ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Fluorescent Dyes , Imaging, Three-Dimensional , Brain/diagnostic imaging , Fluorescent Dyes/chemistry , Imaging, Three-Dimensional/methods , Ionophores , SolventsABSTRACT
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.
Subject(s)
Neurodegenerative Diseases , Parkinson Disease , Animals , Artificial Intelligence , Neurodegenerative Diseases/metabolism , Organoids/metabolism , Parkinson Disease/drug therapy , WorkflowABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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%.
Subject(s)
Bone Density , Osteoporotic Fractures , Absorptiometry, Photon , Female , Humans , Middle Aged , Osteoporotic Fractures/diagnostic imaging , Osteoporotic Fractures/epidemiology , Retrospective Studies , Risk Assessment , Risk FactorsABSTRACT
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.
Subject(s)
Cellular Reprogramming , Epigenesis, Genetic , Histone-Lysine N-Methyltransferase/genetics , Histones/genetics , Human Embryonic Stem Cells/metabolism , Induced Pluripotent Stem Cells/metabolism , Animals , Cell Line , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA (Cytosine-5-)-Methyltransferases/metabolism , Eye Proteins/genetics , Eye Proteins/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , HEK293 Cells , HeLa Cells , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Histones/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Human Embryonic Stem Cells/cytology , Humans , Induced Pluripotent Stem Cells/cytology , Mice , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Octamer Transcription Factors/genetics , Octamer Transcription Factors/metabolism , Otx Transcription Factors/genetics , Otx Transcription Factors/metabolism , Plasmids/chemistry , Plasmids/metabolism , Species Specificity , Transcription, Genetic , Transfection , Homeobox Protein SIX3ABSTRACT
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.
Subject(s)
Adrenal Gland Neoplasms/genetics , Membrane Proteins/genetics , Pheochromocytoma/genetics , Adrenal Gland Neoplasms/epidemiology , Adult , Aged , Aged, 80 and over , Cohort Studies , Databases, Genetic , Female , Genetic Association Studies , Genetic Predisposition to Disease , Genetic Testing , Germ-Line Mutation , Humans , Male , Middle Aged , Pheochromocytoma/epidemiology , Retrospective Studies , Young AdultABSTRACT
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.
Subject(s)
Mesencephalon/cytology , Organoids/cytology , Workflow , Automation , Calcium/metabolism , Cell Lineage , Dopamine/metabolism , Humans , Imaging, Three-Dimensional , Mesencephalon/physiology , Organoids/drug effects , Patch-Clamp Techniques , Pluripotent Stem Cells/cytology , Reproducibility of Results , Sequence Analysis, RNAABSTRACT
Recent advances in induced pluripotent stem cell technologies and phenotypic screening shape the future of bioactive small-molecule discovery. In this review we analyze the impact of small-molecule phenotypic screens on drug discovery as well as on the investigation of human development and disease biology. We further examine the role of 3D spheroid/organoid structures, microfluidic systems, and miniaturized on-a-chip systems for future discovery strategies. In highlighting representative examples, we analyze how recent achievements can translate into future therapies. Finally, we discuss remaining challenges that need to be overcome for the adaptation of the next generation of screening approaches.
Subject(s)
Cell Culture Techniques , Drug Discovery , Induced Pluripotent Stem Cells/cytology , Small Molecule Libraries/chemistry , Animals , Drug Evaluation, Preclinical , Humans , Microfluidic Analytical Techniques , Organoids/chemistry , Phenotype , Spheroids, Cellular/chemistryABSTRACT
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.
