RESUMO
RNA-binding protein (RBP) dysfunction is a fundamental hallmark of amyotrophic lateral sclerosis (ALS) and related neuromuscular disorders. Abnormal neuronal excitability is also a conserved feature in ALS patients and disease models, yet little is known about how activity-dependent processes regulate RBP levels and functions. Mutations in the gene encoding the RBP Matrin 3 (MATR3) cause familial disease, and MATR3 pathology has also been observed in sporadic ALS, suggesting a key role for MATR3 in disease pathogenesis. Here, we show that glutamatergic activity drives MATR3 degradation through an NMDA receptor-, Ca2+-, and calpain-dependent mechanism. The most common pathogenic MATR3 mutation renders it resistant to calpain degradation, suggesting a link between activity-dependent MATR3 regulation and disease. We also demonstrate that Ca2+ regulates MATR3 through a nondegradative process involving the binding of Ca2+/calmodulin to MATR3 and inhibition of its RNA-binding ability. These findings indicate that neuronal activity impacts both the abundance and function of MATR3, underscoring the effect of activity on RBPs and providing a foundation for further study of Ca2+-coupled regulation of RBPs implicated in ALS and related neurological diseases.
Assuntos
Esclerose Lateral Amiotrófica , Humanos , Esclerose Lateral Amiotrófica/metabolismo , Cálcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Calpaína/genética , Calpaína/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismoRESUMO
Drugs that can treat one disease may either be detrimental or beneficial toward another due to possible cross-interactions. Therefore, care in choosing a suitable drug for patients with multiple diseases is crucial in successful patient management. This study explores several currently available ophthalmic drugs used to treat common ocular diseases to understand how they can affect the amyloidogenesis of a transforming growth factor ß-induced protein (TGFBIp) peptide fragment found in abundance in the corneal protein aggregation deposits of lattice corneal dystrophy (LCD) patients. Results from this study provided supporting evidence that some drugs intended to treat other diseases can enhance or inhibit fibrillar aggregation of TGFBIp peptide, which may have potential implication of affecting the disease progression of LCD by either worsening or ameliorating it. Comparisons of the different properties of ophthalmic compounds explored in this study may also provide some guidance for future design of drugs geared toward the treatment of LCD.
Assuntos
Distrofias Hereditárias da Córnea , Proteínas da Matriz Extracelular , Fator de Crescimento Transformador beta , Humanos , Proteínas da Matriz Extracelular/metabolismo , Distrofias Hereditárias da Córnea/metabolismo , Distrofias Hereditárias da Córnea/tratamento farmacológico , Fator de Crescimento Transformador beta/metabolismo , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/metabolismo , Soluções Oftálmicas , Amiloide/metabolismoRESUMO
Mutations in UBQLN2 cause amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neurodegenerations. However, the mechanism by which the UBQLN2 mutations cause disease remains unclear. Alterations in proteins involved in autophagy are prominent in neuronal tissue of human ALS UBQLN2 patients and in a transgenic P497S UBQLN2 mouse model of ALS/FTD, suggesting a pathogenic link. Here, we show UBQLN2 functions in autophagy and that ALS/FTD mutant proteins compromise this function. Inactivation of UBQLN2 expression in HeLa cells reduced autophagic flux and autophagosome acidification. The defect in acidification was rescued by reexpression of wild type (WT) UBQLN2 but not by any of the five different UBQLN2 ALS/FTD mutants tested. Proteomic analysis and immunoblot studies revealed P497S mutant mice and UBQLN2 knockout HeLa and NSC34 cells have reduced expression of ATP6v1g1, a critical subunit of the vacuolar ATPase (V-ATPase) pump. Knockout of UBQLN2 expression in HeLa cells decreased turnover of ATP6v1g1, while overexpression of WT UBQLN2 increased biogenesis of ATP6v1g1 compared with P497S mutant UBQLN2 protein. In vitro interaction studies showed that ATP6v1g1 binds more strongly to WT UBQLN2 than to ALS/FTD mutant UBQLN2 proteins. Intriguingly, overexpression of ATP6v1g1 in UBQLN2 knockout HeLa cells increased autophagosome acidification, suggesting a therapeutic approach to overcome the acidification defect. Taken together, our findings suggest that UBQLN2 mutations drive pathogenesis through a dominant-negative loss-of-function mechanism in autophagy and that UBQLN2 functions as an important regulator of the expression and stability of ATP6v1g1. These findings may have important implications for devising therapies to treat UBQLN2-linked ALS/FTD.