Giant Cell Tumor of Patella with Secondary Aneurysmal Bone Cyst-A Rare Case Report of Anterior Knee Pain in Young Adults.

Introduction: Primary bone tumors of the patella are rare, with an incidence as low as 0.12%. The majority are benign, with giant cell tumors (GCT) being the predominant tumor affecting this sesamoid bone. An associated secondary aneurysmal bone cyst (ABC) component with a primary patellar GCT is rarely seen. Case Report: We report a case of a 20-year-old male with long-standing anterior knee pain for 9 months. Having visited multiple clinics and found no relief, the patient presented to us at our out-patient department. Clinical examination suggested patellar tenderness, and knee radiographs showed a lytic lesion with cortical erosions. Computed tomography and magnetic resonance imaging were suggestive of GCT. A patellectomy with a good extensor repair was performed. A patellar biopsy revealed a mixed picture of a primary GCT with a secondary ABC. The patient was closely followed up, and at 12 months, he recovered completely and recorded an excellent functional outcome. Conclusion: With the incidence of bone tumors on the rise, one should be aware of this relatively rare cause of anterior knee pain. A simple radiograph will help in early diagnosis and will go a long way toward better salvage procedures than more radical procedures like patellectomy.

High grade femoral stem subsidence in uncemented hip hemiarthroplasty - A radiographic analysis and an early prediction while treating femoral neck fractures.

PURPOSE: Femoral component subsidence is a known risk factor affecting almost all hip replacements using a collarless, cement-less stems. High grade subsidence >5mm is functionally limiting to the patient. Early analysis and prediction of this complication on the immediate post-operative radiographs will help surgeons to opt for alternative solutions to mitigate this complication. METHODS: A retrospective study including 116 patients, who underwent cement-less bipolar hemi-arthroplasties treated from 2020-2022 were included in the study. Body Mass Index (BMI) and pre-operative American Society Anesthesiologist (ASA) score was retrieved from the medical records. Post operative radiographs on postoperative day two, at four weeks and at eight weeks were evaluated. Dorr's score, initial subsidence ratio (ISR) , stem angulation, proximal stem-canal fit (PSCF) ratio, distal stem-canal fit (DSCF) ratio, medial flare modifier (MFM) were recorded. RESULTS: A total of 18 patients showed subsidence over 5mm on radiographs evaluated at four weeks. The mean high-grade stem subsidence was 13.5mm +/- 2.67. Evaluating their respective postoperative day two radiographs- ISR was >1 in 16 out of 18 patients (89%), PSCF ratio <0.75 in 83% and DSCF ratio <0.5 in 78% patients. All these patients had a neutral/negative MFM. BMI >25 (p<0.05) and ASA >3 (p<0.001) correlated with a higher degree of stem subsidence. CONCLUSION: A lower BMI and ASA score accompanied by a positive MFM were protective factors against femoral stem subsidence. A higher ISR along with a PSCF ratio <0.75 and DSCF ratio <0.5, were highly predictive of stem subsidence over 5 mm.

EKLF/Klf1 regulates erythroid transcription by its pioneering activity and selective control of RNA Pol II pause-release.

EKLF/Klf1 is a zinc-finger transcription activator essential for erythroid lineage commitment and terminal differentiation. Using ChIP-seq, we investigate EKLF DNA binding and transcription activation mechanisms during mouse embryonic erythropoiesis. We utilize the Nan/+ mouse that expresses the EKLF-E339D (Nan) variant mutated in its conserved zinc-finger region and address the mechanism of hypomorphic and neomorphic changes in downstream gene expression. First, we show that Nan-EKLF limits normal EKLF binding to a subset of its sites. Second, we find that ectopic binding of Nan-EKLF occurs largely at enhancers and activates transcription through pioneering activity. Third, we find that for a subset of ectopic targets, gene activation is achieved in Nan/+ only by Nan-EKLF binding to distal enhancers, leading to RNA polymerase II pause-release. These results have general applicability to understanding how a DNA binding variant factor confers dominant disruptive effects on downstream gene expression even in the presence of its normal counterpart.

Correction notice: Isolation of Healthy F4/80+ Macrophages from Embryonic day E13.5 Mouse Fetal Liver Using Magnetic Nanoparticles for Single Cell Sequencing.

[This corrects the article DOI: 10.21769/BioProtoc.4243.].