Subject(s)
Bone Density , Genetic Association Studies , Genetic Predisposition to Disease , Phenotype , Adolescent , Adult , Aneuploidy , Biomarkers , Child , Chromosomes, Human, Pair 18/genetics , Female , Humans , Male , Young AdultABSTRACT
BACKGROUND Hypercalcemia associated with chronic myeloid leukemia (CML) is an ominous sign. Although rare, several cases have been reported and multiple pathophysiologic mechanisms have been independently proposed. We present a patient case and a literature review of the clinical presentation and mechanisms of CML-associated hypercalcemia. CASE REPORT A 58-year-old male with a past medical history of CML diagnosed six years earlier, presented to the emergency department with one week of acute confusion, disorientation, polyuria, and polydipsia. On physical examination, we observed tachycardia, altered mental status, and dehydration. Blood analysis revealed leukocytosis, thrombocytosis, and marked hypercalcemia (18.6 mg/dL). His chest CT scan showed diffuse lytic lesions and bone destruction concerning for diffuse bone marrow involvement. The patient was diagnosed with hypercalcemia in the context of a CML blast phase. Treatment with hydration, calcitonin, and zoledronic acid lead to control of his symptoms and normalization of his serum calcium levels. After discharged, the patient was maintained on palliative treatment and zoledronic acid management without new episodes of hypercalcemia. However, eight months later, the patient died. CONCLUSIONS Evidence from the literature demonstrates a highly variable clinical presentation of CML-associated hypercalcemia, commonly occurring during an accelerated or a blast phase, and associated with poor survival. Multiple mechanisms could be involved and are not exclusive of each other. Better understanding of the pathophysiologic mechanisms involved in CML-associated hypercalcemia could lead to improvement in clinical and laboratory evaluation of these patients and be the foundation for the development of better management strategies and possibly target-directed therapy to positively improve prognosis.
Subject(s)
Blast Crisis/pathology , Bone Density Conservation Agents/therapeutic use , Calcitonin/therapeutic use , Diphosphonates/therapeutic use , Hypercalcemia/drug therapy , Hypercalcemia/etiology , Imidazoles/therapeutic use , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/complications , Blast Crisis/blood , Disease Progression , Drug Therapy, Combination , Fatal Outcome , Humans , Hypercalcemia/diagnosis , Male , Middle Aged , Zoledronic AcidABSTRACT
Phenotypic drug discovery requires billions of cells for high-throughput screening (HTS) campaigns. Because up to several million different small molecules will be tested in a single HTS campaign, even small variability within the cell populations for screening could easily invalidate an entire campaign. Neurodegenerative assays are particularly challenging because neurons are post-mitotic and cannot be expanded for implementation in HTS. Therefore, HTS for neuroprotective compounds requires a cell type that is robustly expandable and able to differentiate into all of the neuronal subtypes involved in disease pathogenesis. Here, we report the derivation and propagation using only small molecules of human neural progenitor cells (small molecule neural precursor cells; smNPCs). smNPCs are robust, exhibit immortal expansion, and do not require cumbersome manual culture and selection steps. We demonstrate that smNPCs have the potential to clonally and efficiently differentiate into neural tube lineages, including motor neurons (MNs) and midbrain dopaminergic neurons (mDANs) as well as neural crest lineages, including peripheral neurons and mesenchymal cells. These properties are so far only matched by pluripotent stem cells. Finally, to demonstrate the usefulness of smNPCs we show that mDANs differentiated from smNPCs with LRRK2 G2019S are more susceptible to apoptosis in the presence of oxidative stress compared to wild-type. Therefore, smNPCs are a powerful biological tool with properties that are optimal for large-scale disease modeling, phenotypic screening, and studies of early human development.
Subject(s)
Epithelial Cells/cytology , Epithelial Cells/metabolism , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Neurodegenerative Diseases/metabolism , Cell Differentiation/genetics , Cell Differentiation/physiology , Cells, Cultured , Electrophysiology , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Motor Neurons/cytology , Motor Neurons/metabolism , Neural Crest/cytology , Neural Crest/metabolism , Neurodegenerative Diseases/genetics , Neurons/cytology , Neurons/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolismABSTRACT
Melanocytes present in skin and other organs synthesize and store melanin pigment within membrane-delimited organelles called melanosomes. Exposure of human skin to ultraviolet radiation (UV) stimulates melanin production in melanosomes, followed by transfer of melanosomes from melanocytes to neighboring keratinocytes. Melanosomal function is critical for protecting skin against UV radiation, but the mechanisms underlying melanosomal movement and transfer are not well understood. Here we report a novel fluorescent melanosomal marker, which we used to measure real-time melanosomal dynamics in live human epidermal melanocytes (HEMs) and transfer in melanocyte-keratinocyte co-cultures. A fluorescent fusion protein of Ocular Albinism 1 (OA1) localized to melanosomes in both B16-F1 cells and HEMs, and its expression did not significantly alter melanosomal distribution. Live-cell tracking of OA1-GFP-tagged melanosomes revealed a bimodal kinetic profile, with melanosomes exhibiting combinations of slow and fast movement. We also found that exposure to UV radiation increased the fraction of melanosomes exhibiting fast versus slow movement. In addition, using OA1-GFP in live co-cultures, we monitored melanosomal transfer using time-lapse microscopy. These results highlight OA1-GFP as a specific and effective melanosomal marker for live-cell studies, reveal new aspects of melanosomal dynamics and transfer, and are relevant to understanding the skin's physiological response to UV radiation.
