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Open-top light-sheet (OTLS) microscopy offers rapid 3D imaging of large optically cleared specimens. This enables nondestructive 3D pathology, which provides key advantages over conventional slide-based histology including comprehensive sampling without tissue sectioning/destruction and visualization of diagnostically important 3D structures. With 3D pathology, clinical specimens are often labeled with small-molecule stains that broadly target nucleic acids and proteins, mimicking conventional hematoxylin and eosin (H&E) dyes. Tight optical sectioning helps to minimize out-of-focus fluorescence for high-contrast imaging in these densely labeled tissues but has been challenging to achieve in OTLS systems due to trade-offs between optical sectioning and field of view. Here we present an OTLS microscope with voice-coil-based axial sweeping to circumvent this trade-off, achieving 2â µm axial resolution over a 750 × 375â µm field of view. We implement our design in a non-orthogonal dual-objective (NODO) architecture, which enables a 10-mm working distance with minimal sensitivity to refractive index mismatches, for high-contrast 3D imaging of clinical specimens.
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Imageamento Tridimensional , Imageamento Tridimensional/métodos , Humanos , Microscopia/métodos , Coloração e Rotulagem , LuzRESUMO
An important provision of the Minamata Convention on Mercury is to monitor and evaluate the effectiveness of the adopted measures and its implementation. Here, we describe for the first time currently available biotic mercury (Hg) data on a global scale to improve the understanding of global efforts to reduce the impact of Hg pollution on people and the environment. Data from the peer-reviewed literature were compiled in the Global Biotic Mercury Synthesis (GBMS) database (>550,000 data points). These data provide a foundation for establishing a biomonitoring framework needed to track Hg concentrations in biota globally. We describe Hg exposure in the taxa identified by the Minamata Convention: fish, sea turtles, birds, and marine mammals. Based on the GBMS database, Hg concentrations are presented at relevant geographic scales for continents and oceanic basins. We identify some effective regional templates for monitoring methylmercury (MeHg) availability in the environment, but overall illustrate that there is a general lack of regional biomonitoring initiatives around the world, especially in Africa, Australia, Indo-Pacific, Middle East, and South Atlantic and Pacific Oceans. Temporal trend data for Hg in biota are generally limited. Ecologically sensitive sites (where biota have above average MeHg tissue concentrations) have been identified throughout the world. Efforts to model and quantify ecosystem sensitivity locally, regionally, and globally could help establish effective and efficient biomonitoring programs. We present a framework for a global Hg biomonitoring network that includes a three-step continental and oceanic approach to integrate existing biomonitoring efforts and prioritize filling regional data gaps linked with key Hg sources. We describe a standardized approach that builds on an evidence-based evaluation to assess the Minamata Convention's progress to reduce the impact of global Hg pollution on people and the environment.
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Monitoramento Biológico , Monitoramento Ambiental , Mercúrio , Mercúrio/análise , Monitoramento Biológico/métodos , Animais , Monitoramento Ambiental/métodos , Biota , Poluentes Químicos da Água/análise , Aves , Compostos de Metilmercúrio/análise , Peixes/metabolismoRESUMO
Significance: In recent years, we and others have developed non-destructive methods to obtain three-dimensional (3D) pathology datasets of clinical biopsies and surgical specimens. For prostate cancer risk stratification (prognostication), standard-of-care Gleason grading is based on examining the morphology of prostate glands in thin 2D sections. This motivates us to perform 3D segmentation of prostate glands in our 3D pathology datasets for the purposes of computational analysis of 3D glandular features that could offer improved prognostic performance. Aim: To facilitate prostate cancer risk assessment, we developed a computationally efficient and accurate deep learning model for 3D gland segmentation based on open-top light-sheet microscopy datasets of human prostate biopsies stained with a fluorescent analog of hematoxylin and eosin (H&E). Approach: For 3D gland segmentation based on our H&E-analog 3D pathology datasets, we previously developed a hybrid deep learning and computer vision-based pipeline, called image translation-assisted segmentation in 3D (ITAS3D), which required a complex two-stage procedure and tedious manual optimization of parameters. To simplify this procedure, we use the 3D gland-segmentation masks previously generated by ITAS3D as training datasets for a direct end-to-end deep learning-based segmentation model, nnU-Net. The inputs to this model are 3D pathology datasets of prostate biopsies rapidly stained with an inexpensive fluorescent analog of H&E and the outputs are 3D semantic segmentation masks of the gland epithelium, gland lumen, and surrounding stromal compartments within the tissue. Results: nnU-Net demonstrates remarkable accuracy in 3D gland segmentations even with limited training data. Moreover, compared with the previous ITAS3D pipeline, nnU-Net operation is simpler and faster, and it can maintain good accuracy even with lower-resolution inputs. Conclusions: Our trained DL-based 3D segmentation model will facilitate future studies to demonstrate the value of computational 3D pathology for guiding critical treatment decisions for patients with prostate cancer.
