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1.
PLoS One ; 13(6): e0198510, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29879182

RESUMO

Gfi1 is a key molecule in hematopoietic lineage development and mutations in GFI1 cause severe congenital neutropenia (SCN). Neutropenia is associated with low bone mass, but the underlying mechanisms are poorly characterized. Using Gfi1 knock-out mice (Gfi1-ko/ko) as SCN model, we studied the relationship between neutropenia and bone mass upon different pathogen load conditions. Our analysis reveals that Gfi1-ko/ko mice kept under strict specific pathogen free (SPF) conditions demonstrate normal bone mass and survival. However, Gfi1-ko/ko mice with early (nonSPF) or late (SPF+nonSPF) pathogen exposure develop low bone mass. Gfi1-ko/ko mice demonstrate a striking rise of systemic inflammatory markers according to elevated pathogen exposure and reduced bone mass. Elevated inflammatory cytokines include for instance Il-1b, Il-6, and Tnf-alpha that regulate osteoclast development. We conclude that low bone mass, due to low neutrophil counts, is caused by the degree of systemic inflammation promoting osteoclastogenesis.


Assuntos
Proteínas de Ligação a DNA/genética , Neutropenia/congênito , Osteoporose/etiologia , Fatores de Transcrição/genética , Animais , Peso Corporal , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/fisiologia , Diferenciação Celular , Síndrome Congênita de Insuficiência da Medula Óssea , Citocinas/genética , Citocinas/metabolismo , Proteínas de Ligação a DNA/deficiência , Extremidades/patologia , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutropenia/etiologia , Neutropenia/genética , Neutropenia/patologia , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese , Osteoporose/genética , Osteoporose/patologia , Osteoprotegerina/sangue , Pasteurellaceae/patogenicidade , Ligante RANK/sangue , Fatores de Transcrição/deficiência , Trichomonas/patogenicidade
2.
Nano Lett ; 18(6): 3313-3321, 2018 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-29799208

RESUMO

A graphene liquid cell for transmission electron microscopy (TEM) uses one or two graphene sheets to separate the liquid from the vacuum in the microscope. In principle, graphene is an excellent material for such an application because it allows the highest possible spatial resolution, provides a flexible covering foil, and effectively protects the liquid from evaporating. Examples in open literature have demonstrated atomic-resolution TEM using small liquid pockets and the coverage of whole biological cells with graphene sheets. A total of three different basic types of liquid cells are discerned: (i) one graphene sheet is used to cover a liquid sample supported by a thin membrane of another material (for example, silicon nitride, SiN), (ii) two graphene sheets pressed together leaving liquid pockets with graphene at both sides, and (iii) a spacer material with liquid pockets covered at both sides by graphene. A total of four different process flows are available for liquid cell assembly, but there is not yet a consensus on the best routes, and a number of variations exist. The key step is the transfer of graphene to a liquid sample, which is complicated by practical issues that arise from imperfections in the graphene sheets, such as cracks. This review provides an overview of these different approaches to assembling graphene liquid cells and discusses the main obstacles and ideas to overcome them with the prospect of developing the nanoscale technology needed for graphene liquid cells so that they become available on a routine basis for electron microscopy in liquid. It also provides guidance in selecting the appropriate type of graphene liquid cell and the best assembly method for a specific experiment.

3.
Genes (Basel) ; 9(5)2018 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-29748516

RESUMO

Bone is a complex tissue with a variety of functions, such as providing mechanical stability for locomotion, protection of the inner organs, mineral homeostasis and haematopoiesis. To fulfil these diverse roles in the human body, bone consists of a multitude of different cells and an extracellular matrix that is mechanically stable, yet flexible at the same time. Unlike most tissues, bone is under constant renewal facilitated by a coordinated interaction of bone-forming and bone-resorbing cells. It is thus challenging to recreate bone in its complexity in vitro and most current models rather focus on certain aspects of bone biology that are of relevance for the research question addressed. In addition, animal models are still regarded as the gold-standard in the context of bone biology and pathology, especially for the development of novel treatment strategies. However, species-specific differences impede the translation of findings from animal models to humans. The current review summarizes and discusses the latest developments in bone tissue engineering and organoid culture including suitable cell sources, extracellular matrices and microfluidic bioreactor systems. With available technology in mind, a best possible bone model will be hypothesized. Furthermore, the future need and application of such a complex model will be discussed.

