RESUMO
Tartrate-resistant acid phosphatase type 5 (TRAP) exists as two isoforms, 5a and 5b. 5b is a marker of osteoclast number and 5a of chronic inflammation; however, its association with bone resorption is unknown. In this study, a double-TRAP 5a/5b sandwich ELISA measuring 5a and 5b protein in the same sample was developed. TRAP 5a and 5b protein levels were evaluated as osteoclast differentiation/activity markers in serum and in culture, and their correlation to the resorption marker CTX-I was examined. Serum TRAP 5a and 5b concentrations in healthy men were 4.4 ± 0.6 ng/ml and 1.3 ± 0.2 ng/ml, respectively, and they correlated moderately to each other suggesting that their secretion is coupled under healthy conditions. A correlation was also observed between serum TRAP 5a and 5b with CTX-I, suggesting that both TRAP isoforms associate with osteoclast number. During osteoclast differentiation on plastic/bone, predominantly 5b increased in media/lysate from M-CSF/RANKL-stimulated CD14+ PBMCs. However, substantial levels of 5a were detected at later stages suggesting that both isoforms are secreted from differentiating OCs. More TRAP 5b was released on bone indicating a connection to osteoclast resorptive activity, and a peak in TRAP 5b/5a-ratio coincided with rapid CTX-I release. At the end of the culture period of M-CSF + RANKL-stimulated CD14+ PBMCs, there was a correlation between the secretion of TRAP 5a and 5b proteins with CTX-I. The correlation of not only 5b but also 5a with collagen degradation, both in serum and osteoclast cultures indicates that a considerable proportion of the TRAP 5a originates from osteoclasts and may reflect a hitherto undisclosed regulatory mechanism during bone resorption and bone remodeling.
Assuntos
Colágeno Tipo I/metabolismo , Osteoclastos/metabolismo , Peptídeos/metabolismo , Fosfatase Ácida Resistente a Tartarato/metabolismo , Adulto , Idoso , Biomarcadores/análise , Biomarcadores/metabolismo , Células Cultivadas , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Isoenzimas/análise , Isoenzimas/metabolismo , Masculino , Pessoa de Meia-Idade , Proteólise , Via Secretória , Fosfatase Ácida Resistente a Tartarato/análiseRESUMO
The murine macrophage cell line RAW264.7 is extensively used as a progenitor to study osteoclast (OC) differentiation. RAW264.7 is a heterogeneous cell line, containing sub-clones with different abilities to form OCs. The aim of this study was to identify characteristics within the heterogeneous RAW264.7 cells that define sub-clones with an augmented ability to form bone-resorbing OCs (H9), as well as sub-clones representing non-OCs (J8). RAW264.7 sub-clones were isolated by single cell cloning. Selection was based on TRAP/cathepsin K expression in sub-clone cultures without added RANKL. Sub-clones before and after differentiation with RANKL were assayed for multiple OC-characteristics. Sub-clone H9 cells presented a higher expression of OC-markers in cultures without added RANKL compared to the parental RAW264.7. After 6 days of RANKL stimulation, sub-clone H9 cells had equal expression levels of OC-markers with RAW264.7 and formed OCs able to demineralize hydroxyapatite. However, sub-clone H9 cells displayed rapid differentiation of OC already at Day 2 compared to Day 4 from parental RAW264.7, and when cultured on plastic and on bone they were more efficient in resorption. This rapid differentiation was likely due to high initial expression/nuclear translocation of OC master transcription factor, NFATc1. In contrast to H9, J8 cells expressed initially very low levels of OC-markers, and they did not respond to RANKL-stimulation by developing OC-characteristics/OC-marker expression. Hence, H9 is an additional clone suitable for experimental setup requiring rapid differentiation of large numbers of OCs.
