RESUMEN
Disparities in healthy food access and consumption are a major public health concern. This study reports the findings from a two-year randomized control trial conducted at 77 farmers' markets (FMs) in 13 states and the District of Columbia that sought to understand the impact of fruit and vegetable (FV) incentive vouchers, randomly issued at varied incentive levels to Supplemental Nutrition Assistance Program (SNAP) recipients, for use at FMs. Measures included FV and overall household food purchasing; FV consumption; food insecurity; health status; market expenditure; and demographics. A repeated-measures mixed-effects analysis and the Complier Average Causal Effect (CACE) were used to examine outcomes. Despite 82% reporting food insecurity in the prior year, the findings showed that financial incentives at FMs had statistically significant, positive effects on FV consumption; market expenditures increased with added incentives. SNAP recipients receiving an incentive of USD 0.40 for every USD 1.00 in SNAP spent an average of USD 19.03 per transaction, while those receiving USD 2 for every USD 1 (2:1) spent an average of USD 36.28 per transaction. The data showed that the incentive program at the highest level (2:1) maximally increased SNAP FM expenditure and FV consumption, increasing the latter by 0.31 daily cups among those who used their incentive (CACE model).
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Asistencia Alimentaria , Agricultores , Abastecimiento de Alimentos , Frutas , Humanos , Motivación , VerdurasRESUMEN
Granulocyte macrophage colony stimulating factor (GM-CSF) is a key participant in, and a clinical target for, the treatment of inflammatory diseases including rheumatoid arthritis (RA). Therapeutic inhibition of GM-CSF signalling using monoclonal antibodies to the α-subunit of the GM-CSF receptor (GMCSFRα) has shown clear benefit in patients with RA, giant cell arteritis (GCAs) and some efficacy in severe SARS-CoV-2 infection. However, GM-CSF autoantibodies are associated with the development of pulmonary alveolar proteinosis (PAP), a rare lung disease characterised by alveolar macrophage (AM) dysfunction and the accumulation of surfactant lipids. We assessed how the anti-GMCSFRα approach might impact surfactant turnover in the airway. Female C57BL/6J mice received a mouse-GMCSFRα blocking antibody (CAM-3003) twice per week for up to 24 weeks. A parallel, comparator cohort of the mouse PAP model, GM-CSF receptor ß subunit (GMCSFRß) knock-out (KO), was maintained up to 16 weeks. We assessed lung tissue histopathology alongside lung phosphatidylcholine (PC) metabolism using stable isotope lipidomics. GMCSFRß KO mice reproduced the histopathological and biochemical features of PAP, accumulating surfactant PC in both broncho-alveolar lavage fluid (BALF) and lavaged lung tissue. The incorporation pattern of methyl-D9-choline showed impaired catabolism and not enhanced synthesis. In contrast, chronic supra-pharmacological CAM-3003 exposure (100 mg/kg) over 24 weeks did not elicit a histopathological PAP phenotype despite some changes in lung PC catabolism. Lack of significant impairment of AM catabolic function supports clinical observations that therapeutic antibodies to this pathway have not been associated with PAP in clinical trials.
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Artritis Reumatoide/metabolismo , COVID-19/terapia , Proteinosis Alveolar Pulmonar/inmunología , Surfactantes Pulmonares/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidores , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Artritis Reumatoide/terapia , Autoanticuerpos/química , Líquido del Lavado Bronquioalveolar , COVID-19/inmunología , Colina/análogos & derivados , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/química , Inflamación , Interleucina-6/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Proteinosis Alveolar Pulmonar/genética , SARS-CoV-2/inmunología , TensoactivosRESUMEN
Asymmetric stem cell division is a critical mechanism for balancing self-renewal and differentiation. Adult stem cells often orient their mitotic spindle to place one daughter inside the niche and the other outside of it to achieve asymmetric division. It remains unknown whether and how the niche may direct division orientation. Here we discover a novel and evolutionary conserved mechanism that couples cell polarity to cell fate. We show that the cytokine receptor homolog Dome, acting downstream of the niche-derived ligand Upd, directly binds to the microtubule-binding protein Eb1 to regulate spindle orientation in Drosophila male germline stem cells (GSCs). Dome's role in spindle orientation is entirely separable from its known function in self-renewal mediated by the JAK-STAT pathway. We propose that integration of two functions (cell polarity and fate) in a single receptor is a key mechanism to ensure an asymmetric outcome following cell division.
