De-Suppression of Mesenchymal Cell Identities and Variable Phenotypic Outcomes Associated with Knockout of Bbs1

Bardet–Biedl syndrome (BBS) is an archetypal ciliopathy caused by dysfunction of primary cilia. BBS affects multiple tissues, including the kidney, eye and hypothalamic satiety response. Understanding pan-tissue mechanisms of pathogenesis versus those which are tissue-specific, as well as gauging their associated inter-individual variation owing to genetic background and stochastic processes, is of paramount importance in syndromology. The BBSome is a membrane-trafficking and intraflagellar transport (IFT) adaptor protein complex formed by eight BBS proteins, including BBS1, which is the most commonly mutated gene in BBS. To investigate disease pathogenesis, we generated a series of clonal renal collecting duct IMCD3 cell lines carrying defined biallelic nonsense or frameshift mutations in Bbs1, as well as a panel of matching wild-type CRISPR control clones. Using a phenotypic screen and an unbiased multi-omics approach, we note significant clonal variability for all assays, emphasising the importance of analysing panels of genetically defined clones. Our results suggest that BBS1 is required for the suppression of mesenchymal cell identities as the IMCD3 cell passage number increases. This was associated with a failure to express epithelial cell markers and tight junction formation, which was variable amongst clones. Transcriptomic analysis of hypothalamic preparations from BBS mutant mice, as well as BBS patient fibroblasts, suggested that dysregulation of epithelial-to-mesenchymal transition (EMT) genes is a general predisposing feature of BBS across tissues. Collectively, this work suggests that the dynamic stability of the BBSome is essential for the suppression of mesenchymal cell identities as epithelial cells differentiate

Patient-reported outcome measures for hip-related pain: A review of the available evidence and a consensus statement from the International Hip-related Pain Research Network, Zurich 2018

Hip-related pain is a well-recognised complaint among active young and middle-aged active adults. People experiencing hip-related disorders commonly report pain and reduced functional capacity, including difficulties in executing activities of daily living. Patient-reported outcome measures (PROMs) are essential to accurately examine and compare the effects of different treatments on disability in those with hip pain. In November 2018, 38 researchers and clinicians working in the field of hip-related pain met in Zurich, Switzerland for the first International Hip-related Pain Research Network meeting. Prior to the meeting, evidence summaries were developed relating to four prioritised themes. This paper discusses the available evidence and consensus process from which recommendations were made regarding the appropriate use of PROMs to assess disability in young and middle-aged active adults with hip-related pain. Our process to gain consensus had five steps: (1) systematic review of systematic reviews; (2) preliminary discussion within the working group; (3) update of the more recent high-quality systematic review and examination of the psychometric properties of PROMs according to established guidelines; (4) formulation of the recommendations considering the limitations of the PROMs derived from the examination of their quality; and (5

Generating mutant renal cell lines using CRISPR technologies

Gene-editing using the CRISPR/Cas9 system is an extremely efficient approach for generating mutations within the genomic DNA of immortalised cell lines. This procedure begins with a straightforward cloning step to generate a single plasmid encoding the Cas9 enzyme as well as a synthetic guide RNA (sgRNA) which is selected to target specific sites within the genome. This plasmid is transfected into cells either alone, in order to generate random insertion-deletion alleles (‘indels’) at the desired locus via the non-homologous end-joining pathway, or in conjunction with a homology directed repair template oligonucleotide to generate a specific point mutation. Here we describe a procedure to perform gene-editing in IMCD3 and HEK293 cells and to subsequently isolate clonal cell lines carrying mutations of interest

Changes in knee and trunk alignment in people with hip pain and healthy controls when using a decline board during single-leg squat

