Permissive epigenomes endow reprogramming competence to transcriptional regulators.

Identifying molecular and cellular processes that regulate reprogramming competence of transcription factors broadens our understanding of reprogramming mechanisms. In the present study, by a chemical screen targeting major epigenetic pathways in human reprogramming, we discovered that inhibiting specific epigenetic roadblocks including disruptor of telomeric silencing 1-like (DOT1L)-mediated H3K79/K27 methylation, but also other epigenetic pathways, catalyzed by lysine-specific histone demethylase 1A, DNA methyltransferases and histone deacetylases, allows induced pluripotent stem cell generation with almost all OCT factors. We found that simultaneous inhibition of these pathways not only dramatically enhances reprogramming competence of most OCT factors, but in fact enables dismantling of species-dependent reprogramming competence of OCT6, NR5A1, NR5A2, TET1 and GATA3. Harnessing these induced permissive epigenetic states, we performed an additional screen with 98 candidate genes. Thereby, we identified 25 transcriptional regulators (OTX2, SIX3, and so on) that can functionally replace OCT4 in inducing pluripotency. Our findings provide a conceptual framework for understanding how transcription factors elicit reprogramming in dependency of the donor cell epigenome that differs across species.

Descriptive analysis and short-term follow-up clinical results of osteochondral lesions of the distal tibia based on data of the German Cartilage Register (Knorpelregister® DGOU).

INTRODUCTION: An increasing number of ankle injuries with osteochondral lesions (OCL) also include lesions of the distal tibia. Therefore, the German Cartilage Society database is used to describe and examine the characteristics of these lesions and, early on, the results of different surgical therapies on the clinical outcome. MATERIALS AND METHODS: Forty-seven patients out of 844 registered in the German Cartilage Society database met the inclusion criteria showing an OCL of the distal tibia (OLDT). Sixteen of them also presented a 1-year follow-up regarding the Foot and Ankle Ability Measure (FAAM). Further evaluations were included in the follow-up, such as the Foot and Ankle Outcome Score (FAOS) and the Visual Analogue Scale for pain (VAS). RESULTS: The patients' mean age was 35 ± 11 with a mean BMI in the range of overweight (26/27 ± 5 kg/m2). The lesions were equally distributed on the articular surface of the distal tibia. Most patients were operated using anterior ankle arthroscopy [nT 34 (72%); nS 13 (81%)], while some (nT 9; nS 4) converted to open procedures. Almost 90% staged III and IV in the ICRS classification. Debridement, bone marrow stimulation, solid scaffolds, and liquid filler were the treatment choices among the subgroup. All therapies led to a clinical improvement between pre-op and 1-year follow-up but not to a significant level. CONCLUSION: This study presents baseline data of OLDT based on data from a large database. BMS and scaffolds were the treatment of choice but did not present significant improvement after a 1-year follow-up.

Improving stability of atlantoaxial fusion: a biomechanical study.

PURPOSE: The incidence of atlanto-axial injuries is continuously increasing and often requires surgical treatment. Recently, Harati developed a new procedure combining polyaxial transarticular screws with polyaxial atlas massae lateralis screws via a rod system with promising clinical results, yet biomechanical data is lacking. This biomechanical study consequently aims to evaluate the properties of the Harati technique. METHODS: Two groups, each consisting of 7 cervical vertebral segments (C1/2), were formed and provided with a dens axis type 2 fracture according to Alonzo. One group was treated with the Harms and the other with the Harati technique. The specimen was loaded via a lever arm to simulate extension, flexion, lateral flexion and rotation. For statistical analysis, dislocation (°) was measured and compared. RESULTS: For extension and flexion, the Harati technique displayed a mean dislocation of 4.12° ± 2.36° and the Harms technique of 8.48° ± 1.49° (p < 0.01). For lateral flexion, the dislocation was 0.57° ± 0.30° for the Harati and 1.19° ± 0.25° for the Harms group (p < 0.01). The mean dislocation for rotation was 1.09° ± 0.48° for the Harati and 2.10° ± 0.31° for the Harms group (p < 0.01). No implant failure occurred. CONCLUSION: This study found a significant increase in biomechanical stability of the Harati technique when compared to the technique by Harms et al. Consequently, this novel technique can be regarded as a promising alternative for the treatment of atlanto-axial instabilities.