Subject(s)
Fluorescent Dyes/metabolism , Melanosomes/metabolism , Molecular Imaging , Animals , Cell Survival/radiation effects , Coculture Techniques , Diffusion , Dose-Response Relationship, Radiation , Eye Proteins/metabolism , HEK293 Cells , Humans , Keratinocytes/cytology , Melanoma, Experimental/pathology , Melanosomes/radiation effects , Membrane Glycoproteins/metabolism , Mice , Movement/radiation effects , Protein Transport/radiation effects , Receptors, G-Protein-Coupled/metabolism , Ultraviolet Rays , rab GTP-Binding Proteins/metabolism , rab27 GTP-Binding ProteinsABSTRACT
Biomimetic replicas of cellular topography have been utilized to direct neurite outgrowth. Here, we cultured postnatal rat dorsal root ganglion (DRG) explants in the presence of Schwann cell (SC) topography to determine the influence of SC topography on neurite outgrowth. Four distinct poly(dimethyl siloxane) conduits were fabricated within which DRG explants were cultured. To determine the contribution of SC topographical features to neurite guidance, the extent of neurite outgrowth into unpatterned conduits, conduits with randomly oriented SC replicas, and conduits with SC replicas parallel or perpendicular to the conduit long axis was measured. Neurite directionality and outgrowth from DRG were also quantified on two-dimensional SC replicas with orientations corresponding to the four conduit conditions. Additionally, live SC migration and neurite extension from DRG on SC replicas were examined as a first step toward quantification of the interactions between live SC and navigating neurites on SC replicas. DRG neurite outgrowth and morphology within conduits and on two-dimensional SC replicas were directed by the underlying SC topographical features. Maximal neurite outgrowth and alignment to the underlying features were observed into parallel conduits and on parallel two-dimensional substrates, whereas the least extent of outgrowth was observed into perpendicular conduits and on perpendicular two-dimensional replica conditions. Additionally, neurites on perpendicular conditions turned to extend along the direction of underlying SC topography. Neurite outgrowth exceeded SC migration in the direction of the underlying anisotropic SC replica after two days in culture. This finding confirms the critical role that SC have in guiding neurite outgrowth and suggests that the mechanism of neurite alignment to SC replicas depends on direct contact with cellular topography. These results suggest that SC topographical replicas may be used to direct and optimize neurite alignment, and emphasize the importance of SC features in neurite guidance.
Subject(s)
Dimethylpolysiloxanes , Ganglia, Spinal/physiology , Neurites/physiology , Schwann Cells/physiology , Animals , Biomimetics , Cell Movement , Cells, Cultured , Electrodes, Implanted , Immunohistochemistry , Microscopy, Electron, Scanning , Organ Culture Techniques , Rats , Schwann Cells/ultrastructure , Sciatic Nerve/cytology , Sciatic Nerve/physiologyABSTRACT
Little is known about the progression of bone loss during young adulthood and whether it differs between men and women. As part of the San Antonio Family Osteoporosis Study we tested whether bone mineral density (BMD) changed over time in men or women, and whether the rate of BMD change differed between the sexes. BMD of the proximal femur, spine, radius, and whole body was measured in 115 men and 202 pre-menopausal women (ages 25 to 45 years; Mexican American ancestry) by dual-energy x-ray absorptiometry at two time points (5.6 years apart), from which annual percent change-in-BMD was calculated. Likelihood-based methods were used to test whether change-in-BMD differs from zero or differs between men and women. In men, percent change-in-BMD was significantly greater than zero for the 1/3 radius (i.e. indicating a gain of BMD; Bonferroni-adjusted p<0.01), less than zero for the femoral neck, lumbar spine, ultradistal radius, and whole body (i.e. indicating a loss of BMD; p<0.01 for all), and not different than zero for the total hip (p=0.24). In women, percent change-in-BMD was greater than zero for the total hip, 1/3 radius, and whole body (p<0.01 for all), less than zero for the ultradistal radius (p<0.01), and not significantly different than zero for the femoral neck and lumbar spine (p=1.0 for both). For all skeletal sites, men experienced greater decrease in BMD (or less increase in BMD) than women; this result was observed both with and without adjustment for age, BMI, and change-in-BMI (p<0.05 for all). These results suggest that significant bone loss occurs at some skeletal sites in young men and women, and that loss of BMD is occurring significantly faster, or gain of BMD is occurring significantly slower, in young men compared to young women.