4.
J Gen Physiol ; 150(1): 111-125, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29247050

RESUMO

Malignant hyperthermia (MH) is a fatal hypermetabolic state that may occur during general anesthesia in susceptible individuals. It is often caused by mutations in the ryanodine receptor RyR1 that favor drug-induced release of Ca2+ from the sarcoplasmic reticulum. Here, knowing that membrane depolarization triggers Ca2+ release in normal muscle function, we study the cross-influence of membrane potential and anesthetic drugs on Ca2+ release. We used short single muscle fibers of knock-in mice heterozygous for the RyR1 mutation Y524S combined with microfluorimetry to measure intracellular Ca2+ signals. Halothane, a volatile anesthetic used in contracture testing for MH susceptibility, was equilibrated with the solution superfusing the cells by means of a vaporizer system. In the range 0.2 to 3%, the drug causes significantly larger elevations of free myoplasmic [Ca2+] in mutant (YS) compared with wild-type (WT) fibers. Action potential-induced Ca2+ signals exhibit a slowing of their time course of relaxation that can be attributed to a component of delayed Ca2+ release turnoff. In further experiments, we applied halothane to single fibers that were voltage-clamped using two intracellular microelectrodes and studied the effect of small (10-mV) deviations from the holding potential (-80 mV). Untreated WT fibers show essentially no changes in [Ca2+], whereas the Ca2+ level of YS fibers increases and decreases on depolarization and hyperpolarization, respectively. The drug causes a significant enhancement of this response. Depolarizing pulses reveal a substantial negative shift in the voltage dependence of activation of Ca2+ release. This behavior likely results from the allosteric coupling between RyR1 and its transverse tubular voltage sensor. We conclude that the binding of halothane to RyR1 alters the voltage dependence of Ca2+ release in MH-susceptible muscle fibers such that the resting membrane potential becomes a decisive factor for the efficiency of the drug to trigger Ca2+ release.


Assuntos
Potenciais de Ação , Anestésicos Inalatórios/farmacologia , Cálcio/metabolismo , Febre/metabolismo , Halotano/farmacologia , Fibras Musculares Esqueléticas/metabolismo , Animais , Células Cultivadas , Febre/genética , Masculino , Camundongos , Contração Muscular , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/genética
5.
Biomaterials ; 99: 95-108, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27235995

RESUMO

Severe injury to the skeletal muscle often results in the formation of scar tissue, leading to a decline in functional performance. Traditionally, tissue engineering strategies for muscle repair have focused on substrates that promote myogenic differentiation of transplanted cells. In the current study, the reported data indicates that mesenchymal stromal cells (MSCs) transplanted via porous alginate cryogels promote muscle regeneration by secreting bioactive factors that profoundly influence the function of muscle progenitor cells. These cellular functions, which include heightened resistance of muscle progenitor cells to apoptosis, migration to site of injury, and prevention of premature differentiation are highly desirable in the healing cascade after acute muscle trauma. Furthermore, stimulation of MSCs with recombinant growth factors IGF-1 and VEGF165 was found to significantly enhance their paracrine effects on muscle progenitor cells. Multifunctional alginate cryogels were then utilized as synthetic niches that facilitate local stimulation of seeded MSCs by providing a sustained release of growth factors. In a clinically relevant injury model, the modulation of MSC paracrine signaling via engineered niches significantly improved muscle function by remodeling scar tissue and promoting the formation of new myofibers, outperforming standalone cell or growth factor delivery.


Assuntos
Células-Tronco Mesenquimais/citologia , Músculo Esquelético/fisiologia , Cicatrização/fisiologia , Animais , Apoptose , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Feminino , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Fenômenos Mecânicos , Transplante de Células-Tronco Mesenquimais , Camundongos , Desenvolvimento Muscular , Ratos Sprague-Dawley , Regeneração , Engenharia Tecidual , Fator A de Crescimento do Endotélio Vascular/metabolismo
6.
Biomaterials ; 98: 31-40, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27179133

RESUMO

In hip arthroplasty the implants' articulating surfaces can be made of a cobalt-chromium-molybdenum (CoCrMo) alloy. The use of these metal-on-metal (MoM) pairings can lead to the release of wear products such as metallic particles and dissociated metal species, raising concerns regarding their safety amongst orthopedic surgeons and the public. MoM-wear particles are reported to be heterogeneous in their physicochemical properties, are capable of inducing adverse effects on a cellular level and are thought to be involved in relevant clinical problems like aseptic osteolysis. Yet, it remains elusive how MoM-wear affects bone forming cells and their progenitors: bone marrow residing mesenchymal stromal cells (MSCs). This study introduces an assessment of the in vivo exposure to particulate and dissociated Co and Cr and evaluates the effects of MoM-wear on MSCs. The exposure to MoM-wear products in vivo and in vitro leads to a decrease in MSCs' osteogenic matrix mineralization and alkaline phosphatase activity on a cellular and systemic level. In conclusion, MoM-wear products are released in the periprosthetic region and elevate bone marrow Co and Cr concentrations towards levels that impair osteogenic differentiation of MSCs. Therefore, the ongoing use of CoCrMo alloys for articulating surfaces in joint replacement implants needs critical reconsideration.