Combining Automated Organoid Workflows with Artificial Intelligence-Based Analyses: Opportunities to Build a New Generation of Interdisciplinary High-Throughput Screens for Parkinson's Disease and Beyond.

Parkinson's disease (PD) is the second most common neurodegenerative disease and primarily characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain. Despite decades of research and the development of various disease model systems, there is no curative treatment. This could be due to current model systems, including cell culture and animal models, not adequately recapitulating human PD etiology. More complex human disease models, including human midbrain organoids, are maturing technologies that increasingly enable the strategic incorporation of the missing components needed to model PD in vitro. The resulting organoid-based biological complexity provides new opportunities and challenges in data analysis of rich multimodal data sets. Emerging artificial intelligence (AI) capabilities can take advantage of large, broad data sets and even correlate results across disciplines. Current organoid technologies no longer lack the prerequisites for large-scale high-throughput screening (HTS) and can generate complex yet reproducible data suitable for AI-based data mining. We have recently developed a fully scalable and HTS-compatible workflow for the generation, maintenance, and analysis of three-dimensional (3D) microtissues mimicking key characteristics of the human midbrain (called "automated midbrain organoids," AMOs). AMOs build a reproducible, scalable foundation for creating next-generation 3D models of human neural disease that can fuel mechanism-agnostic phenotypic drug discovery in human in vitro PD models and beyond. Here, we explore the opportunities and challenges resulting from the convergence of organoid HTS and AI-driven data analytics and outline potential future avenues toward the discovery of novel mechanisms and drugs in PD research. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

The Use of FRAX in Identifying Women Less Than 65 Years Needing Bone Mineral Density Testing.

The purpose of this study is to determine if the United States Preventive Services Task Force (USPSTF) screening guideline for osteoporosis identifies women under the age of 65 with osteoporosis needing bone mineral density (BMD) testing. If the Fracture Risk Assessment Tool (FRAX) tool fails to identify women under the age of 65 with undiagnosed osteoporosis, then diagnosis and treatment are delayed, potentially leading to increased fractures and morbidity. Another aim of this study is to characterize women under the age of 65 with osteoporosis that FRAX fails to identify and provide descriptive data on our study population. A retrospective chart review was completed between 2012 and 2018. We extracted data for 113 women ≤ 65-years with osteoporosis confirmed by BMD or fractures. Major osteoporotic fracture (MOF) risk calculation without BMD by FRAX of 9.3% or greater (high risk group) was found in 51 (45.1%) of patients. Osteoporosis by T-score < 2.5 was evident in 102 (90%) of patients. Previous osteoporotic fractures were noted in 29 (25.7%) of patients. The average age of women in the high-risk group was 58 years and 55 years in the low-risk group. The sensitivity of FRAX for identifying women with a T-score <-2.5 was 40%. The sensitivity of FRAX for identifying women with a history of fracture was 32%. The sensitivity of FRAX for identifying women with a T-score <-2.5 or identifying women with a history of fracture was 32%. These results demonstrate that the FRAX tool alone (USPSTF recommendation) fails to identify many women under the age of 65 with osteoporosis in need of BMD testing. Over half of women would not have had a BMD performed based on guidelines for screening BMD in women <65. Further study is needed to characterize women under the age of 65 with osteoporosis with a FRAX MOF risk less than 9.3%.

Abnormal bone mineral content and density in people with tetrasomy 18p.

Tetrasomy 18p is a rare chromosomal abnormality, resulting from an additional iso-chromosome composed of two copies of the short arm. It is characterized by craniofacial abnormalities, neuromuscular dysfunction, and developmental delay. The Chromosome 18 Clinical Research Center has established the largest cohort of individuals with this rare genetic condition. Here, we describe a case series of 21 individuals with tetrasomy 18p who have a previously unreported clinical finding: low bone mineral density. Most individuals met criteria for low bone density despite being relatively young (mean age of 21 years). Clinicians providing care to individuals affected by Tetrasomy 18p should be aware of their increased risk for decreased bone density and pathological fractures.