Assuntos
Reabsorção Óssea/genética , Fatores de Transcrição NFATC/genética , Osteoclastos/metabolismo , Animais , Catepsina K/genética , Catepsina K/metabolismo , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Camundongos , Fatores de Transcrição NFATC/metabolismo , Especificidade de Órgãos , Ligante RANK/genética , Ligante RANK/metabolismo , Células RAW 264.7 , Fosfatase Ácida Resistente a Tartarato/genética , Fosfatase Ácida Resistente a Tartarato/metabolismoRESUMO
Inflammatory mediator prostaglandin E2 (PGE2 ) contributes to bone resorption in several inflammatory conditions including periodontitis. The terminal enzyme, microsomal prostaglandin E synthase-1 (mPGES-1) regulating PGE2 synthesis is a promising therapeutic target to reduce inflammatory bone loss. The aim of this study was to investigate effects of mPGES-1 inhibitors, aminothiazoles TH-848 and TH-644, on PGE2 production and osteoclastogenesis in co-cultures of periodontal ligament (PDL) and osteoclast progenitor cells RAW 264.7, stimulated by lipopolysaccharide (LPS), and bone resorption in RANKL-mediated peripheral blood mononuclear cells (PBMCs). PDL and RAW 264.7 cells were cultured separately or co-cultured and treated with LPS alone or in combination with aminothiazoles. Multinucleated cells stained positively for tartrate-resistant acid phosphatase (TRAP) were scored as osteoclast-like cells. Levels of PGE2 , osteoprotegerin (OPG) and interleukin-6, as well as mRNA expression of mPGES-1, OPG and RANKL were analysed in PDL cells. PBMCs were treated with RANKL alone or in combination with aminothiazoles. TRAP-positive multinucleated cells were analysed and bone resorption was measured by the CTX-I assay. Aminothiazoles reduced LPS-stimulated osteoclast-like cell formation both in co-cultures and in RAW 264.7 cells. Additionally, aminothiazoles inhibited PGE2 production in LPS-stimulated cultures, but did not affect LPS-induced mPGES-1, OPG or RANKL mRNA expression in PDL cells. In PBMCs, inhibitors decreased both osteoclast differentiation and bone resorption. In conclusion, aminothiazoles reduced the formation of osteoclast-like cells and decreased the production of PGE2 in co-cultures as well as single-cell cultures. Furthermore, these compounds inhibited RANKL-induced bone resorption and differentiation of PBMCs, suggesting these inhibitors for future treatment of inflammatory bone loss such as periodontitis.
Assuntos
Reabsorção Óssea/tratamento farmacológico , Dinoprostona/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ligamento Periodontal/efeitos dos fármacos , Ligante RANK/metabolismo , Tiazóis/farmacologia , Animais , Reabsorção Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Técnicas de Cocultura/métodos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Interleucina-6/metabolismo , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteoprotegerina/metabolismo , Ligamento Periodontal/metabolismo , Prostaglandina-E Sintases/metabolismo , Células RAW 264.7 , Fosfatase Ácida Resistente a Tartarato/metabolismoRESUMO
Development of fluorescent and electron dense markers is essential for the implementation of correlative light and electron microscopy, as dual-contrast landmarks are required to match the details in the multimodal images. Here, a novel method for correlative microscopy that utilizes fluorescent nanodiamonds (FNDs) as dual-contrast probes is reported. It is demonstrated how the FNDs can be used as dual-contrast labels-and together with automatic image registration tool SuperTomo, for precise image correlation-in high-resolution stimulated emission depletion (STED)/confocal and transmission electron microscopy (TEM) correlative microscopy experiments. It is shown how FNDs can be employed in experiments with both live and fixed cells as well as simple test samples. The fluorescence imaging can be performed either before TEM imaging or after, as the robust FNDs survive the TEM sample preparation and can be imaged with STED and other fluorescence microscopes directly on the TEM grids.
