Canonical PRC2 function is essential for mammary gland development and affects chromatin compaction in mammary organoids.

Distinct transcriptional states are maintained through organization of chromatin, resulting from the sum of numerous repressive and active histone modifications, into tightly packaged heterochromatin versus more accessible euchromatin. Polycomb repressive complex 2 (PRC2) is the main mammalian complex responsible for histone 3 lysine 27 trimethylation (H3K27me3) and is integral to chromatin organization. Using in vitro and in vivo studies, we show that deletion of Suz12, a core component of all PRC2 complexes, results in loss of H3K27me3 and H3K27 dimethylation (H3K27me2), completely blocks normal mammary gland development, and profoundly curtails progenitor activity in 3D organoid cultures. Through the application of mammary organoids to bypass the severe phenotype associated with Suz12 loss in vivo, we have explored gene expression and chromatin structure in wild-type and Suz12-deleted basal-derived organoids. Analysis of organoids led to the identification of lineage-specific changes in gene expression and chromatin structure, inferring cell type-specific PRC2-mediated gene silencing of the chromatin state. These expression changes were accompanied by cell cycle arrest but not lineage infidelity. Together, these data indicate that canonical PRC2 function is essential for development of the mammary gland through the repression of alternate transcription programs and maintenance of chromatin states.

Derivation of a robust mouse mammary organoid system for studying tissue dynamics.

Advances in stem cell research have enabled the generation of 'mini organs' or organoids that recapitulate phenotypic traits of the original biological specimen. Although organoids have been demonstrated for multiple organ systems, there are more limited options for studying mouse mammary gland formation in vitro Here, we have built upon previously described culture assays to define culture conditions that enable the efficient generation of clonal organoid structures from single sorted basal mammary epithelial cells (MECs). Analysis of Confetti-reporter mice revealed the formation of uni-colored structures and thus the clonal nature of these organoids. High-resolution 3D imaging demonstrated that basal cell-derived complex organoids comprised an inner compartment of polarized luminal cells with milk-producing capacity and an outer network of elongated myoepithelial cells. Conversely, structures generated from luminal MECs rarely contained basal/myoepithelial cells. Moreover, flow cytometry and 3D microscopy of organoids generated from lineage-specific reporter mice established the bipotent capacity of basal cells and the restricted potential of luminal cells. In summary, we describe optimized in vitro conditions for the efficient generation of mouse mammary organoids that recapitulate features of mammary tissue architecture and function, and can be applied to understand tissue dynamics and cell-fate decisions.

Evaluation of the Medial Stabilized Knee Design Using Data From National Joint Registries and Current Literature.

BACKGROUND: Various designs of total knee arthroplasty (TKA) have provided satisfactory outcomes for the treatment of knee osteoarthritis for many years. The aim of the study is to evaluate the success and failure rate of the medial stabilized (MS) TKA design through national joint registries and the current literature. MATERIALS AND METHODS: A comprehensive literature review using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses was carried out through PubMed and MEDLINE database. The international registries analyzed included the National Joint Registry, the Australian Orthopedic Association National Joint Replacement Registry, the Dutch Arthroplasty Register, the New Zealand Orthopedic Association Joint Registry, and the Michigan Arthroplasty Registry Collaborative Quality Initiative. We are reporting descriptive data which include means and percentages of survival rates and revision rates and number of years after the primary procedure. The revision rates and the cumulative revision rates are reported separately for each MS implant. RESULTS: Our detailed review identified 5 of 12 registries and 25 of 550 studies reporting on the survivorship of an MS TKA design. There were a total of 3684 procedures between the 25 studies, with an average survivorship free of aseptic loosening of 99% at 6.9 years. There are 26,693 (2.5%) MS TKAs in the current National Joint Registry with a mean cumulative revision rate of 2.63% at 5 years, 3.35% at 10 years, and 4.6% at 15 years. The Australian Orthopedic Association National Joint Replacement Registry provides survivorship data on 19,249 (2.9%) MS TKAs, with a mean cumulative revision rate of 3.34% at 5 years, 7.4% at 10 years, and 8.1% at 15 years. The Dutch Arthroplasty Register contains survivorship data on 1490 MS designs and a mean revision rate of these implants is 0.8% at 1 year, 5.95% at 5 years, and 9.8% at 10 years. The Evolution MP is the only implant reported in the Michigan Arthroplasty Registry Collaborative Quality Initiative and has a revision rate of 2.28% at 3 years. CONCLUSION: The MS TKA design has comparable results to traditional TKA designs across several joint registries and 25 studies in the literature.