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División Celular Asimétrica , Polaridad Celular , Proteínas de Drosophila/metabolismo , Células Germinativas/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Receptores de Interleucina/metabolismo , Células Madre Adultas/fisiología , Animales , Drosophila , Unión Proteica , Huso Acromático/metabolismoRESUMEN
Maintenance of tissue-specific organ lipid compositions characterizes mammalian lipid homeostasis. The lungs and liver synthesize mixed phosphatidylcholine (PC) molecular species that are subsequently tailored for function. The lungs progressively enrich disaturated PC directed to lamellar body surfactant stores before secretion. The liver accumulates polyunsaturated PC directed to very-low-density lipoprotein assembly and secretion, or to triglyceride stores. In each tissue, selective PC species enrichment mechanisms lie at the heart of effective homeostasis. We tested for potential coordination between these spatially separated but possibly complementary phenomena under a major derangement of lung PC metabolism, pulmonary alveolar proteinosis (PAP), which overwhelms homeostasis and leads to excessive surfactant accumulation. Using static and dynamic lipidomics techniques, we compared (1) tissue PC compositions and contents, and (2) in lungs, the absolute rates of synthesis in both control mice and the granulocyte-macrophage colony-stimulating factor knockout model of PAP. Significant disaturated PC accumulation in bronchoalveolar lavage fluid, alveolar macrophage, and lavaged lung tissue occurred alongside increased PC synthesis, consistent with reported defects in alveolar macrophage surfactant turnover. However, microscopy using oil red O staining, coherent anti-Stokes Raman scattering, second harmonic generation, and transmission electron microscopy also revealed neutral-lipid droplet accumulations in alveolar lipofibroblasts of granular macrophage colony-stimulating factor knockout animals, suggesting that lipid homeostasis deficits extend beyond alveolar macrophages. PAP plasma PC composition was significantly polyunsaturated fatty acid enriched, but the content was unchanged and hepatic polyunsaturated fatty acid-enriched PC content increased by 50% with an accompanying micro/macrovesicular steatosis and a fibrotic damage pattern consistent with nonalcoholic fatty liver disease. These data suggest a hepatopulmonary axis of PC metabolism coordination, with wider implications for understanding and managing lipid pathologies in which compromise of one organ has unexpected consequences for another.
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Hígado Graso/metabolismo , Hígado/metabolismo , Macrófagos Alveolares/metabolismo , Fosfatidilcolinas/metabolismo , Proteinosis Alveolar Pulmonar/metabolismo , Alveolos Pulmonares/metabolismo , Animales , Hígado Graso/complicaciones , Hígado Graso/genética , Femenino , Masculino , Ratones , Ratones Noqueados , Especificidad de Órganos/genética , Fosfatidilcolinas/genética , Proteinosis Alveolar Pulmonar/etiología , Proteinosis Alveolar Pulmonar/genéticaRESUMEN
INTRODUCTION: Federal policy supports increased implementation of monetary incentive interventions for chronic disease prevention among low-income populations. This study describes how a Prevention Research Center, working with a dissemination partner, developed and distributed technology to support nationwide implementation and evaluation of healthy food incentive programming focused on Supplemental Nutrition Assistance Program recipients. METHODS: FM Tracks, an iOS-based application and website, was developed to standardize evaluation methods for healthy food incentive program implementation at direct-to-consumer markets. This evaluation examined diffusion and adoption of the technology over 9 months (July 2015-March 2016). Data were analyzed in 2016. RESULTS: FM Tracks was disseminated to 273 markets affiliated with 37 regional networks in 18 states and Washington, DC. All markets adopted the sales transaction data collection feature, with nearly all recording at least one Supplemental Nutrition Assistance Program (99.3%) and healthy food incentive (97.1%) transaction. A total of 43,493 sales transactions were recorded. By the ninth month of technology dissemination, markets were entering individual sales transactions using the application (34.5%) and website (29.9%) and aggregated transactions via website (35.6%) at similar rates. Use of optional evaluation features like recording a customer ID with individual transactions increased successively with a low of 22.2% during the first month to a high of 69.2% in the ninth month. CONCLUSIONS: Systematic and widely used evaluation technology creates possibilities for pragmatic research embedded within ongoing, real-world implementation of food access interventions. Technology dissemination requires supportive technical assistance and continuous refinement that can be advanced through academic-practitioner partnerships.