Uncemented hip revision cup as an alternative for T-type acetabular fractures: A cadaveric study.

BACKGROUND: The current rise in elderly patients with compromised bone quality complicates the surgical treatment of acetabular T-type fractures (AO type 62B2 fractures). There is on ongoing discussion about the treatment options, mostly consisting of an open reduction and internal fixation (ORIF) with or without primary or secondary total hip arthroplasty (THA). Yet, these patients are oftentimes unable to fulfil weight-bearing restrictions and mostly present with an unavailability of a stable anchor site. Consequently, this study investigates the feasibility of a cementless hip revision cup for acetabular T-type fractures and compares its biomechanical properties to ORIF. HYPOTHESIS: The cementless hip revision cup provides sufficient biomechanical stability under the simulation of full weight-bearing. PATIENTS AND METHODS: The study compared two groups of human cadaveric hip bones with T-type fractures, of whom 6 subjects were treated with ORIF (6 male; mean age: 62±17years; mean body weight: 75±15) versus 6 subjects treated with a cementless hip revision cup (2 male; 69±12 years; 73±15kg). The group-assignment was controlled for comparable BMD results (mean BMD: ORIF 110±37 mg Ca-Ha/mL versus hip revision cup 134±32 mg Ca-Ha/mL). To compare for biomechanical stability cyclic loading was applied measuring the force and dislocation of the fracture gap at standardized bone loci using an all-electric testing machine and a 3D-ultrasound measuring system. RESULTS: Comparing superior pubic ramus versus iliac wing (cementless hip revision cup versus ORIF [mean±standard deviation]: 5.8±2.0 versus 7.0±3.2; p=0.032) as well as sacral ala versus iliac wing (4.6±2.2 versus 6.4±3.7; p=0.002), the cementless revision cup achieved a significantly higher stability than the plate osteosynthesis. CONCLUSION: Revision cup and ORIF withstood biomechanical loading forces exceeding full weight-bearing in this biomechanical study. The results of our study suggest that the cementless hip revision cup might be promising alternative to the current standard care of ORIF with or without primary THA. LEVEL OF EVIDENCE: III; case control experimental study.

Evaluation of a New Treatment Strategy for Geriatric Fragility Fractures of the Posterior Pelvic Ring Using Sensor-Supported Insoles: A Proof-of-Concept Study.

The incidence of fragility fractures of the pelvis (FFPs) is currently rising. Surgical treatment, which is performed using sacroiliac screws, is complicated by compromised bone quality, oftentimes resulting in implant failure. The iFuse implant system aims to improve attachment and durability with promising results for sacroiliac dysfunction, though data for its feasibility on FFPs are rare. Consequently, this study aims to evaluate the feasibility of the iFuse for FFPs. A total of 10 patients with FFPs were treated with the iFuse in this study. Pre- and postoperatively, both mobility using an established insole force sensor for an inpatient gait analysis and general well-being and pain using questionnaires were evaluated. When comparing pre- and postoperative findings, this study demonstrated a significant increase in the average (8.14%) and maximum (9.4%) loading (p < 0.001), a reduction in pain, as measured by the visual analog scale (VAS), from 4.60 to 2.80 at rest (p = 0.011) and from 7.00 to 4.40 during movement (p = 0.008), an increase in the Barthel Index by 20 points (p < 0.001) and an increase in the Parker Mobility Score by 2.00 points (p = 0.011). All this contributes to the possibility of early postoperative mobilization and improved general well-being, ultimately preventing the late consequences of postoperative immobilization and maintaining patients autonomy and contentment.

Novel Tape Suture Osteosynthesis for Hyperextension Injuries of the Subaxial Cervical Spine: A Biomechanical Study.

