Bioelectronic Measurement of Target Engagement to a Membrane-Bound Transporter.

The ability to detect and characterize drug binding to a target protein is of high priority in drug discovery research. However, there are inherent challenges when the target of interest is an integral membrane protein (IMP). Assuming successful purification of the IMP, traditional approaches for measuring binding such as surface plasmon resonance (SPR) and fluorescence resonance energy transfer (FRET) have been proven valuable. However, the mass dependence of SPR signals may preclude the detection of binding events when the ligand has a significantly smaller mass than the target protein. In FRET-based experiments, protein labeling through modification may inadvertently alter protein dynamics. Graphene Bio-Electronic Sensing Technology (GBEST) aims to overcome these challenges. Label-free characterization takes place in a microfluidic chamber wherein a fluid lipid membrane is reconstituted directly above the GBEST sensor surface. By leveraging the high conductivity, sensitivity, and electrical properties of monolayer graphene, minute changes in electrostatic charges arising from the binding and unbinding of a ligand to a native IMP target can be detected in real time and in a mass-independent manner. Using crude membrane fractions prepared from cells overexpressing monocarboxylate transporter 1 (MCT1), we demonstrate the ability to (1) form a fluid lipid bilayer enriched with MCT1 directly on top of the GBEST sensor and (2) obtain kinetic binding data for an anti-MCT1 antibody. Further development of this novel technology will enable characterization of target engagement by both low- and high-molecular-weight drug candidates to native IMP targets in a physiologically relevant membrane environment.

Development and Evaluation of Surfactant Nanocapsules for Chemical Enhanced Oil Recovery (EOR) Applications.

The primary objective of this study is the synthesis of nanocapsules (NC) that allow the reduction of the adsorption process of surfactant over the porous media in enhanced oil recovery processes. Nanocapsules were synthesized through the nanoprecipitation method by encapsulating commercial surfactants Span 20 and Petro 50, and using type II resins isolated from vacuum residue as a shell. The NC were characterized using dynamic light scattering, transmission electron microscopy, Fourier transform infrared, solvency tests, softening point measurements and entrapment efficiency. The obtained NC showed spherical geometry with sizes of 71 and 120 nm for encapsulated Span 20 (NCS20), and Petro 50 surfactant (NCP50), respectively. Also, the NCS20 is composed of 90% of surfactant and 10% of type II resins, while the NCP50 material is 94% of surfactant and 6% of the shell. Nanofluids of nanocapsules dispersed in deionized water were prepared for evaluating the nanofluid­sandstone interaction from adsorption phenomena using a batch-mode method, contact angle measurements, and FTIR analysis. The results showed that NC adsorption was null at the different conditions of temperatures evaluated of 25, 50, and 70 °C, and stirring velocities up to 10,000 rpm. IFT measurements showed a reduction from 18 to 1.62 and 0.15 mN/m for the nanofluids with 10 mg/L of NCS20, and NCP50 materials, respectively. Displacements tests were conducted using a 20 °API crude oil in a quarter five-spot pattern micromodel and showed an additional oil recovery of 23% in comparison with that of waterflooding, with fewer pore volumes injected than when using a dissolved surfactant.

Más allá de la victimización de niñas y niños en contextos de conflicto armado: potenciales para la construcción de paz / Beyond Victimizing Children in Contexts of Armed Conflict: Peace-Building Potencials

Resumen El artículo presenta la revisión de estudios correspondiente a una de las fases del proyecto de investigación en el cual se enmarca, que buscó aproximarse al estado del arte del conocimiento producido en Colombia entre 2002 y 2012 sobre la identidad y subjetividad de niños y niñas en torno a la paz y la democracia, en contextos de conflicto armado. Se presenta la afectación de sus derechos en dichos contextos desde las áreas: existencia, desarrollo, participación y protección. Se aporta una lectura crítica a la mirada tradicional centrada en la vulneración de derechos y el posicionamiento de los niños y niñas como víctimas, invitando a identificar los potenciales individuales y colectivos con las que cuentan ellos, ellas y sus familias.

Abstract The article presents the review of studies corresponding to one of the phases of the research project in which it is framed, which sought to approximate the state of the art of knowledge produced in Colombia between 2002 and 2012 on the identity and subjectivity of children around to peace and democracy in contexts of armed conflict. Children and youth rights are affected in these contexts from the areas of existence, development, protection andparticipation. The study provides a critical reading of the traditional view focused on the violation of rights and the positioning of children as victims, inviting to identify the individual and collective potentials with which they and their families are provided.

AMOBES (Active Mobility Very Early After Stroke): A Randomized Controlled Trial.