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Esclerose Lateral Amiotrófica/genética , Autofagossomos/fisiologia , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia/genética , Demência/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Proteínas Relacionadas à Autofagia/genética , Biomarcadores/metabolismo , Linhagem Celular , Demência/metabolismo , Demência/patologia , Predisposição Genética para Doença , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Lisossomal/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Ligação Proteica , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Regulação para Cima , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Mobilization of hematopoietic stem and progenitor cells (HSPCs) has become increasingly important for hematopoietic cell transplantation. Current mobilization approaches are insufficient because they fail to mobilize sufficient numbers of cells in a significant fraction of patients and are biased toward myeloid immune reconstitution. A novel, single drug mobilization agent that allows a more balanced (myeloid and lymphoid) reconstitution would therefore be highly favorable to improve transplantation outcome. In this present study, we tested commercially available IL-33 molecules and engineered novel variants of IL-33. These molecules were tested in cell-based assays in vitro and in mobilization models in vivo. We observed for the first time that IL-33 treatment in mice mobilized HSPCs and common myeloid progenitors more efficiently than clinical mobilizing agents granulocyte colony-stimulating factor (G-CSF) or AMD3100. We engineered several oxidation-resistant IL-33 variants with equal or better in vitro activity. In vivo, these variants mobilized HSPCs and, interestingly, also hematopoietic stem cells, common lymphoid progenitor cells, and endothelial progenitor cells more efficiently than wild-type IL-33 or G-CSF. We then engineered an IL-33-Fc fusion molecule, a single dose of which was sufficient to significantly increase the mobilization of HSPCs after 4 days. In conclusion, our findings suggest that long-acting, oxidation-resistant IL-33 may be a novel approach for HSPC transplantation. IL-33-mobilized HSPCs differ from cells mobilized with G-CSF and AMD3100, and it is possible that these differences may result in better transplantation outcomes.
Assuntos
Fator Estimulador de Colônias de Granulócitos/farmacologia , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Compostos Heterocíclicos/farmacologia , Interleucina-33/farmacologia , Animais , Benzilaminas , Ciclamos , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , CamundongosRESUMO
Flavonoids are a class of plant secondary metabolites that are found ubiquitously in plants and in the human diet. Our objective is to investigate the antiproliferative effects of flavonoids (baicalein, luteolin, genistein, apigenin, scutellarin, galangin, chrysin, and naringenin) toward leukemia cells (HL-60, NB4, U937, K562, Jurkat) as well as the relationship between their antileukemic potencies and molecular structures. At the proteomic level, we evaluate the effects of different flavonoids on the expression levels of various proteins using Protein Pathway Array (PPA) technology. Our results showed a dose-dependent cytotoxicity of flavonoids toward various types of leukemia cells. The results of PPA illustrated that flavonoids, such as baicalein, genistein, and scutellarin affected different proteins in different leukemia cell lines. Cell cycle regulatory proteins, such as CDK4, CDK6, Cyclin D1, Cyclin B1, p-CDC2, and p-RB were affected in different leukemia cells. Furthermore, we found that baicalein suppresses CDK4 and activates p-ERK in most leukemia cells; genistein mainly affects CDK4, p-ERK, p-CDC2, while scutellarin dysregulated the proteins, cell division control protein 42, Notch4, and XIAP. Collectively, a wide variety of dysregulation of key signaling proteins related to apoptosis and cell-cycle regulation contributes to the antileukemic properties of these flavonoids.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/efeitos dos fármacos , Flavonoides/farmacologia , Leucemia/metabolismo , Transdução de Sinais/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Leucemia/patologia , Proteínas de Neoplasias/efeitos dos fármacos , Proteínas de Neoplasias/metabolismoRESUMO