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Próstata , Neoplasias da Próstata , Masculino , Humanos , Próstata/diagnóstico por imagem , Neoplasias da Próstata/diagnóstico por imagem , Biópsia , Corantes , Amarelo de Eosina-(YS)RESUMO
Tissues are active materials where epithelial turnover, immune surveillance, and remodeling of stromal cells such as macrophages all regulate form and function. Scattering modalities such as Brillouin microscopy (BM) can non-invasively access mechanical signatures at GHz. However, our traditional understanding of tissue material properties is derived mainly from modalities which probe mechanical properties at different frequencies. Thus, reconciling measurements amongst these modalities remains an active area. Here, we compare optical tweezer active microrheology (OT-AMR) and Brillouin microscopy (BM) to longitudinally map brain development in the larval zebrafish. We determine that each measurement is able to detect a mechanical signature linked to functional units of the brain. We demonstrate that the corrected BM-Longitudinal modulus using a density factor correlates well with OT-AMR storage modulus at lower frequencies. We also show that the brain tissue mechanical properties are dependent on both the neuronal architecture and the presence of macrophages. Moreover, the BM technique is able to delineate the contributions to mechanical properties of the macrophage from that due to colony stimulating factor 1 receptor (CSF1R) mediated stromal remodeling. Here, our data suggest that macrophage remodeling is instrumental in the maintenance of tissue mechanical homeostasis during development. Moreover, the strong agreement between the OT-AM and BM further demonstrates that scattering-based technique is sensitive to both large and minute structural modification in vivo.
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Recent advances in 3D pathology offer the ability to image orders of magnitude more tissue than conventional pathology methods while also providing a volumetric context that is not achievable with 2D tissue sections, and all without requiring destructive tissue sectioning. Generating high-quality 3D pathology datasets on a consistent basis, however, is not trivial and requires careful attention to a series of details during tissue preparation, imaging and initial data processing, as well as iterative optimization of the entire process. Here, we provide an end-to-end procedure covering all aspects of a 3D pathology workflow (using light-sheet microscopy as an illustrative imaging platform) with sufficient detail to perform well-controlled preclinical and clinical studies. Although 3D pathology is compatible with diverse staining protocols and computationally generated color palettes for visual analysis, this protocol focuses on the use of a fluorescent analog of hematoxylin and eosin, which remains the most common stain used for gold-standard pathological reports. We present our guidelines for a broad range of end users (e.g., biologists, clinical researchers and engineers) in a simple format. The end-to-end workflow requires 3-6 d to complete, bearing in mind that data analysis may take longer.
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Imageamento Tridimensional , Microscopia , Imageamento Tridimensional/métodos , Fluxo de Trabalho , Microscopia/métodos , Corantes , Coloração e RotulagemRESUMO
Dietary uptake is key for transferring potentially toxic contaminants, such as mercury (Hg) and essential dietary nutrients, such as polyunsaturated fatty acids (PUFA), to consumers at higher trophic levels of aquatic food webs. We evaluated the role of diet sources for Hg bioaccumulation and PUFA retention in fish across lake food webs in seven Swedish lakes and two Chinese reservoirs. Fish total Hg (THg) and methyl-Hg (MeHg) differed greatly between the two countries: the Chinese fish contained less than 300 ng g-1 dry weight (d.w.) THg with less than 50% as MeHg, versus the Swedish fishes which contained approximately 2000 ng g-1 d.w. THg and nearly 100% as MeHg. Fatty acids enrichment of linoleic acids (LIN) were more prevalent in the Chinese fishes regardless of size (p < 0.05). Here we examined food web length, fish growth rates, and fatty acids patterns in relation to the quality of fish as a food source for both Hg and FA. Contrary to the expectation that biodilution of Hg throughout the food chain would explain these differences, a more complex picture emerged with high levels of Hg at the base of the food web in the Chinese reservoirs, a decoupling of fatty acid and Hg bioaccumulation, and a major role for both fish stocking and fish feed. It is hoped that this work will provide a nuanced picture of fish quality as a food source in different ecosystems.