RESUMO
Measurement of changes of pH at various intracellular compartments has potential to solve questions concerning the processing of endocytosed material, regulation of the acidification process, and also acidification of vesicles destined for exocytosis. To monitor these events, the nanosized optical pH probes need to provide ratiometric signals in the optically transparent biological window, target to all relevant intracellular compartments, and to facilitate imaging at subcellular resolution without interference from the biological matrix. To meet these criteria we sensitize the surface conjugated pH sensitive indicator via an upconversion process utilizing an energy transfer from the nanoparticle to the indicator. Live cells were imaged with a scanning confocal microscope equipped with a low-energy 980 nm laser excitation, which facilitated high resolution and penetration depth into the specimen, and low phototoxicity needed for long-term imaging. Our upconversion nanoparticle resonance energy transfer based sensor with polyethylenimine-coating provides high colloidal stability, enhanced cellular uptake, and distribution across cellular compartments. This distribution was modulated with membrane integrity perturbing treatment that resulted into total loss of lysosomal compartments and a dramatic pH shift of endosomal compartments. These nanoprobes are well suited for detection of pH changes in in vitro models with high biological background fluorescence and in in vivo applications, e.g., for the bioimaging of small animal models.
Assuntos
Microscopia Confocal , Nanopartículas/química , Polietilenoimina/química , Linhagem Celular Tumoral , Fluoretos/química , Humanos , Concentração de Íons de Hidrogênio , Nanopartículas/metabolismo , Fótons , Espectrofotometria , Ítrio/químicaRESUMO
Osteoclasts are multinucleated bone-resorbing cells with a dynamic actin cytoskeleton. Osteoclasts are derived from circulating mononuclear precursors. Confocal and stimulated emission depletion (STED) super-resolution microscopy was used to investigate peripheral blood-derived human osteoclasts cultured on glass surfaces. STED and confocal microscopy demonstrated that the actin was curved and branched, for instance, in the vicinity of membrane ruffles. The overall architecture of the curved actin network extended from the podosomes to the top of the cell. The other novel finding was that a micrometer-level tube containing actin bridged the osteoclasts well above the level of the culture glass. The actin filaments of the tubes originated from the network of curved actin often surrounding a group of nuclei. Furthermore, nuclei were occasionally located inside the tubes. Our findings demonstrated the accumulation of c-Src, cortactin, cofilin, and actin around nuclei suggesting their role in nuclear processes such as the locomotion of nuclei. ARP2/3 labeling was abundant at the substratum level of osteoclasts and in the branched actin network, where it localized to the branching points. We speculate that the actin-containing tubes of osteoclasts may provide a means of transportation of nuclei, e.g., during the fusion of osteoclasts. These novel findings can pave the way for future studies aiming at the elucidation of the differentiation of multinucleated osteoclasts.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Forma Celular , Microtúbulos/metabolismo , Osteoclastos/metabolismo , Células Cultivadas , Humanos , Osteoclastos/citologiaRESUMO
Thyrotropin or thyroid-stimulating hormone (TSH) is used as a marker for thyroid function. More precise and more sensitive immunoassays are needed to facilitate continuous monitoring of thyroid dysfunctions and to assess the efficacy of the selected therapy and dosage of medication. Moreover, most thyroid diseases are autoimmune diseases making TSH assays very prone to immunoassay interferences due to autoantibodies in the sample matrix. We have developed a super-sensitive TSH immunoassay utilizing nanoparticle labels with a detection limit of 60 nU L-1 in preprocessed serum samples by reducing nonspecific binding. The developed preprocessing step by affinity purification removed interfering compounds and improved the recovery of spiked TSH from serum. The sensitivity enhancement was achieved by stabilization of the protein corona of the nanoparticle bioconjugates and a spot-coated configuration of the active solid-phase that reduced sedimentation of the nanoparticle bioconjugates and their contact time with antibody-coated solid phase, thus making use of the higher association rate of specific binding due to high avidity nanoparticle bioconjugates. Graphical Abstract We were able to decrease the lowest limit of detection and increase sensitivity of TSH immunoassay using Eu(III)-nanoparticles. The improvement was achieved by decreasing binding time of nanoparticle bioconjugates by small capture area and fast circular rotation. Also, we applied a step to stabilize protein corona of the nanoparticles and a serum-preprocessing step with a structurally related antibody.