BACKGROUND: Cervical cancer is the second most common form of cancer among women. There are over 100 different types of human papillomavirus (HPV), 40 of which are frequently detected in anogenital mucosa. HPV is the primary etiological agent of cervical cancer and is present predominantly in cervical cancers. Thirteen commonly recognized high-risk genotypes have oncogenic potential. The most common high-risk HPV (hrHPV) genotypes in cervical cancer are HPV16 and HPV18, which have the greatest malignancies. The objective of this study was to determine the distribution of hrHPV types in patient samples received at the Mount Sinai Medical Center for routine cytology and HPV testing. In addition, the study compared the sensitivity of the Hybrid Capture 2 (HC2) and Roche Linear Array HPV Genotyping assays. METHODS: Cytology specimens with abnormal diagnoses were tested for HPV by the Roche Linear Array HPV Genotyping and Digene Hybrid Capture 2 molecular systems. RESULTS: The Roche Linear Array HPV Genotyping Assay was more sensitive than HC2. Additionally, specimens exhibited higher rates of HPV16, HPV51, and HPV59 infections than HPV16 and HPV18 infections. CONCLUSION: The results demonstrate that geographical distribution of HPV genotypes may play an important role in clinical management of HPV infection, particularly when treating cervical dysplasia and recommending HPV vaccination.
Assuntos
Técnicas de Genotipagem/métodos , Papillomaviridae/genética , Infecções por Papillomavirus/epidemiologia , Displasia do Colo do Útero/virologia , Neoplasias do Colo do Útero/virologia , Adolescente , Adulto , Idoso , Feminino , Genótipo , Humanos , Pessoa de Meia-Idade , Hibridização de Ácido Nucleico/métodos , Infecções por Papillomavirus/complicações , Reação em Cadeia da Polimerase/métodos , Prevalência , Adulto JovemRESUMO
In tissue development and homeostasis, transforming growth factor (TGF)-ß signaling is finely coordinated by latent forms and matrix sequestration. Optogenetics can offer precise and dynamic control of cell signaling. We report the development of an optogenetic human induced pluripotent stem cell system for TGF-ß signaling and demonstrate its utility in directing differentiation into the smooth muscle, tenogenic, and chondrogenic lineages. Light-activated TGF-ß signaling resulted in expression of differentiation markers at levels close to those in soluble factor-treated cultures, with minimal phototoxicity. In a cartilage-bone model, light-patterned TGF-ß gradients allowed the establishment of hyaline-like layer of cartilage tissue at the articular surface while attenuating with depth to enable hypertrophic induction at the osteochondral interface. By selectively activating TGF-ß signaling in co-cultures of light-responsive and non-responsive cells, undifferentiated and differentiated cells were simultaneously maintained in a single culture with shared medium. This platform can enable patient-specific and spatiotemporally precise studies of cellular decision making.
Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Mesenquimais , Humanos , Fator de Crescimento Transformador beta/metabolismo , Optogenética , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Diferenciação Celular , Transdução de Sinais , Condrogênese , Células Cultivadas , CondrócitosRESUMO
Osteoarthritis (OA) is the most common joint disease worldwide, yet we continue to lack an understanding of disease etiology and pathology and effective treatment options. Essential to tissue homeostasis, disease pathogenesis, and therapeutic responses are the stratified organization of cartilage and cross talk at the osteochondral junction. Animal models may capture some of these features, but to establish clinically consistent therapeutics, there remains a need for high-fidelity models of OA that meet all the above requirements in a human patient-specific manner. In vitro bioengineered cartilage-bone tissue models could be developed to recapitulate physiological interactions with human cells and disease-initiating factors. In this study, we highlight human induced pluripotent stem cells (hiPSCs) as the advantageous cell source for these models and review approaches for chondrogenic fate specification from hiPSCs. To achieve native-like stratified cartilage organization with cartilage-bone interactions, spatiotemporal cues mimicking development can be delivered to engineered tissues by patterning of the cells, scaffold, and environment. Once healthy and native-like cartilage-bone tissues are established, an OA-like state can be induced through cytokine challenge or injurious loading. Bioengineered cartilage-bone tissues fall short of recapitulating the full complexity of native tissues, but have demonstrated utility in elucidating some mechanisms of OA progression and enabled screening of candidate therapeutics in patient-specific models. With rapid progress in stem cells, tissue engineering, imaging, and high-throughput omics research in recent years, we propose that advanced human tissue models will soon offer valuable contributions to our understanding and treatment of OA.