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A miniature optical-sectioning fluorescence microscope with high sensitivity and resolution would enable non-invasive and real-time tissue inspection, with potential use cases including early disease detection and intraoperative guidance. Previously, we developed a miniature MEMS-based dual-axis confocal (DAC) microscope that enabled video-rate optically sectioned in vivo microscopy of human tissues. However, the device's clinical utility was limited due to a small field of view, a non-adjustable working distance, and a lack of a sterilization strategy. In our latest design, we have made improvements to achieve a 2x increase in the field of view (600 × 300 µm) and an adjustable working distance range of 150 µm over a wide range of excitation/emission wavelengths (488-750â nm), all while maintaining a high frame rate of 15 frames per second (fps). Furthermore, the device is designed to image through a disposable sterile plastic drape for convenient clinical use. We rigorously characterize the performance of the device and show example images of ex vivo tissues to demonstrate the optical performance of our new design, including fixed mouse skin and human prostate, as well as fresh mouse kidney, mouse intestine, and human head and neck surgical specimens with corresponding H&E histology. These improvements will facilitate clinical testing and translation.
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Peatland vegetation takes up mercury (Hg) from the atmosphere, typically contributing to net production and export of neurotoxic methyl-Hg to downstream ecosystems. Chemical reduction processes can slow down methyl-Hg production by releasing Hg from peat back to the atmosphere. The extent of these processes remains, however, unclear. Here we present results from a comprehensive study covering concentrations and isotopic signatures of Hg in an open boreal peatland system to identify post-depositional Hg redox transformation processes. Isotope mass balances suggest photoreduction of HgII is the predominant process by which 30% of annually deposited Hg is emitted back to the atmosphere. Isotopic analyses indicate that above the water table, dark abiotic oxidation decreases peat soil gaseous Hg0 concentrations. Below the water table, supersaturation of gaseous Hg is likely created more by direct photoreduction of rainfall rather than by reduction and release of Hg from the peat soil. Identification and quantification of these light-driven and dark redox processes advance our understanding of the fate of Hg in peatlands, including the potential for mobilization and methylation of HgII.
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Methylmercury (MeHg) produced in rice paddies is the main source of MeHg accumulation in rice, resulting in high risk of MeHg exposure to humans and wildlife. Net MeHg production is affected by Hg(II) reduction and MeHg demethylation, but it remains unclear to what extent these processes influence net MeHg production, as well as the role of the microbial guilds involved. We used isotopically labeled Hg species and specific microbial inhibitors in microcosm experiments to simultaneously investigate the rates of Hg(II) and MeHg transformations, as well as the key microbial guilds controlling these processes. Results showed that Hg(II) and MeHg reduction rate constants significantly decreased with addition of molybdate or BES, which inhibit sulfate-reduction and methanogenesis, respectively. This suggests that both sulfate-reduction and methanogenesis are important processes controlling Hg(II) and MeHg reduction in rice paddies. Meanwhile, up to 99% of MeHg demethylation was oxidative demethylation (OD) under the incubation conditions, suggesting that OD was the main MeHg degradative pathway in rice paddies. In addition, [202Hg(0)/Me202Hg] from the added 202Hg(NO3)2 was up to 13.9%, suggesting that Hg(II) reduction may constrain Hg(II) methylation in rice paddies at the abandoned Hg mining site. This study improves our understanding of Hg cycling pathways in rice paddies, and more specifically how reduction processes affect net MeHg production and related microbial metabolisms.