Assuntos
Európio/química , Fluorimunoensaio/métodos , Nanopartículas Metálicas/química , Tireotropina/sangue , Biomarcadores , Humanos , Coroa de Proteína , Sensibilidade e Especificidade , Coloração e Rotulagem , Fatores de TempoRESUMO
Many endocytic proteins accumulate in the reserve pool of synaptic vesicles (SVs) in synapses and relocalize to the endocytic periactive zone during neurotransmitter release. Currently little is known about their functions outside the periactive zone. Here we show that in the Drosophila neuromuscular junction (NMJ), the endocytic scaffolding protein Dap160 colocalizes during the SV cycle and forms a functional complex with the SV-associated phosphoprotein synapsin, previously implicated in SV clustering. This direct interaction is strongly enhanced under phosphorylation-promoting conditions and is essential for proper localization of synapsin at NMJs. In a dap160 rescue mutant lacking the interaction between Dap160 and synapsin, perturbed reclustering of SVs during synaptic activity is observed. Our data indicate that in addition to the function in endocytosis, Dap160 is a component of a network of protein-protein interactions that serves for clustering of SVs in conjunction with synapsin. During the SV cycle, Dap160 interacts with synapsin dispersed from SVs and helps direct synapsin back to vesicles. The proteins function in synergy to achieve efficient clustering of SVs in the reserve pool. SIGNIFICANCE STATEMENT: We provide the first evidence for the function of the SH3 domain interaction in synaptic vesicle (SV) organization at the synaptic active zone. Using Drosophila neuromuscular junction as a model synapse, we describe the molecular mechanism that enables the protein implicated in SV clustering, synapsin, to return to the pool of vesicles during neurotransmitter release. We also identify the endocytic scaffolding complex that includes Dap160 as a regulator of the events linking exocytosis and endocytosis in synapses.
Assuntos
Proteínas de Drosophila/fisiologia , Endocitose/fisiologia , Junção Neuromuscular/metabolismo , Sinapsinas/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Análise por Conglomerados , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Exocitose/fisiologia , Feminino , Masculino , Junção Neuromuscular/ultraestrutura , Vesículas Sinápticas/ultraestruturaRESUMO
Nanomedicine is gaining ground worldwide in therapy and diagnostics. Novel nanoscopic imaging probes serve as imaging tools for studying dynamic biological processes in vitro and in vivo. To allow detectability in the physiological environment, the nanostructure-based probes need to be either inherently detectable by biomedical imaging techniques, or serve as carriers for existing imaging agents. In this study, the potential of mesoporous silica nanoparticles carrying commercially available fluorochromes as self-regenerating cell labels for long-term cellular tracking is investigated. The particle surface is organically modified for enhanced cellular uptake, the fluorescence intensity of labeled cells is followed over time both in vitro and in vivo. The particles are not exocytosed and particles which escaped cells due to cell injury or death are degraded and no labeling of nontargeted cell populations are observed. The labeling efficiency is significantly improved as compared to that of quantum dots of similar emission wavelength. Labeled human breast cancer cells are xenotransplanted in nude mice, and the fluorescent cells can be detected in vivo for a period of 1 month. Moreover, ex vivo analysis reveals fluorescently labeled metastatic colonies in lymph node and rib, highlighting the capability of the developed probes for tracking of metastasis.
Assuntos
Rastreamento de Células/métodos , Corantes Fluorescentes/química , Fenômenos Ópticos , Dióxido de Silício/química , Animais , Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Diagnóstico por Imagem , Exocitose , Feminino , Citometria de Fluxo , Fluorescência , Humanos , Camundongos Nus , Nanopartículas/ultraestrutura , Porosidade , Pontos Quânticos/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Biocatalytic pulp fibers were prepared using surface functionalization of bleached kraft pulp with amino groups (F) and further immobilization of a cross-linked glucose oxidase (G*) from Aspergillus niger. The cross-linked enzymes (G*) were characterized using X-ray spectroscopy, Fourier transform infrared spectroscopy, dynamic scanning calorimetry, and dynamic light scattering. According to standard assays, the G* content on the resulting fibers (FG*) was of 11 mg/g of fiber, and enzyme activity was of 215 U/g. The results from confocal- and stimulated emission depletion microscopy techniques demonstrated that glucose oxidase do not penetrate the interlayers of fibers. The benefit of pulp fiber functionalization was evident in the present case, as the introduction of amino groups allowed the immobilization of larger amount of enzymes and rendered more efficient systems. Using the approach described on this paper, several advanced materials from wood pulp fibers and new bioprocesses might be developed by selecting the correct enzyme for the target applications.