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Asistencia Alimentaria/normas , Evaluación de Programas y Proyectos de Salud/métodos , Productos Agrícolas/economía , Publicidad Directa al Consumidor , Abastecimiento de Alimentos/normasRESUMEN
Triterpenoid saponins from Saponinum Album (SA) exert potent lytic effects on eukaryotic cell plasma membranes and, when used at sub-lytic concentrations, significantly augment the cytotoxicity of saporin-based immunotoxins (IT). To help elucidate the mechanism(s) behind these two phenomena we investigated the role of cholesterol to both. Human Daudi lymphoma cells were lipid deprived using a combination of three different approaches. Following treatment, the total cellular lipid content was analyzed by electrospray ionization mass spectrometry (ESI-MS) and plasma membrane (PM) cholesterol content measured using the lipophilic fluorescent probe NR12S. Maximal lipid deprivation of cells resulted in a complete loss of sensitivity to lysis by SA. Similarly augmentation of the anti-CD19 immunotoxin (IT) BU12-SAPORIN by SA was lost but without a concomitant loss of intrinsic IT cytotoxicity. The lytic activity of SA was restored following incubation of lipid deprived Daudi cells with Synthecol or LDL. The augmentative effect of SA on IT cytotoxicity for Daudi cells was restored following repletion of PM cholesterol levels with LDL. NR12S fluorescence and ESI-MS analysis of cellular lipids demonstrated that restoration of SA lytic activity by Synthecol was entirely due to increased PM cholesterol levels. Restoration of cellular and PM cholesterol levels by LDL also restored the augmentative effect of SA for IT, an effect associated with repletion of PM cholesterol with minor changes in some phospholipid species. These results indicate that the lytic and IT augmentative properties of SA are cholesterol-dependent in contrast to intrinsic IT cytotoxicity that is at least partially cholesterol independent.
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Antígenos CD19/inmunología , LDL-Colesterol/fisiología , Inmunotoxinas/farmacología , Linfoma/tratamiento farmacológico , Lípidos de la Membrana/fisiología , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacología , Saponinas/farmacología , Triterpenos/farmacología , Línea Celular Tumoral , Humanos , Linfoma/química , SaporinasRESUMEN
While high-throughput planar patch-clamp instruments are now established to perform whole-cell recordings for drug screening, the conventional micropipette-based approach remains the gold standard for performing cell-attached single-channel recordings. Generally, planar platforms are not well-suited for such studies due to excess noise resulting from low seal resistances and the use of substrates with poor dielectric properties. Since these platforms tend to use the same pore to position a cell by suction and establish a seal, biological debris from the cell suspension can contaminate the pore surface prior to seal formation, reducing the seal resistance. Here, femtosecond laser ablation was used to fabricate dual-pore glass chips optimized for use in cell-attached single-channel recordings that circumvent this problem by using different pores to position a cell and to establish a seal. This dual-pore design also permitted the use of a relatively small patch aperture (D ~ 150 to 300 nm) that is better-suited for establishing high-resistance seals than the micropores used typically in planar patch-clamp setups (D ~ 1 to 2 µm) without compromising the ability of the device to position a cell. Taking advantage of the high seal resistances and low capacitive and dielectric noise realized using glass substrates, patch-clamp experiments with these dual-pore chips consistently achieved high seal resistances (rate of gigaseal formation = 61%, mean seal resistance = 53 GΩ), maintained gigaseals for prolonged durations (up to 6 hours), achieved RMS noise values as low as 0.46 pA at 5 kHz bandwidth, and enabled single-channel recordings in the cell-attached configuration that are comparable to those obtained by conventional patch-clamp.