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Mercúrio , Compostos de Metilmercúrio , Oryza , Humanos , Desmetilação , SulfatosRESUMO
Tree growth in boreal forests is driven by ectomycorrhizal fungal mobilisation of organic nitrogen and mineral nutrients in soils with discrete organic and mineral horizons. However, there are no studies of how ectomycorrhizal mineral weathering and organic nitrogen mobilisation processes are integrated across the soil profile. We studied effects of organic matter (OM) availability on ectomycorrhizal functioning by altering the proportions of natural organic and mineral soil in reconstructed podzol profiles containing Pinus sylvestris plants, using 13 CO2 pulse labelling, patterns of naturally occurring stable isotopes (26 Mg and 15 N) and high-throughput DNA sequencing of fungal amplicons. Reduction in OM resulted in nitrogen limitation of plant growth and decreased allocation of photosynthetically derived carbon and mycelial growth in mineral horizons. Fractionation patterns of 26 Mg indicated that magnesium mobilisation and uptake occurred primarily in the deeper mineral horizon and was driven by carbon allocation to ectomycorrhizal mycelium. In this horizon, relative abundance of ectomycorrhizal fungi, carbon allocation and base cation mobilisation all increased with increased OM availability. Allocation of carbon through ectomycorrhizal fungi integrates organic nitrogen mobilisation and mineral weathering across soil horizons, improving the efficiency of plant nutrient acquisition. Our findings have fundamental implications for sustainable forest management and belowground carbon sequestration.
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Recent advances in 3D pathology offer the ability to image orders-of-magnitude more tissue than conventional pathology while providing a volumetric context that is lacking with 2D tissue sections, all without requiring destructive tissue sectioning. Generating high-quality 3D pathology datasets on a consistent basis is non-trivial, requiring careful attention to many details regarding tissue preparation, imaging, and data/image processing in an iterative process. Here we provide an end-to-end protocol covering all aspects of a 3D pathology workflow (using light-sheet microscopy as an illustrative imaging platform) with sufficient detail to perform well-controlled preclinical and clinical studies. While 3D pathology is compatible with diverse staining protocols and computationally generated color palettes for visual analysis, this protocol will focus on a fluorescent analog of hematoxylin and eosin (H&E), which remains the most common stain for gold-standard diagnostic determinations. We present our guidelines for a broad range of end-users (e.g., biologists, clinical researchers, and engineers) in a simple tutorial format.
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OBJECTIVE: For tumor resections, margin status typically correlates with patient survival but positive margin rates are generally high (up to 45% for head and neck cancer). Frozen section analysis (FSA) is often used to intraoperatively assess the margins of excised tissue, but suffers from severe under-sampling of the actual margin surface, inferior image quality, slow turnaround, and tissue destructiveness. METHODS: Here, we have developed an imaging workflow to generate en face histologic images of freshly excised surgical margin surfaces based on open-top light-sheet (OTLS) microscopy. Key innovations include (1) the ability to generate false-colored H&E-mimicking images of tissue surfaces stained for < 1 min with a single fluorophore, (2) rapid OTLS surface imaging at a rate of 15 min/cm2 followed by real-time post-processing of datasets within RAM at a rate of 5 min/cm2, and (3) rapid digital surface extraction to account for topological irregularities at the tissue surface. RESULTS: In addition to the performance metrics listed above, we show that the image quality generated by our rapid surface-histology method approaches that of gold-standard archival histology. CONCLUSION: OTLS microscopy has the feasibility to provide intraoperative guidance of surgical oncology procedures. SIGNIFICANCE: The reported methods can potentially improve tumor-resection procedures, thereby improving patient outcomes and quality of life.