Assuntos
Celulose/química , Enzimas Imobilizadas/química , Glucose Oxidase/química , Madeira/química , Aspergillus niger/química , Aspergillus niger/enzimologia , Calorimetria , Difusão Dinâmica da Luz , Enzimas Imobilizadas/ultraestrutura , Glucose Oxidase/ultraestrutura , Espectroscopia de Infravermelho com Transformada de Fourier , Madeira/ultraestruturaRESUMO
The single muscle fiber in vitro motility assay (SF-IVMA) is characterized by organized linear motility of actin filaments, i.e., actin filaments motility showing a parallel or anti-parallel direction with similar speed independent of direction in the central part of the flow-cell where density of myosin is high. In contrast, the low myosin density region in the flow-cell exhibits random filament movements, but the mechanisms underlying the organized motility remain unknown. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) imaging techniques have been combined to investigate the morphological features of myosin extracted from single muscle fiber segments in the flow cell. Nanometric scale imaging of myosin filaments in the SF-IVMA showed intact spatial distances between myosin heads being essential for myosin filament function. However, angular spectrum analyses of myosin filaments in the high myosin density region showed organized myosin filament orientation only in small areas, while unorganized filament orientation were dominantly presented when larger areas were analyzed. Thus, parallel myosin filament organization is a less likely mechanism underlying the organized motility of actin filaments and the high myosin density per se is therefore forwarded as the primary "driver" that promotes organized linear motility.
Assuntos
Fibras Musculares Esqueléticas/fisiologia , Miosinas/fisiologia , Animais , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Fibras Musculares Esqueléticas/ultraestrutura , Nanotecnologia , Ratos , Ratos Sprague-DawleyRESUMO
Nanoparticle labels have enhanced the performance of diagnostic, screening, and other measurement applications and hold further promise for more sensitive, precise, and cost-effective assay technologies. Nevertheless, a clear view of the biomolecular interactions on the molecular level is missing. Controlling the ratio of molecular recognition over undesired nonspecific adhesion is the key to improve biosensing with nanoparticles. To improve this ratio with an aim to disallow nonspecific binding, a more detailed perspective into the kinetic differences between the cases is needed. We present the application of two novel methods to determine complex binding kinetics of bioconjugate nanoparticles, interferometry, and force spectroscopy. Force spectroscopy is an atomic force microscopy technique and optical interferometry is a direct method to monitor reaction kinetics in second-hour timescale, both having steadily increasing importance in nanomedicine. The combination is perfectly suited for this purpose, due to the high sensitivity to detect binding events and the ability to investigate biological samples under physiological conditions. We have attached a single biofunctionalized nanoparticle to the outer tip apex and studied the binding behavior of the nanoparticle in a sandwich-type immunoassay using dynamic force spectroscopy in millisecond timescale. Utilization of the two novel methods allowed characterization of binding kinetics in a time range spanning from 50 ms to 4 h. These experiments allowed detection and demonstration of differences between specific and nonspecific binding. Most importantly, nonspecific binding of a nanoparticle was reduced at contact times below 100 ms with the solid-phase surface.
