GLUT2 regulation of p38 MAPK isoform protein expression and p38 phosphorylation in male versus female rat hypothalamic primary astrocyte Cultures.

Recent studies documented regulation of hypothalamic astrocyte mitogen-activated protein kinase (MAPK) pathways, including p38, by the plasma membrane glucose carrier/sensor glucose transporter-2 (GLUT2). Sex-specific GLUT2 control of p38 phosphorylation was observed, but effects on individual p38 family protein profiles were not investigated. Current research employed an established primary astrocyte culture model, gene knockdown tools, and selective primary antisera against p38-alpha, p38-beta, p38-gamma, and p38-delta isoforms to investigate whether GLUT2 governs expression of one or more of these variants in a glucose-dependent manner. Data show that GLUT2 inhibits baseline expression of each p38 protein in male cultures, yet stimulates p38-delta profiles without affecting other p38 proteins in female. Glucose starvation caused selective up-regulation of p38-delta profiles in male versus p38-alpha and -gamma proteins in female; these positive responses were amplified by GLUT2 siRNA pretreatment. GLUT2 opposes or enhances basal p38 phosphorylation in male versus female, respectively. GLUT2 siRNA pretreatment did not affect glucoprivic patterns of phospho-p38 protein expression in either sex. Outcomes document co-expression of the four principal p38 MAPK family proteins in hypothalamic astrocytes, and implicate GLUT2 in regulation of all (male) versus one (female) variant(s). Glucoprivation up-regulated expression of distinctive p38 isoforms in each sex; these stimulatory responses are evidently blunted by GLUT2. Glucoprivic-associated loss of GLUT2 gene silencing effects on p38 phosphorylation infers either that glucose status determines whether this sensor controls phosphorylation, or that decrements in screened glucose in each instance are of sufficient magnitude to abolish GLUT2 regulation of that function.

Sex-dimorphic effects of glucose transporter-2 gene knockdown on hypothalamic primary astrocyte phosphoinositide-3-kinase (PI3K)/protein kinase B (PKB/Akt)/mammalian target of rapamycin (mTOR) cascade protein expression and phosphorylation.

Glucose transporter-2 (GLUT2), a unique high capacity/low affinity, highly efficient membrane transporter and sensor, regulates hypothalamic astrocyte glucose phosphorylation and glycogen metabolism. The phosphoinositide-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway participates in glucose homeostasis, but its sensitivity to glucose-sensory cues is unknown. Current research used a hypothalamic astrocyte primary culture model to investigate whether glucoprivation causes PI3K/Akt/mTOR pathway activation in one or both sexes by GLUT2-dependent mechanisms. Glucoprivation did not alter astrocyte PI3K levels, yet up-regulated both phosphorylated derivatives in female and down-regulated male p60 phosphoprotein expression. GLUT2 siRNA pretreatment diminished glucoprivic patterns of PI3K and phospho-PI3K expression in each sex. Astrocyte Akt and phospho-Akt/Thr308 proteins exhibited divergent, sex-contingent responses to GLUT2 gene knockdown or glucoprivation. GLUT2 siRNA pretreatment exacerbated glucoprivic-associated Akt diminution in the female, and either amplified (male) or reversed (female) glucoprivic regulation of phospho-Akt/Thr308 expression. GLUT2 gene silencing down- (male) or up-(female) regulated mTOR protein, and phospho-mTOR protein in male. Male astrocyte mTOR and phospho-mTOR profile were refractory to glucoprivation, but glucose-deprived females showed GLUT2-independent mTOR inhibition and GLUT2-dependent phospho-mTOR up-augmentation. Results identify a larger number of glucoprivic-sensitive PI3K/Akt/mTOR pathway proteins in female versus male astrocytes, and document divergent responses of common glucose-sensitive targets. GLUT2 stimulates phosphoPI3K protein expression in each sex, but imposes differential control of PI3K, Akt, phospho-Akt/Thr308, mTOR, and phospho-mTOR profiles in male versus female. Data implicate GLUT2 as a driver of distinctive pathway protein responses to glucoprivation in female, but not male.

U-CAN-seq: A Universal Competition Assay by Nanopore Sequencing.