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Margens de Excisão , Microscopia , Humanos , Qualidade de Vida , Técnicas HistológicasRESUMO
Background: Mutations of thyroid hormone receptor α (TRα1) result in resistance to thyroid hormone (RTHα), exhibiting symptoms of retarded growth, delayed bone maturation, anemia, and severe constipation. Using a mouse model of RTHα (Thra1PV/+ mouse), we aimed at understanding the molecular basis underlying the severe constipation observed in patients. Methods: The Thra1PV/+ mouse expresses a strong dominant negative mutant, PV, which has lost T3 binding and transcription activity. Thra1PV/+ mouse faithfully reproduces growth abnormalities and anemia as shown in RTHα patients and therefore is a valid model to examine causes of severe constipation in patients. We used histopathological analysis, confocal fluorescence imaging, transmission electron microscopy (TEM), and gene expression profiles to comprehensively analyze the colonic abnormalities of Thra1PV/+ mouse. Results: We found a significant increase in colonic transit time and decrease stool water content in Thra1PV/+ mouse, mimicking constipation as found in patients. Histopathological analysis showed expanded lamina propria filled with interstitium fluid between crypt columns, enlarged muscularis mucosa, and increased content of collagen in expanded submucosa. The TEM analysis revealed shorter muscle fibers with wider gap junctions between muscle cells, fewer caveolae, and hypoplastic interstitial cells of Cajal (ICC) in the rectal smooth muscles of Thra1PV/+ mice. These abnormal histological manifestations suggested defective intercellular transfer of small molecules, electrolytes, and signals for communication among muscles cells, validated by Lucifer Yellow transferring assays. Expression of key smooth muscle contractility regulators, such as calmodulin, myosin light-chain kinase, and phosphorylated myosin light chain, was markedly lower, and c-KIT signaling in ICC was attenuated, resulting in decreased contractility of the rectal smooth muscles of Thra1PV/+ mice. Collectively, these abnormal histopathological alterations and diminished contractility regulators led to the constipation exhibited in patients. Conclusions: This is the first demonstration that TRα1 mutants could act to cause abnormal rectum smooth muscle organization, defects in intercellular exchange of small molecules, and decreased expression of contractility regulators to weaken the contractility of rectal smooth muscles. These findings provide new insights into the molecular basis underlying constipation found in RTHα patients.
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Anemia , Receptores alfa dos Hormônios Tireóideos , Humanos , Receptores alfa dos Hormônios Tireóideos/genética , Receptores alfa dos Hormônios Tireóideos/metabolismo , Hormônios Tireóideos , Mutação , Constipação Intestinal/genéticaRESUMO
Peatlands are generally important sources of methylmercury (MeHg) to adjacent aquatic ecosystems, increasing the risk of human and wildlife exposure to this highly toxic compound. While microorganisms play important roles in mercury (Hg) geochemical cycles where they directly and indirectly affect MeHg formation in peatlands, potential linkages between net MeHg formation and microbial communities involving these microorganisms remain unclear. To address this gap, microbial community composition and specific marker gene transcripts were investigated along a trophic gradient in a geographically constrained peatland chronosequence. Our results showed a clear spatial pattern in microbial community composition along the gradient that was highly driven by peat soil properties and significantly associated with net MeHg formation as approximated by MeHg concentration and %MeHg of total Hg concentration. Known fermentative, syntrophic, methanogenic and iron-reducing metabolic guilds had the strong positive correlations to net MeHg formation, while methanotrophic and methylotrophic microorganisms were negatively correlated. Our results indicated that sulfate reducers did not have a key role in net MeHg formation. Microbial activity as interpreted from 16S rRNA sequences was significantly correlated with MeHg and %MeHg. Our findings shed new light on the role of microbial community in net MeHg formation of peatlands that undergo ontogenetic change.
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Mercúrio , Compostos de Metilmercúrio , Microbiota , Humanos , Compostos de Metilmercúrio/metabolismo , RNA Ribossômico 16S/genética , Mercúrio/análise , Solo/química , Sulfatos , FerroRESUMO
The microenvironment is an important regulator of intertumoral trafficking and activity of immune cells. Understanding how the immune system can be tailored to maintain anti-tumor killing responses in metastatic disease remains an important goal. Thus, immune mediated eradication of metastasis requires the consideration of organ specific microenvironmental cues. Using a xenograft model of melanoma metastasis in adult zebrafish, we perturbed the dynamic balance between the infiltrating immune cells in the metastatic setting using a suite of different transgenic zebrafish. We employed intravital imaging coupled with metabolism imaging (FLIM) to visualize and map the organ specific metabolism with near simultaneity in multiple metastatic lesions. Of all the MHC complexes examined for brain and skeletal metastases, we determined that there is an organ specific expression of mhc1uba (human ortholog, MR1) for both the melanoma cells and the resident and infiltrating immune cells. Specifically, immune clusters did not express mhc1uba in brain metastatic lesions in immune competent fish. Finally, the differential immune response drove organ specific metabolism where tumor glycolysis was increased in brain metastases compared to skeletal and parental lines as measured using fluorescence lifetime imaging microscopy (FLIM). As MR1 belongs to the MHC class I molecules and is a target of immunotherapeutic drugs, we believe that our data presents an opportunity to understand the relationship between organ specific tumor metabolism and drug efficacy in the metastatic setting.