BACKGROUND/AIM: Anterior tension band injuries are usually the result of high impact hyperextension trauma. Current surgical treatment includes anterior cervical discectomy and fusion bearing the risk of soft tissue irritation, degeneration of adjacent cervical segments, implant failure or iatrogenic spondylodesis. This study examined the biomechanical properties of tape suture constructs reenforcing ligamental stability for the treatment of Association of Osteosynthesis (AO) type B3 injuries compared to anterior fusion. MATERIALS AND METHODS: After creation of an AO type B3 injury in synthetic cervical segments (C5/6, Sawbone®), seven segments were treated with anterior fusion and seven with a tape suture construct, similar to the SpeedBridge™ (Arthrex®). Biomechanical testing was performed, simulating extension, flexion, lateral bending, and rotation. Dislocation (°) and corresponding force (N) were measured and compared. RESULTS: Anterior fusion displayed a mean range of extension, lateral bending, and rotation of 3.60° (SD 1.87°), 2.28° (SD 1.55°), and 2.81° (SD 0.78°), respectively. The tape suture showed a mean range of extension, lateral bending, and rotation of 4.24° (SD 0.81°) (p=0.146), 5.44° (SD 1.56°) (p=0.013), and 5.29° (SD 1.44°) (p<0.01), respectively. No specimen suffered from implant failure. CONCLUSION: The tape suture construct provides sufficient biomechanical stability for the treatment of AO type B3 injuries compared to anterior fusion. Regarding cervical extension, whose limitation is crucial for ligamental healing, the tape suture shows no significant inferiority. Yet, the tape suture approaches physiological mobility in the planes not affected by the injury. Consequently, the tape suture is a promising alternative preventing an iatrogenic spondylodesis.

Three-Dimensional Cell Culture Systems in Pediatric and Adult Brain Tumor Precision Medicine.

Primary brain tumors often possess a high intra- and intertumoral heterogeneity, which fosters insufficient treatment response for high-grade neoplasms, leading to a dismal prognosis. Recent years have seen the emergence of patient-specific three-dimensional in vitro models, including organoids. They can mimic primary parenteral tumors more closely in their histological, transcriptional, and mutational characteristics, thus approximating their intratumoral heterogeneity better. These models have been established for entities including glioblastoma and medulloblastoma. They have proven themselves to be reliable platforms for studying tumor generation, tumor-TME interactions, and prediction of patient-specific responses to establish treatment regimens and new personalized therapeutics. In this review, we outline current 3D cell culture models for adult and pediatric brain tumors, explore their current limitations, and summarize their applications in precision oncology.

Tissue clearing may alter emission and absorption properties of common fluorophores.

In recent years, 3D cell culture has been gaining a more widespread following across many fields of biology. Tissue clearing enables optical analysis of intact 3D samples and investigation of molecular and structural mechanisms by homogenizing the refractive indices of tissues to make them nearly transparent. Here, we describe and quantify that common clearing solutions including benzyl alcohol/benzyl benzoate (BABB), PEG-associated solvent system (PEGASOS), immunolabeling-enabled imaging of solvent-cleared organs (iDISCO), clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC), and ScaleS4 alter the emission spectra of Alexa Fluor fluorophores and fluorescent dyes. Clearing modifies not only the emitted light intensity but also alters the absorption and emission peaks, at times to several tens of nanometers. The resulting shifts depend on the interplay of solvent, fluorophore, and the presence of cells. For biological applications, this increases the risk for unexpected channel crosstalk, as filter sets are usually not optimized for altered fluorophore emission spectra in clearing solutions. This becomes especially problematic in high throughput/high content campaigns, which often rely on multiband excitation to increase acquisition speed. Consequently, researchers relying on clearing in quantitative multiband excitation experiments should crosscheck their fluorescent signal after clearing in order to inform the proper selection of filter sets and fluorophores for analysis.

Generation and Maintenance of Homogeneous Human Midbrain Organoids.