Context: Hip pain is associated with reduced hip muscle strength. range of movement (ROM), and decreased postural stability. Single-leg squat is a reliable and valid method to measure dynamic balance. Objective: To evaluate the influence of physical characteristics and use of a decline board on squat performance in a hip pain population. Design: Cross-sectional study setting. Clinical Patients: In total, 33 individuals scheduled for arthroscopic hip surgery were matched with 33 healthy controls. Interventions: Hip and ankle ROM, hip strength, and trunk endurance were assessed, along with knee and trunk kinematics during squat on flat and 25 degrees decline surfaces. Main Outcome Measures: Between-group and surface differences in alignment, between-group strength and ROM. and associations between alignment and physical characteristics were assessed and determined using mixed model analysis of variance and Pearson R. Results: The hip pain group had significantly less strength and ROM for all directions except abduction strength and ankle dorsiflexion (P > .02). No differences existed between the 2 groups for mink (P < .70) or knee displacement (P < .46) during squat on either surface. When the 2 groups were combined (n = 66), decline squat significantly reduced knee medial displacement in both limbs by approximately 1 cm (P < .01). Decline squat reduced trunk lateral movement on 1 side only (P= .03). Reduced knee displacement during decline squat showed fair association with less hip-extension strength (r= -.29), hip-flexion strength (r= -.25), and less dorsiflexion (r= -.24). Strength and range were not associated with trunk displacement. Conclusions: Decline squat reduced medial knee and lateral trunk displacement regardless of hip pain. Reductions may be greater in those with lesser hip muscle strength and dorsiflexion. Use of a decline board during squat for improving knee and trunk alignment should be considered as a goal of exercise intervention

Associations between type and severity of hip pathology with pre-operative patient reported outcome measures

The relationship between hip pathology and patient reported outcome responses following hip surgery has been previously investigated. No studies have investigated the relationship between pathology and patient reported outcome responses prior to surgery.(1) Determine the prevalence of chondral and labral pathology identified during hip arthroscopy. (2) Determine the association between intra-articular findings and patient reported outcome scores in a pre-arthroscopy hip pain population.Sixty-seven (22 female) participants scheduled for hip arthroscopy after clinical examination and radiographic assessment completed a series of patient reported outcomes (Hip Disability and Osteoarthritis Outcome Score; International Hip Outcome Tool; Pain on Activity; Visual Analogue Scale). Pathology discovered/addressed during arthroscopy was classified. Univariable and multivariable linear regression models were used to assess the relationship between demographics, pathology and patient reported outcome responses.Ninety-one percent of participants had labral pathology; 76% had acetabular chondropathy and 31% had femoral head chondropathy. Across the ten patient reported outcome subscales, severe femoral head chondropathy and large labral tears had the greatest number of significant associations with patient reported outcome scores. The strongest association was with 'Hip Disability and Osteoarthritis Outcome Score symptoms and stiffness' subscale, where severe femoral head chondropathy explained 22% of variability in symptoms and stiffness, when adjusted for Body Mass Index and presence of pincer morphology (p=0.002).Severe femoral head chondropathy and large labral tears along with a high prevalence of labral pathology and acetabular chondropathy were relatively common findings during hip arthroscopy. Severe femoral head chondropathy and large labral tears are most associated with patient reported outcome's, however, at best only explain 22% of the variability

Physical impairments in symptomatic femoroacetabular impingement: a systematic review of the evidence