Assuntos
Cartilagem Articular , Células-Tronco Pluripotentes Induzidas , Osteoartrite , Animais , Osso e Ossos/patologia , Cartilagem/patologia , Cartilagem Articular/patologia , Condrogênese , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Osteoartrite/terapia , Engenharia Tecidual/métodosRESUMO
BACKGROUND: Alzheimer's disease (AD) is the most common cause of dementia characterized by progressive cognitive impairment in the elderly people. The most dramatic abnormalities are those of the cholinergic system. Acetylcholinesterase (AChE) plays a key role in the regulation of the cholinergic system, and hence, inhibition of AChE has emerged as one of the most promising strategies for the treatment of AD. METHODS: In this study, we suggest a workflow for the identification and prioritization of potential compounds targeted against AChE. In order to elucidate the essential structural features for AChE, three-dimensional pharmacophore models were constructed using Discovery Studio 2.5.5 (DS 2.5.5) program based on a set of known AChE inhibitors. RESULTS: The best five-features pharmacophore model, which includes one hydrogen bond donor and four hydrophobic features, was generated from a training set of 62 compounds that yielded a correlation coefficient of R = 0.851 and a high prediction of fit values for a set of 26 test molecules with a correlation of R² = 0.830. Our pharmacophore model also has a high Güner-Henry score and enrichment factor. Virtual screening performed on the NCI database obtained new inhibitors which have the potential to inhibit AChE and to protect neurons from Aß toxicity. The hit compounds were subsequently subjected to molecular docking and evaluated by consensus scoring function, which resulted in 9 compounds with high pharmacophore fit values and predicted biological activity scores. These compounds showed interactions with important residues at the active site. CONCLUSIONS: The information gained from this study may assist in the discovery of potential AChE inhibitors that are highly selective for its dual binding sites.
Assuntos
Acetilcolinesterase/química , Inibidores da Colinesterase/química , Modelos Biológicos , Modelos Moleculares , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/enzimologia , Precursor de Proteína beta-Amiloide/metabolismo , Inibidores da Colinesterase/farmacocinética , Bases de Dados Factuais , Humanos , Neurônios/enzimologiaRESUMO
BACKGROUND: Malignant mesothelioma is a rare cancer caused by exposure to asbestos. Current therapies have limited efficacy and the prognosis is dismal. A better understanding of the underlying mechanism of asbestos-induced malignant transformation will help to identify molecular markers that can be used for diagnosis, prognosis or therapeutic targets. OBJECTIVES: The objectives of this study are (1) to identify altered levels of proteins and phosphoproteins and (2) to establish the interactive network among those proteins in crocidolite-treated benign mesothelial cells and in malignant mesothelial cells. METHODS: Total cellular proteins were extracted from benign mesothelial cells, crocidolite-treated mesothelial cells and malignant mesothelial cells. The expression levels of 112 proteins and phosphoproteins were analyzed using a multiplex immunoblot-based assay followed by computational analysis (Protein Pathway Array). RESULTS: A total of 16 proteins/phosphoproteins (7 down-regulated and 9 up-regulated) were altered after exposure of benign mesothelial cells to crocidolite asbestos and the majority of them are involved in DNA damage repair and cell cycle regulation. In malignant mesothelial cells, 21 proteins/phosphoproteins (5 down-regulated and 16 up-regulated) were dysregulated and majority of them are involved in EGFR/ERK and PI3K/Akt pathways. Within the regulatory network affected by crocidolite, p53 and NF-κB complex are the most important regulators. There was substantial overlap in the regulatory networks between the asbestos-treated cells and malignant mesothelial cells. CONCLUSIONS: Asbestos exposure has extensive effects on regulatory pathways and networks. These altered proteins may be used in the future to identify those with a high risk for developing malignant mesothelioma and as targets for preventing this deadly malignancy.
