conventions to produce names that are informative and readily searchable. The nomenclature builds on historical usage and incorporates phylogenetic relationships, which are strong predictors of structure and function. A key feature is the consistent use of punctuation to represent phylogenetic divergence, making explicit the relationships among variant subtypes that have previously been implicit or unclear. We recommend that by default new histone variants be named with organism-specific paralog-number suffixes that lack phylogenetic implication, while letter suffixes be reserved for structurally distinct clades of variants. For clarity and searchability, we encourage the use of descriptors that are separate from the phylogeny-based variant name to indicate developmental and other properties of variants that may be independent of structure.

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At any time, each cell of the protozoan parasite Trypanosoma brucei expresses a single species of its major antigenic protein, the variant surface glycoprotein (VSG), from a repertoire of >2,000 VSG genes and pseudogenes. The potential to express different VSGs by transcription and recombination allows the parasite to escape the antibody-mediated host immune response, a mechanism known as antigenic variation. The active VSG is transcribed from a sub-telomeric polycistronic unit called the expression site (ES), whose promoter is 40-60 kb upstream of the VSG. While the mechanisms that initiate recombination remain unclear, the resolution phase of these reactions results in the recombinational replacement of the expressed VSG with a donor from one of three distinct chromosomal locations; sub-telomeric loci on the 11 essential chromosomes, on minichromosomes, or at telomere-distal loci. Depending on the type of recombinational replacement (single or double crossover, duplicative gene conversion, etc), several DNA-repair pathways have been thought to play a role. Here we show that VSG recombination relies on at least two distinct DNA-repair pathways, one of which requires RMI1-TOPO3α to suppress recombination and one that is dependent on RAD51 and RMI1. These genetic interactions suggest that both RAD51-dependent and RAD51-independent recombination pathways operate in antigenic switching and that trypanosomes differentially utilize recombination factors for VSG switching, depending on currently unknown parameters within the ES.

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Trypanosoma brucei undergoes major biochemical and morphological changes during its development from the bloodstream form in the mammalian host to the procyclic form in the midgut of its insect host. The underlying regulation of gene expression, however, is poorly understood. More than 60% of the predicted genes remain annotated as hypothetical, and the 5' and 3' untranslated regions important for regulation of gene expression are unknown for >90% of the genes. In this review, we compare the data from four recently published high-throughput RNA sequencing studies in light of the different experimental setups and discuss how these data can enhance genome annotation and give insights into the regulation of gene expression in T. brucei.

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