Femoroacetabular impingement (FAI) and accompanying pathologies are associated with pain and reduced quality of life. Physical impairments can be associated with worse symptoms and may be an important target of rehabilitation programmes in this patient group. Knowledge regarding physical impairments in individuals with symptomatic FAI is limited.In adults aged 18-50 years with symptomatic FAI, to: (1) identify physical impairments in range of motion (ROM), hip muscle function and functional tasks; (2) to compare physical impairments with healthy controls; and (3) to evaluate the effects of interventions targeting physical impairments.Systematic review.A systematic review of the literature was conducted in accordance with the PRISMA statement. The modified Downs and Black checklist was used for quality appraisal. Studies of adults aged 18-50 years with symptomatic FAI that examined ROM, hip muscle function and functional tasks were included. Standardised mean differences (SMDs) were calculated where possible or best evidence synthesis and study conclusions were presented.22 studies fulfilled all inclusion criteria. Methodological quality was varied (47-82% using Downs and Black Appraisal Criteria). Hip joint ROM did not differ in individuals with symptomatic FAI compared with control participants. Individuals with symptomatic FAI demonstrated deficits in hip muscle strength and reduced dynamic balance on 1 leg when compared with control participants. For hip joint ROM, there were no significant within-group differences between preintervention and postintervention time points. Hip muscle strength improved significantly from prehip to posthip arthroscopy in a single case series. No randomised controlled trails evaluated the effect of different types of interventions for symptomatic patients with symptomatic FAI.Individuals with symptomatic FAI demonstrate impairments in hip muscle strength and dynamic single leg balance. This information may assist therapists in providing targeted rehabilitation programmes for individuals with FAI and associated pathology. Further research is needed to determine whether symptomatic FAI affects other aspects of functional performance; and to evaluate whether targeted interventions are effective in symptomatic FAI.This information may assist therapists in providing targeted rehabilitation programmes for individuals with symptomatic FAI

Acute and subacute changes in hip strength and range of movement after arthroscopy to address chondrolabral pathology

Hip pain is associated with reduced muscle strength, range of movement (ROM), and function. Hip arthroscopy is undertaken to address coexistent intra-articular pathologies with the aim of reducing pain and improving function.To evaluate changes in strength and ROM in a cohort with chondrolabral pathology before surgery to 3 and 6 months after hip arthroscopy.Case series; Level of evidence, 4.Sixty-seven individuals with hip pain who were scheduled for hip arthroscopy were matched with 67 healthy controls. Hip strength and ROM were collected preoperatively and at 3 and 6 months postoperatively. Repeated measures analysis of variance evaluated whether strength and ROM differed between limbs and among time points. Bonferroni post hoc tests determined differences in hip strength and ROM among testing times and between the hip pain group and matched controls.Hip extension, internal rotation (IR), external rotation (ER), and adduction ( < .040) strength were greater at 3 months after surgery; all directions, including flexion, abduction, and squeeze, were greater at 6 months ( < .015). Hip flexion ROM was greater at 3 months after surgery ( = .013). Flexion, IR, and ER ROM was greater at 6 months ( < .041). At 6 months, IR ROM ( = .003) and flexion, IR, and ER strength ( < .005) remained less than matched controls.With the exception of squeeze and flexion, all directions of hip strength and hip flexion ROM are significantly improved 3 months after arthroscopy to address chondrolabral pathology. By 6 months after arthroscopy, strength in all directions and flexion and rotation ROM are significantly improved in both limbs, but hip flexion, IR, and ER strength and IR ROM remain significantly less than that of healthy matched controls in both limbs

Crystal structure of intraflagellar transport protein 80 reveals a homodimer required for ciliogenesis.

Oligomeric assemblies of intraflagellar transport (IFT) particles build cilia through sequential recruitment and transport of ciliary cargo proteins within cilia. Here we present the 1.8 Å resolution crystal structure of the Chlamydomonas IFT-B protein IFT80, which reveals the architecture of two N-terminal β-propellers followed by an α-helical extension. The N-terminal β-propeller tethers IFT80 to the IFT-B complex via IFT38 whereas the second β-propeller and the C-terminal αa-helical extension result in IFT80 homo-dimerization. Using CRISPR/Cas to create biallelic Ift80 frameshift mutations in IMCD3 mouse cells, we demonstrate that IFT80 is absolutely required for ciliogenesis. Structural mapping and rescue experiments reveal that human disease-causing missense mutations do not cluster within IFT80 and form functional IFT particles. Unlike missense mutant forms of IFT80, deletion of the C-terminal dimerization domain prevented rescue of ciliogenesis. Taken together our results may provide a first insight into higher order IFT complex formation likely required for IFT train formation