Subject(s)
Bone Resorption/ethnology , Bone Resorption/pathology , Mexican Americans/ethnology , Osteoporosis/ethnology , Osteoporosis/pathology , Sex Characteristics , Adult , Bone Density/physiology , Family , Female , Humans , Male , Middle Aged , Models, Biological , Osteoporosis/physiopathology , Texas , Time FactorsABSTRACT
Bone loss occurs as early as the third decade and its cumulative effect throughout adulthood may impact risk for osteoporosis in later life, however, the genes and environmental factors influencing early bone loss are largely unknown. We investigated the role of genes in the change in bone mineral density (BMD) in participants in the San Antonio Family Osteoporosis Study. BMD change in 327 Mexican Americans (ages 25-45 years) from 32 extended pedigrees was calculated from DXA measurements at baseline and follow-up (3.5 to 8.9 years later). Family-based likelihood methods were used to estimate heritability (h(2)) and perform autosome-wide linkage analysis for BMD change of the proximal femur and forearm and to estimate heritability for BMD change of lumbar spine. BMD change was significantly heritable for total hip, ultradistal radius, and 33% radius (h(2) = 0.34, 0.34, and 0.27, respectively; p < 0.03 for all), modestly heritable for femoral neck (h(2) = 0.22; p = 0.06) and not heritable for spine BMD. Covariates associated with BMD change included age, sex, baseline BMD, menopause, body mass index, and interim BMI change, and accounted for 6% to 24% of phenotype variation. A significant quantitative trait locus (LOD = 3.6) for femoral neck BMD change was observed on chromosome 1q23. In conclusion, we observed that change in BMD in young adults is heritable and performed one of the first linkage studies for BMD change. Linkage to chromosome 1q23 suggests that this region may harbor one or more genes involved in regulating early BMD change of the femoral neck.
Subject(s)
Bone Density/genetics , Chromosomes, Human, Pair 1/genetics , Mexican Americans/genetics , Quantitative Trait Loci , Adult , Bone Diseases, Metabolic/genetics , Chromosome Mapping , Female , Femur Neck/metabolism , Genotype , Humans , Male , Middle AgedABSTRACT
Paget's disease of bone (PDB) is a localized bone disease characterized by excessive bone resorption due to overactive osteoclasts. Seven genetic loci (PDB1-PDB7) have been reported for late-onset PDB. PDB3 is the only locus where a gene, sequestosome 1 (SQSTM1), has been identified. Mutations in SQSTM1 have been associated with both sporadic and hereditary PDB in different populations. However, the SQSTM1 mutation frequency in PDB patients from a more heterogeneous population has never been reported. To investigate this, we determined the frequency of mutations in patients from the United States. Blood was collected from sporadic and hereditary PDB patients in the United States. DNA was isolated from whole blood or from serum. The SQSTM1 sequence was determined for exons and intron/exon junctions from whole blood and serum. A total of 112 (39 hereditary, 73 sporadic) samples were collected. Eight mutations were found in hereditary PDB patients, for a mutation frequency of 20.5% (95% confidence interval [CI] 10.8-35.5%) and did not differ significantly from mutation rates observed in studies in Canada, Great Britain, and The Netherlands. No mutations were found in sporadic patients, for a frequency of 0% (95% CI 0.0-5.0%), which was statistically significantly lower than the mutation rates previously observed in populations from Australia (P = 0.009), Canada (P = 0.008), Great Britain (P = 0.02), and France (P = 0.04) but not compared to rates from Belgium, The Netherlands, and Italy. Four out of five families with the P392L mutation carried it on the H2 haplotype. Mutations in SQSTM1 seem to contribute to the pathogenesis of PDB in hereditary, but not sporadic, patients in the United States.