Three-dimensional cell cultures ("organoids") promise to better recapitulate native tissue physiology than traditional 2D cultures and are becoming increasingly interesting for disease modeling and compound screening efforts. While a number of protocols for the generation of neural organoids have been published, most protocols require extensive manual handling and result in heterogeneous aggregates with high sample-to-sample variation, which can hinder screening-based strategies. We have now developed a fast and efficient protocol for the generation and maintenance of highly homogeneous and reproducible midbrain organoids. The protocol is streamlined for use in fully automated workflows but can also be performed manually without the need for highly specialized equipment. It relies on the aggregation of small molecule neural precursor cells (smNPCs) in standard 96-well V-bottomed plates under static culture conditions without cumbersome matrix embedding. The result is ready-to-assay uniform 3D human midbrain organoids available in freely scalable quantities for downstream analyses in 3D cell culture. Graphic abstract: Automated midbrain organoid generation workflow and timeline.

Fluorescence-based Single-cell Analysis of Whole-mount-stained and Cleared Microtissues and Organoids for High Throughput Screening.

Three-dimensional (3D) cell culture, especially in the form of organ-like microtissues ("organoids"), has emerged as a novel tool potentially mimicking human tissue biology more closely than standard two-dimensional culture. Typically, tissue sectioning is the standard method for immunohistochemical analysis. However, it removes cells from their native niche and can result in the loss of 3D context during analyses. Automated workflows require parallel processing and analysis of hundreds to thousands of samples, and sectioning is mechanically complex, time-intensive, and thus less suited for automated workflows. Here, we present a simple protocol for combined whole-mount immunostaining, tissue-clearing, and optical analysis of large-scale (approx. 1 mm) 3D tissues with single-cell level resolution. While the protocol can be performed manually, it was specifically designed to be compatible with high-throughput applications and automated liquid handling systems. This approach is freely scalable and allows parallel automated processing of large sample numbers in standard labware. We have successfully applied the protocol to human mid- and forebrain organoids, but, in principle, the workflow is suitable for a variety of 3D tissue samples to facilitate the phenotypic discovery of cellular behaviors in 3D cell culture-based high-throughput screens. Graphic abstract: Automatable organoid clearing and high-content analysis workflow and timeline.

Bilateral atypical fractures of the femur: Ten years AFTER ten years of bisphosphonate therapy.

BACKGROUND: Atypical femur fracture (AFF) is a clinically important complication of bisphosphonate (BP) use in the treatment of osteoporosis. The benefits of long-term BP therapy in preventing osteoporotic fractures have been shown to outweigh the risks of treatment. Discontinuation of BPs or "drug holidays" have been implemented as a strategy to reduce the risk of rare complications such as AFF. CASE REPORT: We present the case of a 70-year-old postmenopausal woman who suffered bilateral AFF ten years after discontinuation of BP treatment. Management of this patient included fixation of the complete AFF with an intramedullary rod. A single dose of denosumab was administered prior to referral to endocrinology and seemed to contribute to callus formation. Denosumab was discontinued to prevent progression of the contralateral incomplete AFF. Teriparatide was indicated for the treatment of this patient's osteoporosis and also led to the resolution of the incomplete AFF. CONCLUSION: Patients receiving long-term BP therapy should be periodically reevaluated in order to maximize the benefit and minimize the risk of treatment. Current research supports the implementation of drug holidays to decrease the risk of AFF; however, this case report confirms the need for continued monitoring after discontinuation of BP therapy. Additionally, our review of current literature highlights the need for more specific research regarding duration of BP treatment and drug holidays.

Resomer C212© in vertebroplasty or kyphoplasty: A feasibility study on artificial bones with biomechanical and thermal evaluation.