Mismatch of short straight proximal femur nails with anterior bow of femur in Indian population- A radiological and functional analysis.

INTRODUCTION: Intra-medullary devices are the most common mode of fixation of inter-trochanteric fractures. Short proximal femur nails (PFN) used for fixing these fractures, unlike the long nails, are non-anatomic and are usually straight with no curvature in antero-posterior plane. As a result, there is always a chance of the nail tip impinging against the anterior cortex of femur. MATERIALS AND METHODS: A total of 80 patients with trochanteric fractures (AO 31A2 and 31A3), operated with short PFN, were followed up retrospectively and prospectively from 2019 to 2021, for a period of 6 months. All fractures were fixed with PFNs, with nails ranging from 170 to 250 mm. Radiological analysis was done on hip lateral X-rays (taken at 6 months) using Angle at Distant Axis (ADA) and Nail Tip Position (NTP). Functional outcome analysis was done using Harris Hip Score. Patients were graded into 2 groups according to ADA (ADA>4° and ADA<4°). Incidence of anterior thigh pain was noted in patients on follow up and was statistically evaluated with nail size, nail diameter, ADA and NTP. RESULTS: Mean ADA was 4.19° ± 1.45; mean NTP grade was 1.98 ± 1.11. Mean nail size was 201.87 mm with a mean nail diameter of 9.76 mm. Twenty patients complained of anterior thigh pain on follow-up. Twenty-five patients had NTP grade 3 or above of which 16 complained of anterior thigh pain (p < 0.001). Fifty-five patients had nail diameter of 10 mm or above of which 14 had anterior thigh pain (p < 0.01). Fifty-three patients had a nail length of 200 mm or above of which 16 patients complained of thigh pain on follow up (p < 0.01). CONCLUSION: There is a mismatch of short PFNs with anterior bow of femur. Use of shorter nails with narrow diameters will avoid this mismatch to an extent.

Transcriptional Control of Gene Expression and the Heterogeneous Cellular Identity of Erythroblastic Island Macrophages.

During definitive erythropoiesis, maturation of erythroid progenitors into enucleated reticulocytes requires the erythroblastic island (EBI) niche comprising a central macrophage attached to differentiating erythroid progenitors. Normally, the macrophage provides a nurturing environment for maturation of erythroid cells. Its critical physiologic importance entails aiding in recovery from anemic insults, such as systemic stress or acquired disease. Considerable interest in characterizing the central macrophage of the island niche led to the identification of putative cell surface markers enriched in island macrophages, enabling isolation and characterization. Recent studies focus on bulk and single cell transcriptomics of the island macrophage during adult steady-state erythropoiesis and embryonic erythropoiesis. They reveal that the island macrophage is a distinct cell type but with widespread cellular heterogeneity, likely suggesting distinct developmental origins and biological function. These studies have also uncovered transcriptional programs that drive gene expression in the island macrophage. Strikingly, the master erythroid regulator EKLF/Klf1 seems to also play a major role in specifying gene expression in island macrophages, including a putative EKLF/Klf1-dependent transcription circuit. Our present review and analysis of mouse single cell genetic patterns suggest novel expression characteristics that will enable a clear enrichment of EBI subtypes and resolution of island macrophage heterogeneity. Specifically, the discovery of markers such as Epor, and specific features for EKLF/Klf1-expressing island macrophages such as Sptb and Add2, or for SpiC-expressing island macrophage such as Timd4, or for Maf/Nr1h3-expressing island macrophage such as Vcam1, opens exciting possibilities for further characterization of these unique macrophage cell types in the context of their critical developmental function.

EKLF/KLF1 expression defines a unique macrophage subset during mouse erythropoiesis.

Erythroblastic islands are a specialized niche that contain a central macrophage surrounded by erythroid cells at various stages of maturation. However, identifying the precise genetic and transcriptional control mechanisms in the island macrophage remains difficult due to macrophage heterogeneity. Using unbiased global sequencing and directed genetic approaches focused on early mammalian development, we find that fetal liver macrophages exhibit a unique expression signature that differentiates them from erythroid and adult macrophage cells. The importance of erythroid Krüppel-like factor (EKLF)/KLF1 in this identity is shown by expression analyses in EKLF-/- and in EKLF-marked macrophage cells. Single-cell sequence analysis simplifies heterogeneity and identifies clusters of genes important for EKLF-dependent macrophage function and novel cell surface biomarkers. Remarkably, this singular set of macrophage island cells appears transiently during embryogenesis. Together, these studies provide a detailed perspective on the importance of EKLF in the establishment of the dynamic gene expression network within erythroblastic islands in the developing embryo and provide the means for their efficient isolation.