Assuntos
Imunoensaio , Nanopartículas Metálicas/química , Tireotropina/análise , Animais , Anticorpos Monoclonais/química , Bovinos , Európio/química , Humanos , Interferometria , Cinética , Luz , Nanopartículas Metálicas/ultraestrutura , Microscopia de Força Atômica , Poliestirenos/química , Soroalbumina Bovina/química , Fatores de TempoRESUMO
Sex-specific differences in bone integrity and properties are associated with age as well as the number and activity of cells involved in bone remodeling. The aim of this study was to investigate sex-specific differences in adhesion, proliferation, and differentiation of mouse bone marrow derived cells into osteoclasts. The adherent fraction of bone marrow- derived cells from 12-week-old male and female C57BL/6J mice were assessed for their adhesion, proliferation, and receptor activator of nuclear factor κB (RANKL)-induced differentiation into osteoclasts. Female bone marrow derived macrophages (BMDMs) displayed higher adhesion and proliferation ratio upon macrophage colony stimulating factor (M-CSF) (day 0) and M-CSF + RANKL (day 4) treatment, respectively. On the contrary, male BMDMs differentiated more efficiently into osteoclasts upon RANKL-treatment compared to females (day 5). To further understand these sex-specific differences at the gene expression level, BMDMs treated with M-CSF (day 0) and M-CSF + RANKL (day 4), were assessed for their differential expression of genes through RNA sequencing. M-CSF treatment resulted in 1106 differentially expressed genes, while RANKL-treatment gave 473 differentially expressed genes. Integrin, adhesion, and proliferation-associated genes were elevated in the M-CSF-treated female BMDMs. RANKL-treatment further enhanced the expression of the proliferation- associated genes, and of genes associated with inhibition of osteoclast differentiation in the females, while RANK-signaling-associated genes were upregulated in males. In conclusion, BMDM adhesion, proliferation and differentiation into osteoclasts are sex-specific and may be directed by the PI3K-Akt signaling pathway for proliferation, and the colony stimulating factor 1-receptor and the RANKLsignaling pathway for the differentiation.
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Microgel assembly as void-forming bioinks in 3D bioprinting has evidenced recent success with a highlighted scaffolding performance of these bottom-up biomaterial systems in supporting the viability and function of the laden cells. Here, a ternary-component aqueous emulsion is established as a one-step strategy to integrate the methacrylated gelatin (GelMA) microgel fabrication and assembly through vat photopolymerization in situ using digital light processing (DLP)-based bioprinting. The as-proposed aqueous emulsion is featured with the partitioning of a secondary photo-crosslinkable polysaccharide, methacrylated galactoglucomannan (GGMMA) derived from plant source in both the dispersed phase of GelMA droplets and the continuous phase of dextran (Dex). As an emulgator, GGMMA renders enhanced stability of the aqueous emulsion bioresins. Strategically, the photo-crosslinkable GGMMA adheres the GelMA microgels that are conveniently converted from emulsion droplets to form hydrogel construct in layer-by-layer curing to accommodate the laden cells directly mixed in the aqueous emulsion. The spatially interconnected void space left by the removal of Dex benefits the cell growth under the guidance of the microgel surface and supports cell colonization within the macroscopic porous hydrogel. This work amends a low-concentration and cost-effective bioresin that is highly applicable for facilely fabricating microgel assembly as a porous hydrogel construct in DLP-based bioprinting.
Assuntos
Bioimpressão , Microgéis , Engenharia Tecidual , Emulsões , Materiais Biocompatíveis , Hidrogéis , Gelatina , Alicerces Teciduais , Impressão TridimensionalRESUMO
Spondyloepimetaphyseal dysplasias (SEMDs) are a heterogeneous group of disorders with variable growth failure and skeletal impairments affecting the spine and long bone epiphyses and metaphyses. Here we report on four unrelated families with SEMD in which we identified two monoallelic missense variants and one monoallelic splice site variant in RPL13, encoding the ribosomal protein eL13. In two out of four families, we observed autosomal dominant inheritance with incomplete penetrance and variable clinical expressivity; the phenotypes of the mutation-positive subjects ranged from normal height with or without hip dysplasia to severe SEMD with severe short stature and marked skeletal dysplasia. In vitro studies on patient-derived dermal fibroblasts harboring RPL13 missense mutations demonstrated normal eL13 expression, with proper subcellular localization but reduced colocalization with eL28 (p < 0.001). Cellular functional defects in fibroblasts from mutation-positive subjects indicated a significant increase in the ratio of 60S subunits to 80S ribosomes (p = 0.007) and attenuated global translation (p = 0.017). In line with the human phenotype, our rpl13 mutant zebrafish model, generated by CRISPR-Cas9 editing, showed cartilage deformities at embryonic and juvenile stages. These findings extend the genetic spectrum of RPL13 mutations causing this novel human ribosomopathy with variable skeletal features. Our study underscores for the first time incomplete penetrance and broad phenotypic variability in SEMD-RPL13 type and confirms impaired ribosomal function. Furthermore, the newly generated rpl13 mutant zebrafish model corroborates the role of eL13 in skeletogenesis. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)..