BACKGROUND: Vertebroplasty and kyphoplasty are now well-established methods for treating compression fractures of vertebral bodies (AO type A) as well as vertebral body metastases [1, 2, 3]. However, polymethylmethacrylate (PMMA) augmented vertebrae show fractures of subsequent vertebral bodies due to the increased stability of the augmented vertebral body [4]. Resorbable cements are currently only used experimentally. Many commercially available resorbable calcium phosphate cements do not exhibit sufficient biomechanical stability to treat vertebral body fractures [5]. Resomer C212© (Evonik Industries AG, Essen, Germany) is a slow resorbable poly-ε-caprolactone that has low melting temperatures and good biomechanical properties. OBJECTIVE: This is a feasibility study on how the poly-ε-caprolactone Resomer C212© can be used for kypho- or vertebroplasty, what temperatures are used in the argumentation and how differences in load capacity are measurable compared to conventional PMMA cement. METHODS: 23 Sawbones© blocks (7.5 Open Cell Foam, SKU: 1522-09, laminated on both sides, 4 × 4 × 2.9 cm, Sawbones, Vashon Island, USA) were divided into three groups: 7 without augmentation, 8 augmented with PMMA cement Traumacem V+© (DePuy Synthes, West Chester, USA) and 8 augmented with Resomer C212©. Temperature measurements were made in a 37∘C water bath centrally in the block and on the top and bottom plates. This was followed by a maximum load of up to 2000 N using a universal testing machine (Instron E 10000, Instron Industrial Products, Grove City, USA). RESULTS: In the Resomer C212© test group, the maximum average increase in temperature was 4.15 ± 4.72∘C central, 0.3 ± 0.31∘C at the top and 0.78 ± 1.27∘C at the base. In the cement test group, the average increase in temperature was 9.80 ± 10.65∘C centrally in the test block, 1.50 ± 0.73∘C at the top plate and 1.42 ± 0.66∘C and the base plate. In the axial compression test, the 7 non-kyphoplasted test blocks showed a first loading peak on average at 275.23 ± 80.98 N, a rigidity of 238.47 ± 71.01 N/mm2. In the Traumacem V+© group, the mean peak load was 313.72 ± 46.26 N and rigidity was 353.45 ± 77.23 N/mm2. The Resomer C212© group achieved a peak load of 311.74 ± 52.05 N and a stiffness of 311.30 ± 126.63 N/mm2. A compression to 50% could not be seen in any test block under the load of 2000 N. At 2000 N, Traumacem V+©'s average height reduction was 9.26 ± 2.16 mm and Resomer C212© was 10.93 ± 0.81 mm. CONCLUSIONS: It has been shown that the application of Resomer C212© in kyphoplasty or vertebroplasty is well feasible. Thermal analysis showed significantly lower temperatures and shorter temperature application in the Resomer C212© group. In the biomechanical load up to 2000 N no significant differences could be observed between the individual groups.

Blunt traumatic thoracic aortic injuries: a retrospective cohort analysis of 2 decades of experience.

OBJECTIVES: The aim of this study was to analyse and report the changes in the management of blunt traumatic aortic injuries (BTAIs) in a single centre during the last 2 decades. METHODS: A retrospective analysis of all patients diagnosed with BTAI from January 1999 to January 2020 was performed. Data were collected from electronic/digitalized medical history records. RESULTS: Forty-six patients were included [median age 42.4 years (16-84 years), 71.7% males]. The predominant cause of BTAI was car accidents (54.5%, n = 24) and all patients presented with concomitant injuries (93% bone fractures, 77.8% abdominal and 62.2% pelvic injuries). Over 70% presented grade III or IV BTAI. Urgent repair was performed in 73.8% of patients (n = 31), with a median of 2.75 h between admission and repair. Thoracic endovascular repair (TEVAR) was performed in 87% (n = 49), open surgery (OS) in 10.9% (n = 5) and conservative management in 2.1% (n = 1). Technical success was 82.6% (92.1% TEVAR, 79% OS). In-hospital mortality was 19.5% (17.5% TEVAR, 40% OS). Of these, 3 died from aortic-related causes. Seven (15.2%) required an early vascular reintervention. The median follow-up was 34 months (1-220 months), with 19% of early survivors having a follow-up of >10 years. Only 1 vascular reintervention was necessary during follow-up: secondary TEVAR due to acute graft thrombosis. Of the patients who survived the initial event, 6.7% died during follow-up, none from aortic-related causes. CONCLUSIONS: Even with all the described shortcomings, in our experience TEVAR for BTAI proved to be feasible and effective, with few complications and stable aortic reconstruction at mid-term follow-up. With the current technical expertise and wide availability of a variety of devices, it should be pursued as a first-line therapy in these challenging scenarios.