BACKGROUND AND PURPOSE: Intensive physical therapy (PT) facilitates motor recovery when provided during a subacute stage after stroke. The efficiency of very early intensive PT has been less investigated. We aimed to investigate whether intensive PT conducted within the first 2 weeks could aid recovery of motor control. METHODS: This multicentre randomized controlled trial compared soft PT (20-min/d apart from respiratory needs) and intensive PT (idem+45 minutes of intensive exercises/day) initiated within the first 72 hours after a first hemispheric stroke. The primary outcome was change in motor control between day (D) 90 and D0 assessed by the Fugl-Meyer score. Main secondary outcomes were number of days to walking 10 m unassisted, balance, autonomy, quality of life, and unexpected medical events. All analyses were by intent to treat. RESULTS: We could analyze data for 103 of the 104 included patients (51 control and 52 experimental group; 64 males; median age overall 67 [interquartile range 59-77], 67 right hemispheric lesions, 80 ischemic lesions, National Institutes of Health Stroke Scale score ≥8 for 82%). Fugl-Meyer score increased over time (P<0.0001), with no significant effect of treatment (P=0.29) or interaction between treatment and time (P=0.40). The median change in score between D90 and D0 was 27.5 (12-40) and 22.0 (12-56) for control and experimental groups (P=0.69). Similar results were found for the secondary criteria. CONCLUSIONS: Very early after stroke, intensive exercises may not be efficient in improving motor control. This conclusion may apply to mainly severe stroke. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01520636.

Dynamic Reorganization and Correlation among Lipid Raft Components.

Lipid rafts are widely believed to be an essential organizational motif in cell membranes. However, direct evidence for interactions among lipid and/or protein components believed to be associated with rafts is quite limited owing, in part, to the small size and intrinsically dynamic interactions that lead to raft formation. Here, we exploit the single negative charge on the monosialoganglioside GM1, commonly associated with rafts, to create a gradient of GM1 in response to an electric field applied parallel to a patterned supported lipid bilayer. The composition of this gradient is visualized by imaging mass spectrometry using a NanoSIMS. Using this analytical method, added cholesterol and sphingomyelin, both neutral and not themselves displaced by the electric field, are observed to reorganize with GM1. This dynamic reorganization provides direct evidence for an attractive interaction among these raft components into some sort of cluster. At steady state we obtain an estimate for the composition of this cluster.

Colocalization of the ganglioside G(M1) and cholesterol detected by secondary ion mass spectrometry.

The characterization of the lateral organization of components in biological membranes and the evolution of this arrangement in response to external triggers remain a major challenge. The concept of lipid rafts is widely invoked; however, direct evidence of the existence of these ephemeral entities remains elusive. We report here the use of secondary ion mass spectrometry (SIMS) to image the cholesterol-dependent cohesive phase separation of the ganglioside GM1 into nano- and microscale assemblies in a canonical lipid raft composition of lipids. This assembly of domains was interrogated in a model membrane system composed of palmitoyl sphingomyelin (PSM), cholesterol, and an unsaturated lipid (dioleoylphosphatidylcholine, DOPC). Orthogonal isotopic labeling of every lipid bilayer component and monofluorination of GM1 allowed generation of molecule specific images using a NanoSIMS. Simultaneous detection of six different ion species in SIMS, including secondary electrons, was used to generate ion ratio images whose signal intensity values could be correlated to composition through the use of calibration curves from standard samples. Images of this system provide the first direct, molecule specific, visual evidence for the colocalization of cholesterol and GM1 in supported lipid bilayers and further indicate the presence of three compositionally distinct phases: (1) the interdomain region; (2) micrometer-scale domains (d > 3 µm); (3) nanometer-scale domains (d = 100 nm to 1 µm) localized within the micrometer-scale domains and the interdomain region. PSM-rich, nanometer-scale domains prefer to partition within the more ordered, cholesterol-rich/DOPC-poor/GM1-rich micrometer-scale phase, while GM1-rich, nanometer-scale domains prefer to partition within the surrounding, disordered, cholesterol-poor/PSM-rich/DOPC-rich interdomain phase.

Vesicles tethered to microbubbles by hybridized DNA oligonucleotides: flow cytometry analysis of this new drug delivery vehicle design.

Hybridization of complementary lipid-linked DNA oligonucleotides was used to tether small unilamellar vesicles (SUVs) to the lipid monolayer shells of air-microbubbles, a new attachment design for a drug delivery vehicle to be used in tandem with ultrasound imaging. Flow cytometry was used, and a novel analysis was developed, based upon light scattering and fluorescence intensity, to quantify the fraction of microbubbles of chosen size-ranges with oligonucleotide-tethered fluorescently labeled SUVs. Fluorescence microscopy was used to verify that our methodology results in successful high-density SUV tethering to a similar fraction of the microbubbles when compared to the flow cytometry statistics. The fraction of successful tetherings increased with the concentration of the complementary lipid-linked oligonucleotide as expected and decreased with the time that microbubbles were incubated with SUVs, which was not expected. Also unexpected, a large fraction of microbubbles had only background fluorescence levels while a much smaller fraction (at most one-eighth, for the shortest incubation and highest concentration of lipid-linked oligonucleotide) had oligonucleotide-tethered fluorescently labeled SUVs and, according to our fluorescence microscopy, that small fraction was densely covered with SUVs. Ejection of the lipid-linked oligonucleotide during high surface pressure compression of the monolayer shells of actively shrinking microbubbles subjected to the Laplace overpressure is speculated as a qualitative explanation for the statistics.