Assuntos
Osteocondrodisplasias , Peixe-Zebra , Animais , Variação Biológica da População , Humanos , Proteínas de Neoplasias , Osteocondrodisplasias/diagnóstico por imagem , Osteocondrodisplasias/genética , Linhagem , Proteínas Ribossômicas/genética , Coluna Vertebral , Peixe-Zebra/genéticaRESUMO
BACKGROUND: Tartrate-resistant acid phosphatase (TRAP/ ACP5) belongs to the binuclear metallophosphatase family and is present in two isoforms. The primary translation product is an uncleaved TRAP 5a isoform with low phosphatase activity. TRAP 5a can be post-translationally processed to a cleaved TRAP 5b isoform with high phosphatase activity by e.g. cysteine proteinases, such as Cathepsin K (CtsK). The relevance of the phosphatase activity of TRAP 5b has been demonstrated for proliferation, migration and invasion of cancer cells. TRAP-overexpressing MDA-MB-231 breast cancer cells displayed higher levels of TRAP 5a and efficient processing of TRAP 5a to TRAP 5b protein, but no changes in levels of CtsK when compared to mock-transfected cells. In TRAP-overexpressing cells colocalization of TRAP 5a and proCtsK was augmented, providing a plausible mechanism for generation of TRAP 5b. CtsK expression has been associated with cancer progression and has been pharmacologically targeted in several clinical studies. RESULTS: In the current study, CtsK inhibition with MK-0822/Odanacatib did not abrogate the formation of TRAP 5b, but reversibly increased the intracellular levels of a N-terminal fragment of TRAP 5b and reduced secretion of TRAP 5a reversibly. However, MK-0822 treatment neither altered intracellular TRAP activity nor TRAP-dependent cell migration, suggesting involvement of additional proteases in proteolytic processing of TRAP 5a. Notwithstanding, CtsK was shown to be colocalized with TRAP and to be involved in the regulation of secretion of TRAP 5a in a breast cancer cell line, while it still was not essential for processing of TRAP 5a to TRAP 5b isoform. CONCLUSION: In cancer cells multiple proteases are involved in cleaving TRAP 5a to high-activity phosphatase TRAP 5b. However, CtsK-inhibiting treatment was able to reduce secretion TRAP 5a from TRAP-overexpressing cancer cells.
Assuntos
Neoplasias da Mama/metabolismo , Catepsina K , Fosfatase Ácida Resistente a Tartarato , Catepsina K/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Fosfatase Ácida Resistente a Tartarato/metabolismoRESUMO
Intracellular drug delivery by mesoporous silica nanoparticles (MSNs) carrying hydrophilic and hydrophobic fluorophores as model drug cargo is demonstrated on 2D cellular and 3D tumor organoid level. Two different MSN designs, chosen on the basis of the characteristics of the loaded cargo, were used: MSNs with a surface-grown poly(ethylene imine), PEI, coating only for hydrophobic cargo and MSNs with lipid bilayers covalently coupled to the PEI layer as a diffusion barrier for hydrophilic cargo. First, the effect of hydrophobicity corresponding to loading degree (hydrophobic cargo) as well as surface charge (hydrophilic cargo) on intracellular drug release was studied on the cellular level. All incorporated agents were able to release to varying degrees from the endosomes into the cytoplasm in a loading degree (hydrophobic) or surface charge (hydrophilic) dependent manner as detected by live cell imaging. When administered to organotypic 3D tumor models, the hydrophilic versus hydrophobic cargo-carrying MSNs showed remarkable differences in labeling efficiency, which in this case also corresponds to drug delivery efficacy in 3D. The obtained results could thus indicate design aspects to be taken into account for the development of efficacious intracellular drug delivery systems, especially in the translation from standard 2D culture to more biologically relevant organotypic 3D cultures.