Isolation of Healthy F4/80+ Macrophages from Embryonic day E13.5 Mouse Fetal Liver Using Magnetic Nanoparticles for Single Cell Sequencing.

In vivo erythropoiesis occurs in the erythroblast island niche (EBI), comprising of a central macrophage that attaches to and aids the maturation of erythroid progenitors into mature reticulocytes. Macrophages in hematopoietic tissue such as embryonic fetal liver are heterogeneous and express the cell surface protein F4/80. Earlier methods of isolating F4/80+ macrophages from hematopoietic tissue relied on FACS sorting, but the relatively low numbers of F4/80+ cells obtained after FACS sometimes led to poor RNA quality. Additionally, since EBI macrophages are attached to erythroblasts, care must be taken to avoid contamination with bound erythroblasts. We have developed a novel method for isolating F4/80+ cells from E13.5 mouse fetal liver using magnetic nanoparticles, which can be performed on the lab bench. During cell suspension and homogenization, we also add a peptide that disrupts erythroid macrophage interactions and generates F4/80+ single cells free of erythroid contamination. Thus, our protocol generates a population enriched in F4/80+ cells that are healthy and ready for sensitive techniques such as single cell sequencing.

mmi1 and rep2 mRNAs are novel RNA targets of the Mei2 RNA-binding protein during early meiosis in Schizosaccharomyces pombe.

The RNA-binding protein Mei2 is crucial for meiosis in Schizosaccharomyces pombe. In mei2 mutants, pre-meiotic S-phase is blocked, along with meiosis. Mei2 binds a long non-coding RNA (lncRNA) called meiRNA, which is a 'sponge RNA' for the meiotic inhibitor protein Mmi1. The interaction between Mei2, meiRNA and Mmi1 protein is essential for meiosis. But mei2 mutants have stronger and different phenotypes than meiRNA mutants, since mei2Δ arrests before pre-meiotic S, while the meiRNA mutant arrests after pre-meiotic S but before meiosis. This suggests Mei2 may bind additional RNAs. To identify novel RNA targets of Mei2, which might explain how Mei2 regulates pre-meiotic S, we used RNA immunoprecipitation and cross-linking immunoprecipitation. In addition to meiRNA, we found the mRNAs for mmi1 (which encodes Mmi1) and for the S-phase transcription factor rep2 There were also three other RNAs of uncertain relevance. We suggest that at meiotic initiation, Mei2 may sequester rep2 mRNA to help allow pre-meiotic S, and then may bind both meiRNA and mmi1 mRNA to inactivate Mmi1 at two levels, the protein level (as previously known), and also the mRNA level, allowing meiosis. We call Mei2-meiRNA a 'double sponge' (i.e. binding both an mRNA and its encoded protein).

Relative contributions of the structural and catalytic roles of Rrp6 in exosomal degradation of individual mRNAs.

The RNA exosome is a conserved complex for RNA degradation with two ribonucleolytic subunits, Dis3 and Rrp6. Rrp6 is a 3'-5' exonuclease, but it also has a structural role in helping target RNAs to the Dis3 activity. The relative importance of the exonuclease activity and the targeting activity probably differs between different RNA substrates, but this is poorly understood. To understand the relative contributions of the exonuclease and the targeting activities to the degradation of individual RNA substrates in Schizosaccharomyces pombe, we compared RNA levels in an rrp6 null mutant to those in an rrp6 point mutant specifically defective in exonuclease activity. A wide range of effects was found, with some RNAs dependent mainly on the structural role of Rrp6 ("protein-dependent" targets), other RNAs dependent mainly on the catalytic role ("activity-dependent" targets), and some RNAs dependent on both. Some protein-dependent RNAs contained motifs targeted via the RNA-binding protein Mmi1, while others contained a motif possibly involved in response to iron. In these and other cases Rrp6 may act as a structural adapter to target specific RNAs to the exosome by interacting with sequence-specific RNA-binding proteins.