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Vidrio , Porosidad , Impedancia Eléctrica , Células HEK293 , Humanos , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Técnicas de Placa-Clamp/instrumentación , Técnicas de Placa-Clamp/métodosRESUMEN
The alveolar type II epithelial (ATII) cell is highly specialised for the synthesis and storage, in intracellular lamellar bodies, of phospholipid destined for secretion as pulmonary surfactant into the alveolus. Regulation of the enzymology of surfactant phospholipid synthesis and metabolism has been extensively characterised at both molecular and functional levels, but understanding of surfactant phospholipid metabolism in vivo in either healthy or, especially, diseased lungs is still relatively poorly understood. This review will integrate recent advances in the enzymology of surfactant phospholipid metabolism with metabolic studies in vivo in both experimental animals and human subjects. It will highlight developments in the application of stable isotope-labelled precursor substrates and mass spectrometry to probe lung phospholipid metabolism in terms of individual molecular lipid species and identify areas where a more comprehensive metabolic model would have considerable potential for direct application to disease states.
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Fosfolípidos/metabolismo , Surfactantes Pulmonares/metabolismo , Animales , HumanosRESUMEN
Phosphatidylinositol transfer proteins (PITPs) are versatile proteins required for signal transduction and membrane traffic. The best characterized mammalian PITPs are the Class I PITPs, PITPα (PITPNA) and PITPß (PITPNB), which are single domain proteins with a hydrophobic cavity that binds a phosphatidylinositol (PI) or phosphatidylcholine molecule. In this study, we report the lipid binding properties of an uncharacterized soluble PITP, phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1) (alternative name, RdgBß), of the Class II family. We show that the lipid binding properties of this protein are distinct to Class I PITPs because, besides PI, RdgBß binds and transfers phosphatidic acid (PA) but hardly binds phosphatidylcholine. RdgBß when purified from Escherichia coli is preloaded with PA and phosphatidylglycerol. When RdgBß was incubated with permeabilized HL60 cells, phosphatidylglycerol was released, and PA and PI were now incorporated into RdgBß. After an increase in PA levels following activation of endogenous phospholipase D or after addition of bacterial phospholipase D, binding of PA to RdgBß was greater at the expense of PI binding. We propose that RdgBß, when containing PA, regulates an effector protein or can facilitate lipid transfer between membrane compartments.
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Proteínas de Transporte de Membrana/fisiología , Ácidos Fosfatidicos/química , Animales , Citosol/metabolismo , Escherichia coli/metabolismo , Células HL-60 , Humanos , Lípidos/química , Espectrometría de Masas/métodos , Proteínas de Transporte de Membrana/metabolismo , Modelos Biológicos , Neovascularización Patológica , Fosfatidilgliceroles/química , Fosfolipasa D/química , Fosfolípidos/química , Unión Proteica , Proteínas Recombinantes/química , Transducción de SeñalRESUMEN
Bacterial antibiotic resistance is one of the major concerns of modern healthcare worldwide, and the development of rapid, growth-based, antimicrobial susceptibility tests is key for addressing it. The cover image shows a self-assembled asynchronous magnetic bead rotation (AMBR) biosensor developed for rapid detection of bacterial growth. Using the biosensors, the minimum inhibitory concentration of a clinical E. coli isolate can be measured within two hours, where currently tests take 6-24 hours. A 16-well prototype is also constructed for simple and robust observation of the self-assembled AMBR biosensors.