The dystroglycan receptor maintains glioma stem cells in the vascular niche.

Glioblastomas (GBMs) are malignant central nervous system (CNS) neoplasms with a very poor prognosis. They display cellular hierarchies containing self-renewing tumourigenic glioma stem cells (GSCs) in a complex heterogeneous microenvironment. One proposed GSC niche is the extracellular matrix (ECM)-rich perivascular bed of the tumour. Here, we report that the ECM binding dystroglycan (DG) receptor is expressed and functionally glycosylated on GSCs residing in the perivascular niche. Glycosylated αDG is highly expressed and functional on the most aggressive mesenchymal-like (MES-like) GBM tumour compartment. Furthermore, we found that DG acts to maintain an MES-like state via tight control of MAPK activation. Antibody-based blockade of αDG induces robust ERK-mediated differentiation leading to reduced GSC potential. DG was shown to be required for tumour initiation in MES-like GBM, with constitutive loss significantly delaying or preventing tumourigenic potential in-vivo. These findings reveal a central role of the DG receptor, not only as a structural element, but also as a critical factor promoting MES-like GBM and the maintenance of GSCs residing in the perivascular niche.

Acetabular Version Increases During Adolescence Secondary to Reduced Anterior Femoral Head Coverage.

BACKGROUND: Acetabular version influences joint mechanics and the risk of impingement. Cross-sectional studies have reported an increase in acetabular version during adolescence; however, to our knowledge no longitudinal study has assessed version or how the change in version occurs. Knowing this would be important because characterizing the normal developmental process of the acetabulum would allow for easier recognition of a morphologic abnormality. QUESTIONS/PURPOSES: To determine (1) how acetabular version changes during adolescence, (2) calculate how acetabular coverage of the femoral head changed during this period, and (3) to identify whether demographic factors or hip ROM are associated with acetabular development. METHODS: This retrospective analysis of data from a longitudinal study included 17 volunteers (34 hips) with a mean (± SD) age of 11 ± 2 years; seven were male and 10 were female. The participants underwent a clinical examination of BMI and ROM and MRIs of both hips at recruitment and at follow-up (6 ± 2 years). MR images were assessed to determine maturation of the triradiate cartilage complex, acetabular version, and degree of the anterior, posterior, and superior acetabular sector angles (reflecting degree of femoral head coverage provided by the acetabulum anteriorly, posteriorly and superiorly respectively). An orthopaedic fellow (GG) and a senior orthopaedic resident (PJ) performed all readings in consensus; 20 scans were re-analyzed for intraobserver reliability. Thereafter, a musculoskeletal radiologist (KR) repeated measurements in 10 scans to test interobserver reliability. The intra- and interobserver interclass correlation coefficients for absolute agreement were 0.85 (95% CI 0.76 to 0.91; p < 0.001) and 0.77 (95% CI 0.70 to 0.84), respectively. All volunteers underwent a clinical examination by a senior orthopaedic resident (PJ) to assess their range of internal rotation (in 90° of flexion) in the supine and prone positions using a goniometer. We tested investigated whether the change in anteversion and sector angles differed between genders and whether the changes were correlated with BMI or ROM using Pearson's coefficient. The triradiate cartilage complex was open (Grade I) at baseline and closed (Grade III) at follow-up in all hips. RESULTS: The acetabular anteversion increased, moving caudally further away from the roof at both timepoints. The mean (range) anteversion angle increased from 7° ± 4° (0 to 18) at baseline to 12° ± 4° (5 to 22) at the follow-up examination (p < 0.001). The mean (range) anterior sector angle decreased from 72° ± 8° (57 to 87) at baseline to 65° ± 8° (50 to 81) at the final follow-up (p = 0.002). The mean (range) posterior (98° ± 5° [86 to 111] versus 97° ± 5° [89 to 109]; p = 0.8) and superior (121° ± 4° [114 to 129] to 124° ± 5° [111 to 134]; p = 0.07) sector angles remained unchanged. The change in the anterior sector angle correlated with the change in version (rho = 0.5; p = 0.02). The change in version was not associated with any of the tested patient factors (BMI, ROM). CONCLUSIONS: With skeletal maturity, acetabular version increases, especially rostrally. This increase is associated with, and is likely a result of, a reduced anterior acetabular sector angle (that is, less coverage anteriorly, while the degree of coverage posteriorly remained the same). Thus, in patients were the normal developmental process is disturbed, a rim-trim might be an appropriate surgical solution, since the degree of posterior coverage is sufficient and no reorientation osteotomy would be necessary. However, further study on patients with retroversion (of various degrees) is necessary to characterize these observations further. The changes in version were not associated with any of the tested patient factors; however, further study with greater power is needed. LEVEL OF EVIDENCE: Level II, prognostic study.