RNA viruses quickly evolve subtle genotypic changes that can have major impacts on viral fitness and host range, with potential consequences for human health. It is therefore important to understand the evolutionary fitness of novel viral variants relative to well-studied genotypes of epidemic viruses. Competition assays are an effective and rigorous system with which to assess the relative fitness of viral genotypes. However, it is challenging to quickly and cheaply distinguish and quantify fitness differences between very similar viral genotypes. Here, we describe a protocol for using reverse transcription PCR in combination with commercial nanopore sequencing services to perform competition assays on untagged RNA viruses. Our assay, called the Universal Competition Assay by Nanopore Sequencing (U-CAN-seq), is relatively cheap and highly sensitive. We used a well-studied N24A mutation in the chikungunya virus (CHIKV) nsp3 gene to confirm that we could detect a competitive disadvantage using U-CAN-seq. We also used this approach to show that mutations to the CHIKV 5' conserved sequence element that disrupt sequence but not structure did not affect the fitness of CHIKV. However, similar mutations to an adjacent CHIKV stem loop (SL3) did cause a fitness disadvantage compared to wild-type CHIKV, suggesting that structure-independent, primary sequence determinants in this loop play an important role in CHIKV biology. Our novel findings illustrate the utility of the U-CAN-seq competition assay.

Posibles beneficios médicos de los tocotrienoles de la palma en la prevención y tratamiento del cáncer de mama / Potential health benefits of palm tocotrienols in the prevention and treatment of breast cancer

Tocoferoles y tocotrienoles representan los dos subgrupos en la familia de compuestos de la vitamina E, pero sólo los tocotrienoles presentan fuerte actividad anticancerosa en dosis que tienen muy poco o ningún efecto en el desarrollo o la función normales de las células. Los tocotrienoles son fuertes antioxidantes, pero la actividad antitumoral es independiente de la actividad antioxidante. No es del todo clara la razón por la que los tocotrienoles son más potentes que los tocoferoles, pero al menos eso se debe (en parte) a una mayor acumulación celular. Además, estudios realizados acerca de dosis-respuesta indican que las dosis de tocotrienoles inhibidoras del desarrollo son de 5 a 6 veces menores que sus correspondientes dosis letales, lo cual sugiere que en los efectos antiproliferantes y citotóxicos de los tocotrienoles intervienen diferentes mecanismos. Estudios recientes indican que la muerte celular programada inducida por tocotrienoles (apoptosis) se debe a la activación de proteasas cisteínicas intracelulares específicas (caspasas) asociadas con activación del receptor de muerte y la transducción de señales. Además, los inhibidores específicos de caspasa en tratamiento combinado bloquean los efectos citotóxicos de los tocotrienoles en las células epiteliales mamarias malignas. Por el contrario, la inhibición de muerte celular provocada por tocotrienol parece incluir la supresión de hormonas y de las vías de señalamiento mitógeno del factor-receptor de desarrollo. Aunque es preciso realizar estudios complementarios para esclarecer los mecanismos intracelulares que intervienen en los efectos anticancerosos de los tocotrienoles, la evidencia experimental sugiere firmemente que el suplemento dietético con tocotrienoles puede ofrecer beneficios médicos significativos, ya que disminuye en las mujeres el riesgo de cáncer de mama...

Tocopherols and tocotrienol represent the two subgroups within the vitamin E family of compounds, but only tocotrienols display potent anticancer activity at doses that have no effect on normal cell growth or function. Tocotrienols are potent antioxidants, but antitumor activity is independent of antioxidant activity. The exact reason why tocotrienols are more potent than tocopherols is not completely understood, but at least part of the reason is because of greater cellular accumulation. Furthermore, dose response studies show that growth inhibitory doses of tocotrienols are 5-6 times lower than their corresponding lethal doses, suggesting that the antiproliferative and cytotoxic effects of tocotrienols are mediated by different mechanisms. Recent studies show that tocotrienol-induced programmed cell death (apoptosis) results from the activation of specific intracellular cysteine proteases (caspases) associated with death receptor activation and signal transduction. Furthermore, combined treatment specific caspase inhibitors block the cytotoxic effects of tocotrienols in malignant mammary epithelial cells. In contrast, tocotrienol inhibition of cell death appears to involve the suppression of hormone - and growth factor-receptor mitogenic signaling pathways. Although additional studies are required to clarify the intracellular mechanisms mediating the anticancer effects of tocotrienols, experimental evidence strongly suggests that dietary supplementation of tocotrienols may provide significant health benefits in lowering the risk of breast cancer in women...