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Antiinfecciosos/farmacología , Técnicas Biosensibles/instrumentación , Escherichia coli/crecimiento & desarrollo , Magnetismo/instrumentación , Pruebas de Sensibilidad Microbiana/instrumentación , Pruebas de Sensibilidad Microbiana/métodos , Microesferas , Escherichia coli/efectos de los fármacos , Escherichia coli/aislamiento & purificación , RotaciónRESUMEN
Pulmonary inflammation is associated with altered lipid synthesis and clearance related to diabetes, obesity, and various inherited metabolic disorders. In many tissues, lipogenesis is regulated at the transcriptional level by the activity of sterol-response element-binding proteins (SREBP). The role of SREBP activation in the regulation of lipid metabolism in the lung was assessed in mice in which both Insig1 and Insig2 genes, encoding proteins that bind and inhibit SREBPs in the endoplasmic reticulum, were deleted in alveolar type 2 cells. Although deletion of either Insig1 or Insig2 did not alter SREBP activity or lipid homeostasis, deletion of both genes (Insig1/2(Δ/Δ) mice) activated SREBP1, causing marked accumulation of lipids that consisted primarily of cholesterol esters and triglycerides in type 2 epithelial cells and alveolar macrophages. Neutral lipids accumulated in type 2 cells in association with the increase in mRNAs regulating fatty acid, cholesterol synthesis, and inflammation. Although bronchoalveolar lavage fluid phosphatidylcholine was modestly decreased, lung phospholipid content and lung function were maintained. Insig1/2(Δ/Δ) mice developed lung inflammation and airspace abnormalities associated with the accumulation of lipids in alveolar type 2 cells, alveolar macrophages, and within alveolar spaces. Deletion of Insig1/2 activated SREBP-enhancing lipogenesis in respiratory epithelial cells resulting in lipotoxicity-related lung inflammation and tissue remodeling.
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Lipogénesis , Proteínas de la Membrana/metabolismo , Neumonía/metabolismo , Alveolos Pulmonares/metabolismo , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo , Animales , Ésteres del Colesterol/genética , Ésteres del Colesterol/metabolismo , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patología , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Neumonía/genética , Neumonía/patología , Alveolos Pulmonares/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Proteínas de Unión a los Elementos Reguladores de Esteroles/genética , Triglicéridos/genética , Triglicéridos/metabolismoRESUMEN
Microtubule assembly is vital for many fundamental cellular processes. Current models for microtubule assembly kinetics assume that the subunit dissociation rate from a microtubule tip is independent of free subunit concentration. Total-Internal-Reflection-Fluorescence (TIRF) microscopy experiments and data from a laser tweezers assay that measures in vitro microtubule assembly with nanometer resolution, provides evidence that the subunit dissociation rate from a microtubule tip increases as the free subunit concentration increases. These data are consistent with a two-dimensional model for microtubule assembly, and are explained by a shift in microtubule tip structure from a relatively blunt shape at low free concentrations to relatively tapered at high free concentrations. We find that because both the association and the dissociation rates increase at higher free subunit concentrations, the kinetics of microtubule assembly are an order-of-magnitude higher than currently estimated in the literature.
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Microtúbulos/metabolismo , Animales , Línea Celular , Cinética , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Biológicos , Porcinos , Tubulina (Proteína)/metabolismoRESUMEN
Stable isotope labelling of lipid precursors coupled with mass spectrometry-based lipidomic analyses and determination of isotope enrichment in substrate, intermediate and product pools provide the parameters needed to determine absolute flux rates through lipid pathways in vivo. Here, as an illustration of the power of such analyses we investigated lung phosphatidylcholine (PC) synthesis in Surfactant Protein-D (SP-D) null mice. These animals develop emphysema, foamy alveolar macrophages and an alveolar lipoproteinosis with increasing age. We used the incorporation of methyl-9-[(2)H] choline chloride coupled with ESI-MS/MS to quantify absolute rates of lung surfactant PC synthesis and secretion in an SP-D(-/-) mouse model, together with an analysis of the molecular specificity of lung PC synthesis. PC synthetic rates were comparable in control (0.52 µmol/lung/h) and SP-D(-/-) (0.69 µmol/lung/h) mice, as were rates of surfactant PC secretion (29.8 and 30.6 nmol/lung/h, respectively). Increased lung PC in the SP-D(-/-) mouse was due to impaired catabolism, with a rate of accumulation of 0.057 µmol/lung/h. The relatively low rates of surfactant PC secretion compared with total lung PC synthesis were compatible with a suggested ABCA1-mediated basolateral lipid efflux from alveolar type II epithelial cells. Finally, PC molecular species analysis suggested that a proportion of newly synthesised PC is secreted rapidly into the lung air spaces in both control and SP-D(-/-) mice before significant PC acyl remodelling occurs.