Assuntos
Prótese de Quadril/efeitos adversos , Células-Tronco Mesenquimais/metabolismo , Próteses Articulares Metal-Metal/efeitos adversos , Material Particulado/efeitos adversos , Idoso , Fosfatase Alcalina/sangue , Matriz Óssea/metabolismo , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/patologia , Pessoa de Meia-Idade , Minerais/metabolismo , Osteogênese
7.
Methods Enzymol ; 567: 129-56, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26794353

RESUMO

For many in vitro studies, purified membrane proteins need to be reconstituted from detergent micelles into lipid bilayers to regain their native structures and functions. Stoichiometric complexation of detergent by cyclodextrin provides a tightly controllable strategy for detergent extraction. Here, we describe a practical approach making use of isothermal titration calorimetry to obtain a complete set of thermodynamic parameters that allows for quantitative prediction of the transition from micelles to bilayer membranes during reconstitution. These parameters include the dissociation constant of the cyclodextrin/detergent inclusion complex, the critical micellar concentration of the detergent, and the phase boundaries of the lipid/detergent phase diagram. The underlying theoretical framework involves linked equilibria among all pseudophases, as described previously (Textor, Vargas, & Keller, 2015). This chapter focuses on practical aspects of the approach and discusses caveats and calorimetry-specific details of data analysis. With the entire parameter set at hand, exploration of different reconstitution trajectories within the lipid/detergent phase diagram is possible. Together with the straightforward control over the rate of detergent extraction offered by cyclodextrin complexation, this opens the possibility of systematically tuning and optimizing the reconstitution process of membrane proteins. Provided some particular precautions are taken, the approach can be adapted to many other combinations of proteins, lipids, detergents, and cyclodextrins.


Assuntos
Calorimetria/métodos , Detergentes/química , Ciclodextrinas/química , Proteínas de Membrana/química , Termodinâmica
8.
Anal Biochem ; 485: 119-21, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26079704

RESUMO

Determination of the critical micellar concentration of surfactants and of the heat of demicellization by means of isothermal titration calorimetry usually involves either calculation of the first derivative of the heat of demicellization with respect to surfactant concentration or application of a generic sigmoidal fit to the demicellization isotherm. Here, we show that a combination of both approaches provides an unbiased and reproducible data analysis strategy without the need for user input other than the calorimetric data proper. The approach is explained and exemplified using demicellization isotherms of the fluorinated surfactant F6OPC (3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluoro-n-octylphosphocholine) and the zwitterionic detergent CHAPSO (3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate).


Assuntos
Ácidos Cólicos/química , Micelas , Tensoativos/química , Automação , Calorimetria
9.
Eur Biophys J ; 44(7): 503-12, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26016666

RESUMO

Bilayer mechanical properties are not only of crucial importance to the mechanism of action of mechanosensation in lipid membranes but also affect preparative laboratory tasks such as membrane-protein refolding. We report this for coupled refolding and bilayer insertion of outer membrane phospholipase A (OmpLA), an integral membrane enzyme that catalyses the hydrolytic cleavage of glycerophospholipids. OmpLA can be refolded into a variety of detergent micelles and unilamellar vesicles composed of short-chain phospholipids but, in the absence of chemical or molecular chaperones, not into thicker membranes. Controlled modulation of bilayer mechanical properties by judicious use of subsolubilising concentrations of detergents induces monolayer curvature strain, acyl chain fluidisation, membrane thinning, and transient aqueous bilayer defects. This enables quantitative and functional refolding of OmpLA even into bilayer membranes composed of long-chain phospholipids to yield enzymatically active proteoliposomes without requiring membrane solubilisation.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Bicamadas Lipídicas/metabolismo , Fosfolipases A1/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Fenômenos Biomecânicos , Detergentes/farmacologia , Bicamadas Lipídicas/química , Fosfolipases A1/química , Dobramento de Proteína , Lipossomas Unilamelares/metabolismo
10.
Methods ; 76: 183-193, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25583305