Assuntos
Asbesto Crocidolita/farmacologia , Células Epiteliais/metabolismo , Mesotelioma/metabolismo , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Transdução de Sinais/efeitos dos fármacosRESUMO
Accumulating evidence suggests X-linked dominant mutations in UBQLN2 cause amyotrophic lateral sclerosis (ALS) with frontotemporal dementia (FTD) through both loss- and gain-of-function mechanisms. However, the mechanisms by which the mutations cause disease are still unclear. The goal of the study was to uncover the possible pathomechanism(s) by which UBQLN2 mutations cause ALS/FTD. An analysis of proteomic changes in neuronal tissue was used to identify proteins with altered accumulation in the P497S UBQLN2 transgenic mouse model of ALS/FTD. We then used immunocytochemistry and biochemical techniques to confirm protein changes in the mutant P497S mice. Additionally, we used cell lines inactivated of UBQLN2 expression to determine whether its loss underlies the alteration in the proteins seen in P497S mice. The proteome screen identified a dramatic alteration of serine protease inhibitor (serpin) proteins in the mutant P497S animals. Double immunofluorescent staining of brain and spinal cord tissues of the mutant and control mice revealed an age-dependent change in accumulation of Serpin A1, C1, and I1 in puncta whose staining colocalized with UBQLN2 puncta in the mutant P497S mice. Serpin A1 aggregation in P497S animals was confirmed by biochemical extraction and filter retardation assays. A similar phenomenon of serpin protein aggregation was found in HeLa and NSC34 motor neuron cells with inactivated UBQLN2 expression. We found aberrant aggregation of serpin proteins, particularly Serpin A1, in the brain and spinal cord of the P497S UBQLN2 mouse model of ALS/FTD. Similar aggregation of serpin proteins was found in UBQLN2 knockout cells suggesting that serpin aggregation in the mutant P497S animals may stem from loss of UBQLN2 function. Because serpin aggregation is known to cause disease through both loss- and gain-of-function mechanisms, we speculate that their accumulation in the P497S mouse model of ALS/FTD may contribute to disease pathogenesis through similar mechanism(s).
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Proteínas Relacionadas à Autofagia/metabolismo , Demência Frontotemporal/patologia , Serpinas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Proteínas Relacionadas à Autofagia/genética , Modelos Animais de Doenças , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Serpinas/metabolismo , Medula Espinal/patologiaRESUMO
Cataract is known as one of the leading causes of vision impairment worldwide. While the detailed mechanism of cataratogenesis remains unclear, cataract is believed to be correlated with the aggregation and/or misfolding of human ocular lens proteins called crystallins. A 173-residue structural protein human γD-crystallin is a major γ-crystallin protein in the human eye lens and associated with the development of juvenile and mature-onset cataracts. This work is aimed at investigating the effect of a small molecule, e.g., ortho-vanillin, on human γD-crystallin aggregation upon exposure to ultraviolet-C irradiation. According to the findings of right-angle light scattering, transmission electron microscopy, and gel electrophoresis, ortho-vanillin was demonstrated to dose-dependently suppress ultraviolet-C-triggered aggregation of human γD-crystallin. Results from the synchronous fluorescence spectroscopy, tryptophan fluorescence quenching, and molecular docking studies revealed the structural change of γD-crystallin induced by the interaction/binding between ortho-vanillin and protein. We believe the outcome from this work may contribute to the development of potential therapeutics for cataract.
Assuntos
Catarata , Cristalino , gama-Cristalinas , Benzaldeídos , Humanos , Simulação de Acoplamento MolecularRESUMO
Spine stereotactic body radiation therapy frequently involves the delivery of high doses to targets in proximity to the spinal cord; thus, the radiation must be delivered with great spatial accuracy. Monitoring for large shifts in target and cord position that might occur during dose delivery is a challenge for clinics equipped with a conventional C-arm Linac. Treatment must be halted, then imaging and registration must be done to determine whether a significant shift has occurred. In this retrospective study of 1019 spine SBRT treatments, we investigated the number of target shifts >2 mm in any direction that occurred in carefully immobilized patients. Orthogonal kV images were acquired 3 to 5 times during each session using in an in-room imaging system. Although the likelihood of large intrafractional shifts was found to be very low, they did occur in 6 treatment sessions. Intrafractional monitoring was found to be an important safety component of treatment delivery.