Factors Affecting Wear of Constrained Polyethylene Tibial Inserts in Total Knee Arthroplasty.

BACKGROUND: Wear in posterior-stabilized (PS) polyethylene tibial inserts depends on component position, limb alignment, and ligament balancing. Valgus-varus constrained (VVC) inserts are likely to be affected similarly. We aimed to compare wear characteristics of VVC and PS inserts and identify associated factors. METHODS: This was a retrieval analysis (macroscopic and radiographic) of all 18 VVC liners collected from patients who underwent revision surgery from 1999 to 2011. These patients were matched to another group with PS inserts who underwent revision in the same time period. RESULTS: There was significantly more damage in the posts of the VVC group (13.0 ± 5.0, compared to 4.7 ± 1.9 in the PS group; P < .001). Within the VVC group, the total damage score and cold flow damage were significantly higher with excessive joint line changes (≥5 mm; P = .01). The excessive joint line elevation was associated with rotational wear pattern of the post (P = .004). Damage scores were increased with femoral component malposition (P = .04), anterior tibial slope (P = .04), and tibial component malposition (P = .04). CONCLUSION: Joint line elevation, femoral and tibial component malposition, and anterior tibial slope resulted in significantly more wear in the VVC inserts. Joint line elevation of >5 mm resulted in wear of the medial and lateral aspects of the post, femoral valgus resulted in cold flow wear of the anterior post, and tibial valgus and anterior slope resulted in wear of lateral aspect of the post.

Integration of microRNA signatures of distinct mammary epithelial cell types with their gene expression and epigenetic portraits.

INTRODUCTION: MicroRNAs (miRNAs) have been implicated in governing lineage specification and differentiation in multiple organs; however, little is known about their specific roles in mammopoiesis. We have determined the global miRNA expression profiles of functionally distinct epithelial subpopulations in mouse and human mammary tissue, and compared these to their cognate transcriptomes and epigenomes. Finally, the human miRNA signatures were used to interrogate the different subtypes of breast cancer, with a view to determining miRNA networks deregulated during oncogenesis. METHODS: RNA from sorted mouse and human mammary cell subpopulations was subjected to miRNA expression analysis using the TaqMan MicroRNA Array. Differentially expressed (DE) miRNAs were correlated with gene expression and histone methylation profiles. Analysis of miRNA signatures of the intrinsic subtypes of breast cancer in The Cancer Genome Atlas (TCGA) database versus those of normal human epithelial subpopulations was performed. RESULTS: Unique miRNA signatures characterized each subset (mammary stem cell (MaSC)/basal, luminal progenitor, mature luminal, stromal), with a high degree of conservation across species. Comparison of miRNA and transcriptome profiles for the epithelial subtypes revealed an inverse relationship and pinpointed key developmental genes. Interestingly, expression of the primate-specific miRNA cluster (19q13.4) was found to be restricted to the MaSC/basal subset. Comparative analysis of miRNA signatures with H3 lysine modification maps of the different epithelial subsets revealed a tight correlation between active or repressive marks for the top DE miRNAs, including derepression of miRNAs in Ezh2-deficient cellular subsets. Interrogation of TCGA-identified miRNA profiles with the miRNA signatures of different human subsets revealed specific relationships. CONCLUSIONS: The derivation of global miRNA expression profiles for the different mammary subpopulations provides a comprehensive resource for understanding the interplay between miRNA networks and target gene expression. These data have highlighted lineage-specific miRNAs and potential miRNA-mRNA networks, some of which are disrupted in neoplasia. Furthermore, our findings suggest that key developmental miRNAs are regulated by global changes in histone modification, thus linking the mammary epigenome with genome-wide changes in the expression of genes and miRNAs. Comparative miRNA signature analyses between normal breast epithelial cells and breast tumors confirmed an important linkage between luminal progenitor cells and basal-like tumors.

Retrieval analysis of posterior stabilized polyethylene tibial inserts and its clinical relevance.

This was a retrieval analysis of 83 PS inserts to assess the effect of limb alignment, implant position and joint line position on the pattern of wear in posterior stabilized (PS) tibial inserts. The total damage score was significantly higher in knees with postoperative varus alignment more than 3° (P = 0.03). The total damage score to the post was significantly more in knees with joint line elevation more than 5 mm (9.7 ± 3.9, compared to 6.5 ± 3.7 in knees with less joint line elevation) (P = 0.05). Limb malalignment and joint line elevation resulted in more damage in PS inserts. An external rotation subluxation damage pattern was found in joint line elevation.

