Maitotoxin-induced membrane blebbing and cell death in bovine aortic endothelial cells

BACKGROUND: Maitotoxin, a potent cytolytic agent, causes an increase in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) via activation of Ca(2+)-permeable, non-selective cation channels (CaNSC). Channel activation is followed by formation of large endogenous pores that allow ethidium and propidium-based vital dyes to enter the cell. Although activation of these cytolytic/oncotic pores, or COP, precedes release of lactate dehydrogenase, an indication of oncotic cell death, the relationship between CaNSC, COP, membrane lysis, and the associated changes in cell morphology has not been clearly defined. In the present study, the effect maitotoxin on [Ca(2+)](i), vital dye uptake, lactate dehydrogenase release, and membrane blebbing was examined in bovine aortic endothelial cells. RESULTS: Maitotoxin produced a concentration-dependent increase in [Ca(2+)](i) followed by a biphasic uptake of ethidium. Comparison of ethidium (M(w) 314 Da), YO-PRO-1 (M(w) 375 Da), and POPO-3 (M(w) 715 Da) showed that the rate of dye uptake during the first phase was inversely proportional to molecular weight, whereas the second phase appeared to be all-or-nothing. The second phase of dye uptake correlated in time with the release of lactate dehydrogenase. Uptake of vital dyes at the single cell level, determined by time-lapse videomicroscopy, was also biphasic. The first phase was associated with formation of small membrane blebs, whereas the second phase was associated with dramatic bleb dilation. CONCLUSIONS: These results suggest that maitotoxin-induced Ca(2+) influx in bovine aortic endothelial cells is followed by activation of COP. COP formation is associated with controlled membrane blebbing which ultimately gives rise to uncontrolled bleb dilation, lactate dehydrogenase release, and oncotic cell death

Blockade of maitotoxin-induced oncotic cell death reveals zeiosis

BACKGROUND: Maitotoxin (MTX) initiates cell death by sequentially activating 1) Ca(2+) influx via non-selective cation channels, 2) uptake of vital dyes via formation of large pores, and 3) release of lactate dehydrogenase, an indication of cell lysis. MTX also causes formation of membrane blebs, which dramatically dilate during the cytolysis phase. To determine the role of phospholipase C (PLC) in the cell death cascade, U73122, a specific inhibitor of PLC, and U73343, an inactive analog, were examined on MTX-induced responses in bovine aortic endothelial cells. RESULTS: Addition of either U73122 or U73343, prior to MTX, produced a concentration-dependent inhibition of the cell death cascade (IC(50) ≈ 1.9 and 0.66 μM, respectively) suggesting that the effect of these agents was independent of PLC. Addition of U73343 shortly after MTX, prevented or attenuated the effects of the toxin, but addition at later times had little or no effect. Time-lapse videomicroscopy showed that U73343 dramatically altered the blebbing profile of MTX-treated cells. Specifically, U73343 blocked bleb dilation and converted the initial blebbing event into "zeiosis", a type of membrane blebbing commonly associated with apoptosis. Cells challenged with MTX and rescued by subsequent addition of U73343, showed enhanced caspase-3 activity 48 hr after the initial insult, consistent with activation of the apoptotic program. CONCLUSIONS: Within minutes of MTX addition, endothelial cells die by oncosis. Rescue by addition of U73343 shortly after MTX showed that a small percentage of cells are destined to die by oncosis, but that a larger percentage survive; cells that survive the initial insult exhibit zeiosis and may ultimately die by apoptotic mechanisms

Refuerzo de subrasante con aplicación de geomalla – sistema viario 1 – PK 2+700 AL PK 3+120 – Mirones bajos – Lima – 2018

Este estudio demostró que el uso de geomalla para el refuerzo de subrasante es una técnica factible, para eso tomamos como área de estudio 420 metros del Sistema Viario 1 entre las progresivas PK 2+700 al PK 3+120 – Mirones Bajos, por medio de calicatas se pudo analizar las características presentadas por el terreno, donde se obtuvieron CBR menores al 6% era necesario plantear un mejoramiento. Con este tipo de refuerzo, se logró disminuir el tamaño de la capa de la subrasante para lo cual se utilizó el software desarrollado por tensar SpectraPave 4 Pro en la obtención del grosor mínimo requerido. En ese sentido, se simularon varios escenarios dentro de los cuales sin geomalla se obtuvo un resultado de 0.53 m, con la geomalla BX 1100 se obtuvo un espesor de mejoramiento de 0.37 m lo que representa un 30% de reducción, aplicando la BX 1200 obtenemos un espesor de mejoramiento de 0.24 m lo que representa un 55% y para la geomalla Triaxial TX 140 presenta un espesor de mejoramiento de 0.25 m equivalente a un 53 % de reducción y aplicando la geomalla Triaxial TX 160 presenta un espesor de mejoramiento de 0.15 m equivalente a un 72 % de reducción. Los geosintéticos vienen demostrando que su uso, en diferentes trabajos de ingeniería es de mucha utilidad ya que permite generar un refuerzo estructural a los suelos con una fácil y rápida instalación