Assuntos
Lesões por Radiação/prevenção & controle , Radiocirurgia/métodos , Planejamento da Radioterapia Assistida por Computador/métodos , Radioterapia de Intensidade Modulada/métodos , Neoplasias da Coluna Vertebral/radioterapia , Tomografia Computadorizada de Feixe Cônico , Fracionamento da Dose de Radiação , Humanos , Movimento , Órgãos em Risco/diagnóstico por imagem , Aceleradores de Partículas , Posicionamento do Paciente , Lesões por Radiação/etiologia , Radiocirurgia/instrumentação , Planejamento da Radioterapia Assistida por Computador/instrumentação , Radioterapia de Intensidade Modulada/instrumentação , Estudos Retrospectivos , Medula Espinal/diagnóstico por imagem , Medula Espinal/efeitos da radiação , Neoplasias da Coluna Vertebral/diagnóstico por imagem , Fluxo de TrabalhoRESUMO
In vivo tissue deposition of fibrillar protein aggregates is the cause of several degenerative diseases. Evidence suggests that interfering with the pathology-associated amyloid fibrillogenesis by inhibitory molecules is envisaged as the primary therapeutic strategy. Amyloid fibril formation of proteins has been demonstrated to be influenced by nanoparticles/nanomaterials. As compared with their molecular form counterpart, this work examined the effect of sucrose-terminated nanoparticles on the in vitro amyloid fibrillogenesis and structural properties of ß-lactoglobulin at pH 2.0 and 80 °C. ThT binding and electron microscopy results demonstrated that sucrose-terminated nanoparticles were able to suppress ß-lactoglobulin fibrillogenesis in a concentration-dependent fashion. Importantly, sucrose-terminated nanoparticles showed better ß-lactoglobulin fibril-inhibiting ability than sucrose molecules. ANS fluorescence and right-angle light scattering results showed reduced solvent exposure and decreased aggregation, respectively, in the ß-lactoglobulin samples upon treatment with sucrose-terminated nanoparticles. Moreover, fluorescence quenching analyses revealed that the static quenching mechanism and formation of a non-fluorescent fluorophore-nanoparticle complex are involved in the nanoparticle-ß-lactoglobulin interaction. We believe that the results from this study may suggest that the nanoparticle form of biocompatible sugar-related osmolytes may serve as effective inhibiting/suppressing agents toward protein fibrillogenesis.
Assuntos
Amiloide/química , Lactoglobulinas/química , Nanopartículas/química , Sacarose/química , Amiloide/ultraestrutura , Animais , Bovinos , Temperatura Alta , Concentração de Íons de Hidrogênio , Nanopartículas/ultraestruturaRESUMO
Joint disorders can be detrimental to quality of life. There is an unmet need for precise functional reconstruction of native-like cartilage and bone tissues in the craniofacial space and particularly for the temporomandibular joint (TMJ). Current surgical methods suffer from lack of precision and comorbidities and frequently involve multiple operations. Studies have sought to improve craniofacial bone grafts without addressing the cartilage, which is essential to TMJ function. For the human-sized TMJ in the Yucatan minipig model, we engineered autologous, biologically, and anatomically matched cartilage-bone grafts for repairing the ramus-condyle unit (RCU), a geometrically intricate structure subjected to complex loading forces. Using image-guided micromilling, anatomically precise scaffolds were created from decellularized bone matrix and infused with autologous adipose-derived chondrogenic and osteogenic progenitor cells. The resulting constructs were cultured in a dual perfusion bioreactor for 5 weeks before implantation. Six months after implantation, the bioengineered RCUs maintained their predefined anatomical structure and regenerated full-thickness, stratified, and mechanically robust cartilage over the underlying bone, to a greater extent than either autologous bone-only engineered grafts or acellular scaffolds. Tracking of implanted cells and parallel bioreactor studies enabled additional insights into the progression of cartilage and bone regeneration. This study demonstrates the feasibility of TMJ regeneration using anatomically precise, autologous, living cartilage-bone grafts for functional, personalized total joint replacement. Inclusion of the adjacent tissues such as soft connective tissues and the TMJ disc could further extend the functional integration of engineered RCUs with the host.