Microbubbles coated with disaturated lipids and DSPE-PEG2000: phase behavior, collapse transitions, and permeability.

Saturated diacyl (disaturated) phosphatidylcholine (PC) mixed with the lipopolymer distearoylphosphatidylethanolamine (DSPE)-polyethyleneglycol molecular weight 2000 (PEG2000) self-assemble as a monolayer at the air-water interface of air-in-water micrometer-scale bubbles (microbubbles), similar to coatings (shells) on leading medical ultrasound contrast agents (UCAs). This system is characterized here to study the impact of the DSPE-PEG2000 species and PC chain-length on the monolayer coating phase behavior, collapse, shedding, and air transport resistance and microbubble dissolution rate and surface contour. Using fluorescence microscopy of dissolving microbubbles, we found that film microstructure and collapse behavior for all chain lengths (n = 14-20) was indicative of primarily condensed phase monolayers, unlike similar coatings containing polyethyleneglycol 40 stearate (PEG40S) that are either expanded phase or coexisting expanded-condensed phase monolayers. Additionally, we observed a new surface buckling type of behavior with all chain lengths, by bright field microscopy, where the air-water interface continuously appears rough (rather than cyclically rough and smooth), with this behavior most frequently observed for n = 16. In correlating the statistical frequency of this behavior with the monolayer microstructure, we propose that it arises from a slowed nucleation rate of collapse structures at condensed-condensed phase interfaces, not present in systems containing PEG40S. By modeling the dissolution (radius vs time) data, we obtained, for each chain length, the film air transport resistance (R(shell)) that was then fit to a chain-length-dependent energy barrier model. Importantly, the pre-exponential factor was approximately 10 x higher and the microbubbles persisted approximately 4 x longer (from 15 microm at a fixed dissolved oxygen content) in comparison to previously studied films containing PEG40S. We attribute the unique stability properties of microbubble coatings containing DSPE-PEG2000 to the propensity of this molecule to form a condensed-phase monolayer, such that the monolayer coatings approach the properties of one continuous condensed domain.

Long-term stability by lipid coating monodisperse microbubbles formed by a flow-focusing device.

In this letter, the long-term stabilization of monodisperse microbubbles produced by flow focusing is demonstrated using lipid encapsulation. Fluorescence microscopy, high-speed camera imaging, and particle size analysis were used to investigate the roles of lipid phase behavior, dissolution, Ostwald ripening, and coalescence in the stability of microbubbles formed by flow focusing. It was found that these behaviors were controlled through compositional changes with respect to lipid, emulsifier, and viscosity agents. Microbubbles coated with lipid and PEG emulsifier in a viscous solution were found to contain an extremely narrow size distribution (diameter(av) = 51 microm, standard deviation = 4 microm), which was maintained for up to several months.

Simultaneous detection of double-stranded RNA-induced protein kinase and its specific mRNA by single cell analysis.

Fluorescent in situ hybridization was combined with flow cytometry to detect the expression of the double-stranded-RNA-induced protein kinase (PKR) in single cells. Labeled anti-sense oligonucleotide was used to target the specific mRNA while the protein was targeted with an antibody. It was demonstrated that the PKR-mRNA signal could be protected through a lengthy immunostaining procedure. The expression pattern of the PKR-mRNA with respect to DNA content was shown to be comparable to that of 18S ribosomal RNA.

Composición química del pan tradicional e industrial y su aporte de energía y proteína en la población del Noroeste de México / Chemical composition of a typical bread from Northwest of Mexico

El pan elaborado a base de trigo, es uno de los productos de mayor consumo en la dieta de la población mexicana siendo un importante aportador de energía y proteína. En este estudio se determinó la composición proximal y contenido de minerales de pan preparado en forma artesanal e industrial. Asimismo se hizo la comparación sobre el costo por gramo de proteína y de energía en cada uno de los tratamientos. Se analizaron siete tipos de panes (pan blanco: bolillo (T), virginia y de caja; pan dulce: cochita, cuernos, bollos y donas). En la determinación de la composición proximal (humedad, proteína, grasa, ceniza y carbohidratos por diferencia) se utilizó la metodología analítica oficial (AOAC, 1984) y el contenido de minerales (Na, K, Ca, Mg, Fe y Zn) por espectrofotometría de absorción atómica. Basado en encuestas de recordatorio de 24 hrs se calculó el consumo diario de cada tipo de pan y se determinó el porcentaje de la recomendación calórica y proteica cubierta por consumo de pan. Se encontró que existen diferencias significativas (p<0.05) con respecto al contenido de macro y micronutrimentos, entre los panes elaborados por los dos tipos de industria panificadora. También se encontró, en la mayoría de los casos, que el pan blanco y pan dulce de la panadería industrial tienen mayor costo por gramo de proteína y energía que el pan tradicional (p<0.05). El contenido de grasa de algunos panes en ambas panaderías fue superior al establecido en la norma oficial Mexicana. El contenido de Ca y Na fue mayor en el pan industrial mientras que el resto de los minerales (K,Mg, Fe, Zn) fueron mas elevados en el pan tradicional