Cell-Type-Specific High Throughput Toxicity Testing in Human Midbrain Organoids.

Toxicity testing is a crucial step in the development and approval of chemical compounds for human contact and consumption. However, existing model systems often fall short in their prediction of human toxicity in vivo because they may not sufficiently recapitulate human physiology. The complexity of three-dimensional (3D) human organ-like cell culture systems ("organoids") can generate potentially more relevant models of human physiology and disease, including toxicity predictions. However, so far, the inherent biological heterogeneity and cumbersome generation and analysis of organoids has rendered efficient, unbiased, high throughput evaluation of toxic effects in these systems challenging. Recent advances in both standardization and quantitative fluorescent imaging enabled us to dissect the toxicities of compound exposure to separate cellular subpopulations within human organoids at the single-cell level in a framework that is compatible with high throughput approaches. Screening a library of 84 compounds in standardized human automated midbrain organoids (AMOs) generated from two independent cell lines correctly recognized known nigrostriatal toxicants. This approach further identified the flame retardant 3,3',5,5'-tetrabromobisphenol A (TBBPA) as a selective toxicant for dopaminergic neurons in the context of human midbrain-like tissues for the first time. Results were verified with high reproducibility in more detailed dose-response experiments. Further, we demonstrate higher sensitivity in 3D AMOs than in 2D cultures to the known neurotoxic effects of the pesticide lindane. Overall, the automated nature of our workflow is freely scalable and demonstrates the feasibility of quantitatively assessing cell-type-specific toxicity in human organoids in vitro.

Cementless hip revision cup for the primary fixation of osteoporotic acetabular fractures in geriatric patients.

BACKGROUND: Elderly patients suffering from hip fractures are usually not able to fulfil postoperative weight-bearing restrictions. Therefore, the operative fixation construct has to be as stable as possible. Aim of the present study was to determine (1) whether a therapeutic advantage could be achieved when using hip arthroplasty to treat acetabular fractures in geriatric patients; (2) whether an acetabular revision cup would be suitable for achieving fast postoperative mobilization and full weight-bearing; and (3) when a treatment with an uncemented hip revision cup for the primary fixation of osteoporotic acetabular fractures in geriatric patients is indicated. MATERIALS AND METHODS: The functional outcome of THA using a reconstruction cup for an acetabular fracture was evaluated in ten patients using standardized scoring instruments. In addition, an analysis of the preexisting literature referring to total hip replacement in geriatric acetabular fractures was conducted and an algorithm for standardizing the treatment approach for geriatric patients with acetabular fractures was developed. RESULTS: The mean EQ-5D-3L quality of life score 0.7. The mean VAS Score was 58.2. The average Barthel Index was 80.0 points [range: 0-100]. The mean HHS was 72.0 points, while the MHH Score yielded an average of 63.4 points. The average AP Score was 7.5. The literature analysis showed that total hip arthroplasty could be a feasible option for geriatric acetabular fractures. CONCLUSION: Primary hip arthroplasty using uncemented revision cup fixed with angular stable screws showed good results and is a feasible treatment option of acetabular fractures in geriatric patients. The approach is especially beneficial in patients with poor bone stock and allows postoperative full weight-bearing. The presented treatment algorithm could be a useful tool for identifying the most appropriate treatment option. LEVEL OF EVIDENCE: IIb.

Genotype-Phenotype Features of Germline Variants of the TMEM127 Pheochromocytoma Susceptibility Gene: A 10-Year Update.