Assuntos
Qualidade de Vida , Engenharia Tecidual , Animais , Cartilagem , Humanos , Suínos , Porco Miniatura , Articulação Temporomandibular , Alicerces TeciduaisRESUMO
Since current microbiology methods are not suitable to detect Clostridium perfringens in formalin-fixed, paraffin-embedded tissue samples, we developed a PCR assay to detect toxin-encoding genes and the 16S rRNA gene of C. perfringens. We successfully detected and genotyped C. perfringens in tissue sections from two autopsy cases.
Assuntos
Toxinas Bacterianas/genética , Infecções por Clostridium/diagnóstico , Clostridium perfringens/genética , Reação em Cadeia da Polimerase/métodos , Preservação de Tecido/métodos , Toxinas Bacterianas/biossíntese , Clostridium perfringens/isolamento & purificação , DNA Bacteriano/genética , DNA Ribossômico/genética , Fixadores/farmacologia , Formaldeído/farmacologia , Genótipo , Humanos , Inclusão em Parafina , RNA Ribossômico 16S/genéticaRESUMO
Mutations in ATP13A2 cause Kufor-Rakeb syndrome (KRS), a juvenile form of Parkinson's disease (PD) with dementia. However, the mechanisms by which mutations in ATP13A2 cause KRS is not understood. The mutations lead to misfolding of the translated Atp13a2 protein and its premature degradation in the endoplasmic reticulum, never reaching the lysosome where the protein is thought to function. Atp13a2 is a P-type ATPase, a class of proteins that function in ion transport. Indeed, studies of human, mouse, and yeast Atp13a2 proteins suggest a possible involvement in regulation of heavy metal toxicity. Here we report on the cytoprotective function of Atp13a2 on HeLa cells and dopamine neurons of Caenorhabditis elegans (C. elegans). HeLa cells stably overexpressing V5- tagged Atp13a2Isoform-1 protein were more resistant to elevated manganese exposure and to starvation-induced cell death compared to cells not overexpressing the protein. Because PD is characterized by loss of dopamine neurons, we generated transgenic C. elegans expressing GFP-tagged human Atp13a2 protein in dopamine neurons. The transgenic animals exhibited higher resistance to dopamine neuron degeneration after acute exposure to manganese compared to nematodes that expressed GFP alone. The results suggest Atp13a2 Isoform-1 protein confers cytoprotection against toxic insults, including those that cause PD syndromes.
Assuntos
Morte Celular , Manganês/toxicidade , ATPases Translocadoras de Prótons/farmacologia , Inanição , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Neurônios Dopaminérgicos/efeitos dos fármacos , Células HeLa , Humanos , Doença de Parkinson/prevenção & controle , Substâncias Protetoras/metabolismo , Isoformas de Proteínas , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismoRESUMO
More than thirty human proteins and/or peptides can aggregate to form amyloid deposits that are linked to several amyloid diseases including clinical syndrome injection-localized amyloidosis, which is correlated with the aggregation of the 51-residue polypeptide insulin. While no cure is currently available toward tackling amyloid diseases, prevention or suppression of amyloid ï¬brillization is considered as the primary therapeutic strategy. Nanomaterials have been demonstrated to possess great potential in the fields of biomedical diagnosis and drug delivery, they are also able to affect the amyloid aggregation of proteins. This work explores the effects of three different magnetic nanoparticles coated with dextran-based polymers on the in vitro amyloid fibrillogenesis of human insulin. Surface modification of nanoparticles with dextran-based polymers was used to improve the biocompatibility of maghemite nanoparticles. We demonstrated that insulin fibrillization may be mitigated by the studied nanoparticles in a concentration-dependent fashion as verified by ThT binding assay and transmission electron microscopy. The extent of inhibitory activity against human insulin fibril formation was found to be associated with the physico-chemical properties of nanoparticles, with the highest inhibitory activity observed for diethylaminoethyl-dextran-coated nanoparticles. Using circular dichroism spectroscopy, ANS fluorescence spectroscopy, and right-angle light scattering, we probed the structural/conformational changes and investigated the aggregating behavior of insulin upon treatment with nanoparticles. This work demonstrates that nanoparticles with an appropriate surface modification can be utilized to suppress or even inhibit amyloid fibril formation of proteins.