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ZRSR2 (zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2) is an essential splicing factor involved in 3' splice-site recognition as a component of both the major and minor spliceosomes that mediate the splicing of U2-type (major) and U12-type (minor) introns, respectively. Studies of ZRSR2-depleted cell lines and ZRSR2-mutated patient samples revealed its essential role in the U12-dependent minor spliceosome. However, the role of ZRSR2 during embryonic development is not clear, as its function is compensated for by Zrsr1 in mice. Here, we utilized the zebrafish model to investigate the role of zrsr2 during embryonic development. Using CRISPR/Cas9 technology, we generated a zrsr2-knockout zebrafish line, termed zrsr2hg129/hg129 (p.Trp167Argfs*9) and examined embryo development in the homozygous mutant embryos. zrsr2hg129/hg129 embryos displayed multiple developmental defects starting at 4 days post fertilization (dpf) and died after 8 dpf, suggesting that proper Zrsr2 function is required during embryonic development. The global transcriptome analysis of 3 dpf zrsr2hg129/hg129 embryos revealed that the loss of Zrsr2 results in the downregulation of essential metabolic pathways and the aberrant retention of minor introns in about one-third of all minor intron-containing genes in zebrafish. Overall, our study has demonstrated that the role of Zrsr2 as a component of the minor spliceosome is conserved and critical for proper embryonic development in zebrafish.
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Splicing de RNA , Ribonucleoproteínas , Peixe-Zebra , Animais , Camundongos , Desenvolvimento Embrionário , Íntrons/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Sítios de Splice de RNA , Splicing de RNA/genética , Fatores de Processamento de RNA/genética , Spliceossomos/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by improper biogenesis of lysosome-related organelles (LROs). Lung fibrosis is the leading cause of death among adults with HPS-1 and HPS-4 genetic types, which are associated with defects in the biogenesis of lysosome-related organelles complex-3 (BLOC-3), a guanine exchange factor (GEF) for a small GTPase, Rab32. LROs are not ubiquitously present in all cell types, and specific cells utilize LROs to accomplish dedicated functions. Fibroblasts are not known to contain LROs, and the function of BLOC-3 in fibroblasts is unclear. Here, we report that lung fibroblasts isolated from patients with HPS-1 have increased migration capacity. Silencing HPS-1 in normal lung fibroblasts similarly leads to increased migration. We also show that the increased migration is driven by elevated levels of Myosin IIB. Silencing HPS1 or RAB32 in normal lung fibroblasts leads to increased MYOSIN IIB levels. MYOSIN IIB is downstream of p38-MAPK, which is a known target of angiotensin receptor signaling. Treatment with losartan, an angiotensin receptor inhibitor, decreases MYOSIN IIB levels and impedes HPS lung fibroblast migration in vitro. Furthermore, pharmacologic inhibition of angiotensin receptor with losartan seemed to decrease migration of HPS lung fibroblasts in vivo in a zebrafish xenotransplantation model. Taken together, we demonstrate that BLOC-3 plays an important role in MYOSIN IIB regulation within lung fibroblasts and contributes to fibroblast migration.
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Síndrome de Hermanski-Pudlak , Albinismo , Animais , Movimento Celular , Fibroblastos/metabolismo , Transtornos Hemorrágicos , Síndrome de Hermanski-Pudlak/genética , Humanos , Losartan/metabolismo , Pulmão/metabolismo , Miosina não Muscular Tipo IIB/metabolismo , Receptores de Angiotensina , Peixe-ZebraRESUMO
The use of zebrafish in functional genomics and disease modeling has become popular due to the ease of targeted mutagenesis with genome editing nucleases, i.e., zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9). These nucleases, specifically CRISPR/Cas9, are routinely used to generate gene knockout mutants by causing a double stranded break at the desired site in the target gene and selecting for frameshift insertions or deletions (indels) caused by the errors during the repair process. Thus, a variety of methods have been developed to identify fish with indels during the process of mutant generation and phenotypic analysis. These methods range from PCR and gel-based low-throughput methods to high-throughput methods requiring specific reagents and/or equipment. Here, we provide a comprehensive review of currently used indel detection methods in zebrafish. By discussing the molecular basis for each method as well as their pros and cons, we hope that this review will serve as a comprehensive resource for zebrafish researchers, allowing them to choose the most appropriate method depending upon their budget, access to required equipment and the throughput needs of the projects.