PURPOSE: This work aimed to evaluate genotype-phenotype associations in individuals carrying germline variants of transmembrane protein 127 gene (TMEM127), a poorly known gene that confers susceptibility to pheochromocytoma (PHEO) and paraganglioma (PGL). DESIGN: Data were collected from a registry of probands with TMEM127 variants, published reports, and public databases. MAIN OUTCOME ANALYSIS: Clinical, genetic, and functional associations were determined. RESULTS: The cohort comprised 110 index patients (111 variants) with a mean age of 45 years (range, 21-84 years). Females were predominant (76 vs 34, P < .001). Most patients had PHEO (n = 94; 85.5%), although PGL (n = 10; 9%) and renal cell carcinoma (RCC, n = 6; 5.4%) were also detected, either alone or in combination with PHEO. One-third of the cases had multiple tumors, and known family history was reported in 15.4%. Metastatic PHEO/PGL was rare (2.8%). Epinephrine alone, or combined with norepinephrine, accounted for 82% of the catecholamine profiles of PHEO/PGLs. Most variants (n = 63) occurred only once and 13 were recurrent (2-12 times). Although nontruncating variants were less frequent than truncating changes overall, they were predominant in non-PHEO clinical presentations (36% PHEO-only vs 69% other, P < .001) and clustered disproportionately within transmembrane regions (P < .01), underscoring the relevance of these domains for TMEM127 function. Integration of clinical and previous experimental data supported classification of variants into 4 groups based on mutation type, localization, and predicted disruption. CONCLUSIONS: Patients with TMEM127 variants often resemble sporadic nonmetastatic PHEOs. PGL and RCC may also co-occur, although their causal link requires further evaluation. We propose a new classification to predict variant pathogenicity and assist with carrier surveillance.

A fully automated high-throughput workflow for 3D-based chemical screening in human midbrain organoids.

Three-dimensional (3D) culture systems have fueled hopes to bring about the next generation of more physiologically relevant high-throughput screens (HTS). However, current protocols yield either complex but highly heterogeneous aggregates ('organoids') or 3D structures with less physiological relevance ('spheroids'). Here, we present a scalable, HTS-compatible workflow for the automated generation, maintenance, and optical analysis of human midbrain organoids in standard 96-well-plates. The resulting organoids possess a highly homogeneous morphology, size, global gene expression, cellular composition, and structure. They present significant features of the human midbrain and display spontaneous aggregate-wide synchronized neural activity. By automating the entire workflow from generation to analysis, we enhance the intra- and inter-batch reproducibility as demonstrated via RNA sequencing and quantitative whole mount high-content imaging. This allows assessing drug effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow.

In 1907, the American zoologist Ross Granville Harrison developed the first technique to artificially grow animal cells outside the body in a liquid medium. Cells are still grown in much the same way in modern laboratories: a single layer of cells is placed in a warm incubator with nutrient-rich broth. These cell layers are often used to test new drugs, but they cannot recapitulate the complexity of a real organ made from multiple cell types within a living, breathing human body. Growing three-dimensional miniature organs or 'organoids' that behave in a similar way to real organs is the next step towards creating better platforms for drug screening, but there are several difficulties inherent to this process. For one thing, it is hard to recreate the multitude of cell types that make up an organ. For another, the cells that do grow often fail to connect and communicate with each other in biologically realistic ways. It is also tough to grow a large number of organoids that all behave in the same way, making it hard to know whether a particular drug works or whether it is just being tested on a 'good' organoid. Renner et al. have been able to overcome these issues by using robotic technology to create thousands of identical, mid-brain organoids from human cells in the lab. The robots perform a series of precisely controlled tasks ­ including dispensing the initial cells into wells, feeding organoids as they grow and testing them at different stages of development. These mini-brains, which are the size of the head of a pin, mimic the part of the brain where Parkinson's disease first manifests. They can be used to test new drugs for Parkinson's, and to better understand the biology of the brain. Perhaps more importantly, other types of organoids can be created using the same technique to model diseases that affect other areas of the brain, or other organs altogether. For example, Renner et al. also generated forebrain organoids using an automated approach for both generation and analysis. This research, which shows that organoids can be grown and tested in a fully automated, reproducible and scalable way, creates a platform to quickly, cheaply and easily test thousands of drugs for Parkinson's and other difficult-to-treat diseases in a human setting. This approach has the potential to reduce research waste by increasing the chances that a drug that works in the lab will also ultimately work in a patient; and reduce animal experiments, as drugs that do not work in human tissues will not proceed to animal testing.