RESUMO

Reconstitution from detergent micelles into lipid bilayer membranes is a prerequisite for many in vitro studies on purified membrane proteins. Complexation by cyclodextrins offers an efficient and tightly controllable way of removing detergents for membrane-protein reconstitution, since cyclodextrins sequester detergents at defined stoichiometries and with tuneable affinities. To fully exploit the potential advantages of cyclodextrin for membrane-protein reconstitution, we establish a quantitative model for predicting the supramolecular transition from mixed micelles to vesicles during cyclodextrin-mediated detergent extraction. The model is based on a set of linked equilibria among all pseudophases present in the course of the reconstitution process. Various isothermal titration-calorimetric protocols are used for quantifying a detergent's self-association as well as its colloidal and stoichiometric interactions with lipid and cyclodextrin, respectively. The detergent's critical micellar concentration, the phase boundaries in the lipid/detergent phase diagram, and the dissociation constant of the cyclodextrin/detergent complex thus obtained provide all thermodynamic parameters necessary for a quantitative prediction of the transition from micelles to bilayer membranes during cyclodextrin-driven reconstitution. This is exemplified and validated by stepwise complexation of the detergent lauryldimethylamine N-oxide in mixtures with the phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine upon titration with 2-hydroxypropyl-ß-cyclodextrin, both in the presence and in the absence of the membrane protein Mistic. The calorimetric approach presented herein quantitatively predicts the onset and completion of the reconstitution process, thus obviating cumbersome trial-and-error efforts and facilitating the rational optimisation of reconstitution protocols, and can be adapted to different cyclodextrin/lipid/detergent combinations.


Assuntos
Calorimetria/métodos , Ciclodextrinas/química , Detergentes/química , Lipídeos de Membrana/química , Modelos Moleculares , Bicamadas Lipídicas/química , Micelas , Termodinâmica
11.
PLoS One ; 7(12): e52700, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23285157

RESUMO

Mesenchymal stromal cells (MSCs) are of high relevance for the regeneration of mesenchymal tissues such as bone and cartilage. The promising role of MSCs in cell-based therapies and tissue engineering appears to be limited due to a decline of their regenerative potential with increasing donor age, their limited availability in human tissues and the need of in vitro expansion prior to treatment. We therefore aimed to determine to which degree in vitro aging and chronological aging may be similar processes or if in vitro culture-related changes at the cellular and molecular level are at least altered as a function of donor age. For that purpose we established MSCs cultures from young (yMSCs) and aged (aMSCs) rats that were cultured for more than 100 passages. These long-term MSCs cultures were non-tumorigenic and exhibited similar surface marker patterns as primary MSCs of passage 2. During in vitro expansion, but not during chronological aging, MSCs progressively lose their progenitor characteristics, e.g., complete loss of osteogenic differentiation potential, diminished adipogenic differentiation, altered cell morphology and increased susceptibility towards senescence. Transcriptome analysis revealed that long-term in vitro MSCs cultivation leads to down-regulation of genes involved in cell differentiation, focal adhesion organization, cytoskeleton turnover and mitochondria function. Accordingly, functional analysis demonstrated altered mitochondrial morphology, decreased antioxidant capacities and elevated ROS levels in long-term cultivated yMSCs as well as aMSCs. Notably, only the MSC migration potential and their antioxidative capacity were altered by in vitro as well as chronological aging. Based on specific differences observed between the impact of chronological and in vitro MSC aging we conclude that both are distinct processes.


Assuntos
Senescência Celular , Citoesqueleto/metabolismo , Células-Tronco Mesenquimais/metabolismo , Mitocôndrias/metabolismo , Animais , Diferenciação Celular , Movimento Celular , Células Cultivadas , Senescência Celular/genética , Citoesqueleto/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Masculino , Células-Tronco Mesenquimais/citologia , Mitocôndrias/genética , Osteogênese , Ratos
12.
Rev. neuropsiquiatr ; 55(3): 167-182, sept. 1992.
Artigo em Espanhol | LILACS, LIPECS | ID: lil-484063

RESUMO

Se presenta en primer lugar el desarrollo de la psicoterapia durante las dos últimas décadas. Se enfatiza la rápida multiplicación de enfoques y las consecuencias negativas de la formación de "escuelas" de psicoterapia: 1) La investigación de "factores no específicos de tratamiento" que se encuentran presentes en todos los enfoques de psicoterapia y causan el cambio comportamental; 2) La búsqueda de un lenguaje común, en el cual se da énfasis especial a la psicología cognitiva; 3) Eclecticismo, por el cual los clínicos emplean conceptos y técnicas de diferentes enfoques de psicoterapia; 4) La combinación y fusión de las dos mayores orientaciones psicoterapéuticas (psicoanálisis y conductismo); y, 5) La psicoterapia integrativa que intenta la confluencia de todos los enfoques psicoterapéuticos importantes en una teoría comprehensiva.


Assuntos
Humanos , Psicoterapia/classificação , Psicoterapia/história , Psicoterapia/tendências
13.
Am J Orthopsychiatry ; 57(4): 495-507, 1987 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-3314535

RESUMO

Major causes of drug abuse identified in the literature are outlined. Contributing factors are located on individual, interpersonal, and social levels, with the family of origin established as very important. An integrated approach to family therapy that takes all these levels into account is described employing concepts, hypotheses, and techniques from different kinds of therapy.


Assuntos
Terapia Familiar/métodos , Transtornos Relacionados ao Uso de Substâncias/reabilitação , Família , Humanos , Transtornos Relacionados ao Uso de Substâncias/psicologia
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