Assuntos
Amiloide/metabolismo , Materiais Revestidos Biocompatíveis/farmacologia , Dextranos/farmacologia , Insulina/metabolismo , Nanopartículas/química , Benzotiazóis/metabolismo , Dicroísmo Circular , Difusão Dinâmica da Luz , Humanos , Insulina/química , Nanopartículas/ultraestrutura , Agregados Proteicos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espectrometria de Fluorescência , Eletricidade EstáticaRESUMO
The 129-residue lysozyme has been shown to form amyloid fibrils in vitro. While methylene blue (MB), a compound in the phenothiazinium family, has been shown to dissemble tau fibril formation, its anti-fibrillogenic effect has not been thoroughly characterized in other proteins/peptides. This study examines the effects of MB on the in vitro fibrillogenesis of lysozyme at pHâ¯2.0 and 55⯰C. Our results demonstrated that, upon 7-day incubation, the plateau ThT fluorescence of the sample was found to be ~8.69% or ~2.98% of the control when the molar ratio of lysozyme to MB was at 1:1.11 or 1:3.33, respectively, indicating that the inhibitory potency of MB against lysozyme fibrillogenesis is positively correlated with its concentration. We also found that MB is able to destabilize the preformed lysozyme fibrils. Moreover, molecular docking and molecular dynamics simulations results revealed that MB's mechanism of fibril formation inhibition may be triggered by binding with lysozyme's aggregation-prone region. Results reported here provide solid support for MB's effect on amyloid fibrillogenesis. We believe the additional insights gained herein may pave way to the discovery of other small molecules that may have similar action toward amyloid fibril formation and its associated diseases.
Assuntos
Amiloide/química , Azul de Metileno/química , Muramidase/química , Agregados Proteicos , Amiloide/metabolismo , Amiloide/ultraestrutura , Amiloidose , Azul de Metileno/farmacologia , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Muramidase/metabolismo , Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas , Ligação Proteica/efeitos dos fármacos , Análise Espectral , Relação Estrutura-AtividadeRESUMO
Amyloid aggregates of proteins are one of the most abundant and important naturally occurring self-associated assemblies. Formation of poly/peptide amyloid aggregates is also associated with the widely spread diseases, so called amyloidosis, which include Alzheimer's disease, diabetes mellitus and lysozyme amyloidosis. These disorders are still incurable and novel therapeutical approaches are focused on using small molecules for inhibition of amyloid aggregation. We have observed effect of three structurally distinct groups of tacrine/acridone - coumarin heterodimers on hen egg white (HEW) lysozyme fibrillization in vitro. The ability of heterodimers to interfere with lysozyme amyloid aggregation was examined using Thioflavin T fluorescence assay, atomic force microscopy and docking method. The obtained data suggest that inhibitory effect of heterodimers on lysozyme fibrillization depends on their composition. We have shown that tacrine-coumarin heterodimers with alkylenediamine linker are the most effective inhibitors of lysozyme fibrillization. The inhibitory activities were quantified through IC50 values; the most potent heterodimers interfere with lysozyme aggregation in the scale of micromolar concentrations (19.2⯵M-105.4⯵M). The molecular docking showed that the modes of possible interactions involved in the binding are mainly hydrophobic interactions, hydrogen bonding and van der Waals interactions. Studied heterodimers had none or weak cytotoxic effect on human neuroblastoma cells. The obtained results can be helpful for the design and development of new therapeutics for amyloid-related diseases.