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Fosfatidilcolinas/metabolismo , Surfactantes Pulmonares/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem , Animales , Líquido del Lavado Bronquioalveolar , Femenino , Marcaje Isotópico , Masculino , Ratones , Ratones Transgénicos , Fosfatidilcolinas/biosíntesis , Fosfatidilcolinas/química , Alveolos Pulmonares/metabolismo , Proteína D Asociada a Surfactante Pulmonar/deficiencia , Proteína D Asociada a Surfactante Pulmonar/genética , Surfactantes Pulmonares/química , Eliminación de SecuenciaRESUMEN
During cell division, chromosomes must faithfully segregate to maintain genome integrity, and this dynamic mechanical process is driven by the macromolecular machinery of the mitotic spindle. However, little is known about spindle mechanics. For example, spindle microtubules are organized by numerous cross-linking proteins yet the mechanical properties of those cross-links remain unexplored. To examine the mechanical properties of microtubule cross-links we applied optical trapping to mitotic asters that form in mammalian mitotic extracts. These asters are foci of microtubules, motors, and microtubule-associated proteins that reflect many of the functional properties of spindle poles and represent centrosome-independent spindle-pole analogs. We observed bidirectional motor-driven microtubule movements, showing that microtubule linkages within asters are remarkably compliant (mean stiffness 0.025 pN/nm) and mediated by only a handful of cross-links. Depleting the motor Eg5 reduced this stiffness, indicating that Eg5 contributes to the mechanical properties of microtubule asters in a manner consistent with its localization to spindle poles in cells. We propose that compliant linkages among microtubules provide a mechanical architecture capable of accommodating microtubule movements and distributing force among microtubules without loss of pole integrity-a mechanical paradigm that may be important throughout the spindle.
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Mitosis , Huso Acromático/metabolismo , Fenómenos Biomecánicos , Células HeLa , Humanos , Microtúbulos/metabolismo , Modelos BiológicosRESUMEN
Many stem cells divide asymmetrically to balance self-renewal and differentiation. In Drosophila testes, two stem cell populations, germline stem cells (GSCs) and somatic cyst stem cells (CySCs), cohere and regulate one another. Here, we report that CySCs divide asymmetrically through repositioning the mitotic spindle around anaphase. CySC spindle repositioning requires functional centrosomes, Dynein and the actin-membrane linker Moesin. Anaphase spindle repositioning is required to achieve high-fidelity asymmetric divisions in CySCs, thus maintaining both GSC and CySC numbers. We propose that dynamic spindle repositioning allows CySCs to divide asymmetrically while accommodating the structure of the GSCs they encapsulate.
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División Celular , Huso Acromático/metabolismo , Células Madre/citología , Testículo/citología , Anafase , Animales , Centrosoma , Drosophila , Dineínas , Células Germinativas , Masculino , Proteínas de Microfilamentos , Huso Acromático/fisiologíaRESUMEN
Single femtosecond pulsed laser damage can be confined radially to regions smaller than the focus spot size due to the highly nonlinear mechanisms for energy absorption and ablation in transparent dielectrics. Along the propagation axis, however, we show that channels can be machined much deeper than the Rayleigh range of the laser focus. Using focused ion beam cross sections and acetate imprints, we analyze these channels and show that spherical aberration is not the primary source for this elongated damage, which is likely caused by microscale filamentation.