Estrategias narrativas implementadas en el spot publicitario digital "Pizza con piña" en Facebook, como parte de la campaña "Amistad Sin Diferencias" de Pilsen, para generar inclusión en temáticas LGBT

La innovación de estrategias de marketing en el Perú encuentra diversificación respecto a las diversas temáticas sociales inscritas en el contenido publicitario. El gay advertising o publicidad LGBT empieza a ocupar espacio en la malla de contenido y comunicación de marcas que lideran el mercado peruano. Pilsen Callao tomó la iniciativa de transformarse renovando su identidad de marca, buscando generar conciencia sobre equidad de género a través de la campaña “Amistad Sin Diferencias”. Esta campaña presentó el concepto de amistad desde la perspectiva del respeto y la igualdad. Debido al auge de la creación de marcas que compiten en las mismas categorías, nace la necesidad de estrategias de marketing que permitan generar afinidad con el consumidor. Uno de los métodos más conocidos es el storytelling, dicha técnica narrativa permite desarrollar la identidad de una marca, que tiene como objetivo crear empatía y llamar la atención del consumidor. Ante este contexto, el storytelling, se emplea como táctica publicitaria para abarcar diversos segmentos y nichos de mercado. En base a ello, el presente trabajo de investigación académica tiene como objetivo analizar dichas herramientas empleadas —permitiendo de este modo renovar su discurso— en la campaña digital "Amistad Sin Diferencias" de Pilsen efectuada en la plataforma Facebook. El desarrollo de la investigación se lleva a cabo mediante una metodología cualitativa, basada en la revisión de bibliografía y entrevistas realizadas a especialistas del rubro publicitario.The innovation of marketing strategies in Peru finds diversification with respect to the various social themes inscribed in the advertising content. Gay advertising or LGBT advertising begins to occupy space in the content and communication mesh of brands that lead the Peruvian market. Pilsen Callao took the initiative to transform itself by renewing its brand identity, seeking to raise awareness about gender equality through the “Friendship Without Differences” campaign. This campaign presented the concept of friendship from the perspective of respect and equality. Due to the rise in the creation of brands that compete in the same categories, the need arises for marketing strategies that allow generating affinity with the consumer. One of the best-known methods is storytelling, this narrative technique allows developing the identity of a brand, which aims to create empathy and attract the attention of the consumer. In this context, storytelling is used as an advertising tactic to cover various market segments and niches. Based on this, the present academic research work aims to identify these tools used - thus allowing them to renew their discourse - in Pilsen's "Friendship Without Differences" digital campaign carried out on the Facebook platform. The development of the research is carried out through a qualitative methodology, based on the literature review and interviews with specialists in the advertising field.Tesi

Estrategias narrativas implementadas en el spot publicitario digital "Pizza con piña" en Facebook, como parte de la campaña "Amistad Sin Diferencias" de Pilsen, para generar inclusión en temáticas LGBT

La innovación de estrategias de marketing en el Perú encuentra diversificación respecto a las diversas temáticas sociales inscritas en el contenido publicitario. El gay advertising o publicidad LGBT empieza a ocupar espacio en la malla de contenido y comunicación de marcas que lideran el mercado peruano. Pilsen Callao tomó la iniciativa de transformarse renovando su identidad de marca, buscando generar conciencia sobre equidad de género a través de la campaña “Amistad Sin Diferencias”. Esta campaña presentó el concepto de amistad desde la perspectiva del respeto y la igualdad. Debido al auge de la creación de marcas que compiten en las mismas categorías, nace la necesidad de estrategias de marketing que permitan generar afinidad con el consumidor. Uno de los métodos más conocidos es el storytelling, dicha técnica narrativa permite desarrollar la identidad de una marca, que tiene como objetivo crear empatía y llamar la atención del consumidor. Ante este contexto, el storytelling, se emplea como táctica publicitaria para abarcar diversos segmentos y nichos de mercado. En base a ello, el presente trabajo de investigación académica tiene como objetivo analizar dichas herramientas empleadas —permitiendo de este modo renovar su discurso— en la campaña digital "Amistad Sin Diferencias" de Pilsen efectuada en la plataforma Facebook. El desarrollo de la investigación se lleva a cabo mediante una metodología cualitativa, basada en la revisión de bibliografía y entrevistas realizadas a especialistas del rubro publicitario.The innovation of marketing strategies in Peru finds diversification with respect to the various social themes inscribed in the advertising content. Gay advertising or LGBT advertising begins to occupy space in the content and communication mesh of brands that lead the Peruvian market. Pilsen Callao took the initiative to transform itself by renewing its brand identity, seeking to raise awareness about gender equality through the “Friendship Without Differences” campaign. This campaign presented the concept of friendship from the perspective of respect and equality. Due to the rise in the creation of brands that compete in the same categories, the need arises for marketing strategies that allow generating affinity with the consumer. One of the best-known methods is storytelling, this narrative technique allows developing the identity of a brand, which aims to create empathy and attract the attention of the consumer. In this context, storytelling is used as an advertising tactic to cover various market segments and niches. Based on this, the present academic research work aims to identify these tools used - thus allowing them to renew their discourse - in Pilsen's "Friendship Without Differences" digital campaign carried out on the Facebook platform. The development of the research is carried out through a qualitative methodology, based on the literature review and interviews with specialists in the advertising field.Trabajo de investigació