Ureteric trauma caused by urethral catheter insertion: case report and literature review.

Urethral catheterization is one of the most common procedures in medical practice. Catheterization is not only restricted for urological purposes, but also used for many other indications. For instance, urethral catheterization could be used for intensive care unit patients, trauma and multiple fracture injuries, and advanced neurological condition e.g. multiple sclerosis. Therefore, it may be performed by both well trained and not fully trained medical professionals resulting in complications. We present an 82-year-old female presented to A&E with hematuria, abdominal pain and low catheter output drainage after recent catheter exchange by the district nurse. Interestingly, non-contrast computed tomography (CT) scan showed the catheter inserted into the left ureter and the catheter balloon was inflated at the level of the mid-ureter. Later contrast CT study showed extravasation confirming ureteric wall partial disruption injury. The patient was managed conservatively without apparent complications in the follow-up.

Publisher Correction: A reference collection of patient-derived cell line and xenograft models of proneural, classical and mesenchymal glioblastoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The diverse origins of New Zealand house mice.

Molecular markers and morphological characters can help infer the colonization history of organisms. A combination of mitochondrial (mt) D-loop DNA sequences, nuclear DNA data, external measurements and skull characteristics shows that house mice (Mus musculus) in New Zealand and its outlying islands are descended from very diverse sources. The predominant genome is Mus musculus domesticus (from western Europe), but Mus musculus musculus (from central Europe) and Mus musculus castaneus (from southern Asia) are also represented genetically. These subspecies have hybridized to produce combinations of musculus and domesticus nuclear DNA coupled with domesticus mtDNA, and castaneus or musculus mtDNA with domesticus nuclear DNA. The majority of the mice with domesticus mtDNA that we sampled had D-loop sequences identical to two haplotypes common in Britain. This is consistent with long-term British-New Zealand cultural linkages. The origins of the castaneus mtDNA sequences widespread in New Zealand are less easy to identify.

Patellar maltracking: an update on the diagnosis and treatment strategies.

Patellar maltracking occurs as a result of an imbalance in the dynamic relationship between the patella and trochlea. This is often secondary to an underlying structural abnormality. The clinical evaluation can provide useful clues for the presence of such entity; however, the diagnosis can often be challenging especially in the absence of a documented history of patellar dislocation. Imaging, particularly MRI, can detect subtle features that could lead to the diagnosis, probably even more importantly when there is no clear history of patellar dislocation or before its development. This can provide a road map for formulating a treatment strategy that would be primarily aimed at stabilizing the patellofemoral joint to halt or slow the progression of articular cartilage loss. The purpose of this article is to discuss the clinical and radiologic evaluation of patellar maltracking providing an update on the cross-sectional imaging assessment and also a synopsis of the management options.

High-resolution 3D imaging of fixed and cleared organoids.

In vitro 3D organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition of organoids and demonstrated organoids' phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor organoids, as well as on mouse mammary gland organoids. It includes a simple clearing method utilizing a homemade fructose-glycerol clearing agent that captures 3D organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From organoid harvest to image analysis, the protocol takes 3 d.

A reference collection of patient-derived cell line and xenograft models of proneural, classical and mesenchymal glioblastoma.

Low-passage, serum-free cell lines cultured from patient tumour tissue are the gold-standard for preclinical studies and cellular investigations of glioblastoma (GBM) biology, yet entrenched, poorly-representative cell line models are still widely used, compromising the significance of much GBM research. We submit that greater adoption of these critical resources will be promoted by the provision of a suitably-sized, meaningfully-described reference collection along with appropriate tools for working with them. Consequently, we present a curated panel of 12 readily-usable, genetically-diverse, tumourigenic, patient-derived, low-passage, serum-free cell lines representing the spectrum of molecular subtypes of IDH-wildtype GBM along with their detailed phenotypic characterisation plus a bespoke set of lentiviral plasmids for bioluminescent/fluorescent labelling, gene expression and CRISPR/Cas9-mediated gene inactivation. The cell lines and all accompanying data are readily-accessible via a single website, Q-Cell (qimrberghofer.edu.au/q-cell/) and all plasmids are available from Addgene. These resources should prove valuable to investigators seeking readily-usable, well-characterised, clinically-relevant, gold-standard models of GBM.

EphA3 Pay-Loaded Antibody Therapeutics for the Treatment of Glioblastoma.