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Rayos Láser , Nanoestructuras , Nanotecnología/métodos , Pulso ArterialRESUMEN
Microtubule (MT) polymerization dynamics, which are crucial to eukaryotic life and are the target of important anticancer agents, result from the addition and loss of 8-nm-long tubulin-dimer subunits. Addition and loss of one or a few subunits cannot be observed at the spatiotemporal resolution of conventional microscopy, and requires development of approaches with higher resolution. Here we describe an assay in which one end of an MT abuts a barrier, and MT length changes are coupled to the movement of an optically trapped bead, the motion of which is tracked with high resolution. We detail assay execution, including preparation of the experimental chamber and orientation of the MT against the barrier. We describe design requirements for the experimental apparatus and barriers, and preparation of materials including stable, biotinylated MT seeds from which growth is initiated and NeutrAvidin-coated beads. Finally, we discuss advantages of moving the optical trap such that it applies a constant force (force clamping), detection limits, the importance of high temporal resolution, data analysis, and potential sources of experimental artifacts.
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Microtecnología/instrumentación , Microtecnología/métodos , Microtúbulos/química , Microtúbulos/metabolismo , Pinzas Ópticas , Multimerización de Proteína , Animales , Técnicas de Laboratorio Clínico , Diseño de Equipo/instrumentación , Diseño de Equipo/métodos , Falla de Equipo , Humanos , Límite de Detección , Nanoestructuras/análisis , Nanoestructuras/química , Pinzas Ópticas/estadística & datos numéricosRESUMEN
Nanofluidic devices make use of molecular-level forces and phenomena to increase their density, speed and accuracy. However, fabrication is challenging, because dissimilar materials need to be integrated in three dimensions with nanoscale precision. Here, we report a three-dimensional nanoscale liquid glass electrode made from monolithic substrates without conductive materials by femtosecond-laser nanomachining. The electrode consists of a nanochannel terminating at a nanoscale glass tip that becomes a conductor in the presence of high electric fields through dielectric breakdown, and returns to being an insulator when this field is removed. This reversibility relies on control of nanoampere breakdown currents and extremely fast heat dissipation at nanoscale volumes. We use the nanoscale liquid glass electrode to fabricate a nano-injector that includes an electrokinetic pump, 4 microm across with 0.6 microm channels, which is capable of producing well-controlled flow rates below 1 fl s(-1). The electrode can be integrated easily into other nanodevices and fluidic systems, including actuators and sensors.
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Vidrio/química , Nanoestructuras/química , Nanotecnología , Técnicas Electroquímicas , Electrodos , Eritrocitos , Humanos , Técnicas Analíticas Microfluídicas , Nanoestructuras/ultraestructura , Nanotecnología/instrumentación , Nanotecnología/métodosRESUMEN
ATP-binding cassette A3 (ABCA3) is a lipid transport protein required for synthesis and storage of pulmonary surfactant in type II cells in the alveoli. Abca3 was conditionally deleted in respiratory epithelial cells (Abca3(Δ/Δ)) in vivo. The majority of mice in which Abca3 was deleted in alveolar type II cells died shortly after birth from respiratory distress related to surfactant deficiency. Approximately 30% of the Abca3(Δ/Δ) mice survived after birth. Surviving Abca3(Δ/Δ) mice developed emphysema in the absence of significant pulmonary inflammation. Staining of lung tissue and mRNA isolated from alveolar type II cells demonstrated that â¼50% of alveolar type II cells lacked ABCA3. Phospholipid content and composition were altered in lung tissue, lamellar bodies, and bronchoalveolar lavage fluid from adult Abca3(Δ/Δ) mice. In adult Abca3(Δ/Δ) mice, cells lacking ABCA3 had decreased expression of mRNAs associated with lipid synthesis and transport. FOXA2 and CCAAT enhancer-binding protein-α, transcription factors known to regulate genes regulating lung lipid metabolism, were markedly decreased in cells lacking ABCA3. Deletion of Abca3 disrupted surfactant lipid synthesis in a cell-autonomous manner. Compensatory surfactant synthesis was initiated in ABCA3-sufficient type II cells, indicating that surfactant homeostasis is a highly regulated process that includes sensing and coregulation among alveolar type II cells.