RESUMO
Current diagnostic immunotechnologies are universally based on the measurement of the bound label-antibody fraction in direct binding or sandwich-assay type approaches with various detection techniques (e.g. enzyme-linked immunosorbent assay or ELISA) on solid stationary phase surface. Here an alternative reciprocal approach is presented based on the detection of the non-bound fraction of nanoparticle-labelled antibodies using microparticles as solid support. The advantage of detecting the non-bound fraction of the labelled antibody instead of the bound fraction is the high dynamics and the suggested increased flexibility in the selection of the detection mode. No actual washing steps are required as the bound and non-bound fractions of the detection nanoparticle label are separated using physical separation rather than consecutive washing repeats. The quantitative proof-of-concept set-up was demonstrated through blood-based detection of C-reactive protein (CRP). A blood sample containing CRP was diluted 1/50 and measured in 15-min resulting in a linear response at a range from 1 to 30µg/ml. The lowest limit of detection was below 0.03µg/ml and the assay coefficient of variation ranged from 0.3 to 9%. The nanoparticle-based residual label detection outperformed the corresponding molecular label method providing wider applicability with nearly an order of magnitude higher signal-to-background ratio for novel assay configurations in clinical diagnostics practices.
Assuntos
Anticorpos Monoclonais/química , Técnicas Biossensoriais/métodos , Proteína C-Reativa/análise , Európio/química , Imunoensaio/métodos , Nanopartículas/química , Humanos , Imunoconjugados/química , Limite de Detecção , Medições Luminescentes/métodosRESUMO
To elucidate processes in the osteoclastic bone resorption, visualise resorption and related actin reorganisation, a combination of imaging technologies and an applicable in vitro model is needed. Nanosized bone powder from matching species is deposited on any biocompatible surface in order to form a thin, translucent, smooth and elastic representation of injured bone. Osteoclasts cultured on the layer expressed matching morphology to ones cultured on sawed cortical bone slices. Resorption pits were easily identified by reflectance microscopy. The coating allowed actin structures on the bone interface to be visualised with super-resolution microscopy along with a detailed interlinked actin networks and actin branching in conjunction with V-ATPase, dynamin and Arp2/3 at actin patches. Furthermore, we measured the timescale of an adaptive osteoclast adhesion to bone by force spectroscopy experiments on live osteoclasts with bone-coated AFM cantilevers. Utilising the in vitro model and the advanced imaging technologies we localised immunofluorescence signals in respect to bone with high precision and detected resorption at its early stages. Put together, our data supports a cyclic model for resorption in human osteoclasts.
Assuntos
Reabsorção Óssea/metabolismo , Modelos Biológicos , Osteoclastos/metabolismo , Reabsorção Óssea/patologia , Feminino , Humanos , Masculino , Microscopia de Força Atômica , Microscopia de Interferência , Osteoclastos/ultraestruturaRESUMO
Small interfering RNA (siRNA) is a highly potent drug in gene-based therapy with the challenge being to deliver it in a sustained manner. The combination of mesoporous silica nanoparticles (MSNs) and polycations in the confined pore space allows for incorporation and controlled release of therapeutic siRNA payloads. We hereby constructed MSNs with expanded mesopores and pore-surface-hyperbranched poly(ethyleneimine) (PEI) tethered with redox-cleavable linkers that could carry a high payload of siRNA (120 mg·g-1). The developed nanocarriers were efficiently taken up by cancer cells and were subsequently able to escape to the cytoplasm from the endosomes, most likely owing to the integrated PEI. Triggered by the intracellular redox conditions, the siRNA was sustainably released inside the cells over a period of several days. Functionality of siRNAs was demonstrated by using cell-killing siRNA as cargo. Despite not being the aim of the developed system, in vitro experiments using cell-killing siRNAs showed that the efficacy of siRNA transfection was comparable to the commercial in vitro transfection agent Lipofectamine. Consequently, the developed MSN-based delivery system offers a potential approach to hybrid nanocarriers for more efficient and long-term siRNA delivery and, in a longer perspective, in vivo gene silencing for RNA interference (RNAi) therapy.