The EphA3 receptor has recently emerged as a functional tumour-specific therapeutic target in glioblastoma (GBM). EphA3 is significantly elevated in recurrent disease, is most highly expressed on glioma stem cells (GSCs), and has a functional role in maintaining self-renewal and tumourigenesis. An unlabelled EphA3-targeting therapeutic antibody is currently under clinical assessment in recurrent GBM patients. In this study, we assessed the efficacy of EphA3 antibody drug conjugate (ADC) and radioimmunotherapy (RIT) approaches using orthotopic animal xenograft models. Brain uptake studies, using positron emission tomography/computed tomography (PET/CT) imaging, show EphA3 antibodies are effectively delivered across the blood-tumour barrier and accumulate at the tumour site with no observed normal brain reactivity. A robust anti-tumour response, with no toxicity, was observed using EphA3, ADC, and RIT approaches, leading to a significant increase in overall survival. Our current research provides evidence that GBM patients may benefit from pay-loaded EphA3 antibody therapies.

Identification of quiescent and spatially restricted mammary stem cells that are hormone responsive.

Despite accumulating evidence for a mammary differentiation hierarchy, the basal compartment comprising stem cells remains poorly characterized. Through gene expression profiling of Lgr5+ basal epithelial cells, we identify a new marker, Tetraspanin8 (Tspan8). Fractionation based on Tspan8 and Lgr5 expression uncovered three distinct mammary stem cell (MaSC) subsets in the adult mammary gland. These exist in a largely quiescent state but differ in their reconstituting ability, spatial localization, and their molecular and epigenetic signatures. Interestingly, the deeply quiescent MaSC subset (Lgr5+Tspan8hi) resides within the proximal region throughout life, and has a transcriptome strikingly similar to that of claudin-low tumours. Lgr5+Tspan8hi cells appear to originate from the embryonic mammary primordia before switching to a quiescent state postnatally but can be activated by ovarian hormones. Our findings reveal an unexpected degree of complexity within the adult MaSC compartment and identify a dormant subset poised for activation in response to physiological stimuli.

Construction of developmental lineage relationships in the mouse mammary gland by single-cell RNA profiling.

The mammary epithelium comprises two primary cellular lineages, but the degree of heterogeneity within these compartments and their lineage relationships during development remain an open question. Here we report single-cell RNA profiling of mouse mammary epithelial cells spanning four developmental stages in the post-natal gland. Notably, the epithelium undergoes a large-scale shift in gene expression from a relatively homogeneous basal-like program in pre-puberty to distinct lineage-restricted programs in puberty. Interrogation of single-cell transcriptomes reveals different levels of diversity within the luminal and basal compartments, and identifies an early progenitor subset marked by CD55. Moreover, we uncover a luminal transit population and a rare mixed-lineage cluster amongst basal cells in the adult mammary gland. Together these findings point to a developmental hierarchy in which a basal-like gene expression program prevails in the early post-natal gland prior to the specification of distinct lineage signatures, and the presence of cellular intermediates that may serve as transit or lineage-primed cells.

Essential role for a novel population of binucleated mammary epithelial cells in lactation.

The mammary gland represents a unique tissue to study organogenesis as it predominantly develops in the post-natal animal and undergoes dramatic morphogenetic changes during puberty and the reproductive cycle. The physiological function of the mammary gland is to produce milk to sustain the newborn. Here we view the lactating gland through three-dimensional confocal imaging of intact tissue. We observed that the majority of secretory alveolar cells are binucleated. These cells first arise in very late pregnancy due to failure of cytokinesis and are larger than mononucleated cells. Augmented expression of Aurora kinase-A and Polo-like kinase-1 at the lactogenic switch likely mediates the formation of binucleated cells. Our findings demonstrate an important physiological role for polyploid mammary epithelial cells in lactation, and based on their presence in five different species, suggest that binucleated cells evolved to maximize milk production and promote the survival of offspring across all mammalian species.

Nuclear factor one B (NFIB) encodes a subtype-specific tumour suppressor in glioblastoma.

Glioblastoma (GBM) is an essentially incurable and rapidly fatal cancer, with few markers predicting a favourable prognosis. Here we report that the transcription factor NFIB is associated with significantly improved survival in GBM. NFIB expression correlates inversely with astrocytoma grade and is lowest in mesenchymal GBM. Ectopic expression of NFIB in low-passage, patient-derived classical and mesenchymal subtype GBM cells inhibits tumourigenesis. Ectopic NFIB expression activated phospho-STAT3 signalling only in classical and mesenchymal GBM cells, suggesting a mechanism through which NFIB may exert its context-dependent tumour suppressor activity. Finally, NFIB expression can be induced in GBM cells by drug treatment with beneficial effects.