Inhibition of native TRPC6 channel activity by phosphatidylinositol 4,5-bisphosphate in mesenteric artery myocytes

The present work investigates the effect of phosphatidylinositol-4,5-bisphosphate (PIP2) on native TRPC6 channel activity in freshly dispersed rabbit mesenteric artery myocytes using patch clamp recording and co-immunoprecipitation methods. Inclusion of 100 μm diC8-PIP2 in the patch pipette and bathing solutions, respectively, inhibited angiotensin II (Ang II)-evoked whole-cell cation currents and TRPC6 channel activity by over 90%. In inside-out patches diC8-PIP2 also inhibited TRPC6 activity induced by the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) with an IC50 of 7.6 μm. Anti-PIP2 antibodies potentiated Ang II- and OAG-evoked TRPC6 activity by about 2-fold. Depleters of tissue PIP2 wortmannin and LY294002 stimulated TRPC6 activity, as did the polycation PIP2 scavenger poly-l-lysine. Wortmannin reduced Ang II-evoked TRPC6 activity by over 75% but increased OAG-induced TRPC6 activity by over 50-fold. Co-immunoprecipitation studies demonstrated association between PIP2 and TRPC6 proteins in tissue lysates. Pre-treatment with Ang II, OAG and wortmannin reduced TRPC6 association with PIP2. These results provide for the first time compelling evidence that constitutively produced PIP2 exerts a powerful inhibitory action on native TRPC6 channels

Paroxysmal extreme pain disorder M1627K mutation in human Nav1.7 renders DRG neurons hyperexcitable

Background: Paroxysmal extreme pain disorder (PEPD) is an autosomal dominant painful neuropathy with many, but not all, cases linked to gain-of-function mutations in SCN9A which encodes voltage-gated sodium channel Nav1.7. Severe pain episodes and skin flushing start in infancy and are induced by perianal probing or bowl movement, and pain progresses to ocular and mandibular areas with age. Carbamazepine has been effective in relieving symptoms, while other drugs including other anti-epileptics are less effective. Results: Sequencing of SCN9A coding exons from an English patient, diagnosed with PEPD, has identified a methionine 1627 to lysine (M1627K) substitution in the linker joining segments S4 and S5 in domain IV. We confirm that M1627K depolarizes the voltage-dependence of fast-inactivation without substantially altering activation or slow-inactivation, and inactivates from the open state with slower kinetics. We show here that M1627K does not alter development of closed-state inactivation, and that M1627K channels recover from fast-inactivation faster than wild type channels, and produce larger currents in response to a slow ramp stimulus. Using current-clamp recordings, we also show that the M1627K mutant channel reduces the threshold for single action potentials in DRG neurons and increases the number of action potentials in response to graded stimuli. Conclusion: M1627K mutation was previously identified in a sporadic case of PEPD from France, and we now report it in an English family. We confirm the initial characterization of mutant M1627K effect on fast-inactivation of Nav1.7 and extend the analysis to other gating properties of the channel. We also show that M1627K mutant channels render DRG neurons hyperexcitable. Our new data provide a link between altered channel biophysics and pain in PEPD patients

Characterization of a de novo SCN8A mutation in a patient with epileptic encephalopathy

Objective Recently, de novo SCN8A missense mutations have been identified as a rare dominant cause of epileptic encephalopathies. Functional studies on the first described case demonstrated gain-of-function effects of the mutation. We describe a novel de novo mutation of SCN8A in a patient with epileptic encephalopathy, and functional characterization of the mutant protein. Design Whole exome sequencing was used to discover the variant. We generated a mutant cDNA, transfected HEK293 cells, and performed Western blotting to assess protein stability. To study channel functional properties, patch-clamp experiments were carried out in transfected neuronal ND7/23 cells. Results The proband exhibited seizure onset at 6 months of age, diffuse brain atrophy, and more profound developmental impairment than the original case. The mutation p.Arg233Gly in the voltage sensing transmembrane segment D1S4 was present in the proband and absent in both parents. This mutation results in a temperature-sensitive reduction in protein expression as well as reduced sodium current amplitude and density and a relative increased response to a slow ramp stimulus, though this did not result in an absolute increased current at physiological temperatures. Conclusion The new de novo SCN8A mutation is clearly deleterious, resulting in an unstable protein with reduced channel activity. This differs from the gain-of-function attributes of the first SCN8A mutation in epileptic encephalopathy, pointing to heterogeneity of mechanisms. Since Nav1.6 is expressed in both excitatory and inhibitory neurons, a differential effect of a loss-of-function of Nav1.6 Arg223Gly on inhibitory interneurons may underlie the epilepsy phenotype in this patient