Enhanced Ca2+ Entry Sustains the Activation of Akt in Glucose Deprived SH-SY5Y Cells

The two crucial cellular insults that take place during cerebral ischemia are the loss of oxygen and loss of glucose, which can both activate a cascade of events leading to neuronal death. In addition, the toxic overactivation of neuronal excitatory receptors, leading to Ca²⁺ overload, may contribute to ischemic neuronal injury. Brain ischemia can be simulated in vitro by oxygen/glucose deprivation, which can be reversible by the re-establishment of physiological conditions. Accordingly, we examined the effects of glucose deprivation on the PI3K/Akt survival signaling pathway and its crosstalk with HIF-1α and Ca²⁺ homeostasis in SH-SY5Y human neuroblastoma cells. It was found that glucose withdrawal decreased HIF-1α protein levels even in the presence of the ischemia-mimicking CoCl_2. On the contrary, and despite neuronal death, we identified a strong activation of the master pro-survival kinase Akt, a finding that was also confirmed by the increased phosphorylation of GSK3, a direct target of p-Akt. Remarkably, the elevated Ca²⁺ influx recorded was found to promptly trigger the activation of Akt, while a re-addition of glucose resulted in rapid restoration of both Ca²⁺ entry and p-Akt levels, highlighting the plasticity of neurons to respond to ischemic challenges and the important role of glucose homeostasis for multiple neurological disorders.     

Aberrant PLN-R14del Protein Interactions Intensify SERCA2a Inhibition,Driving Impaired Ca2+ Handling and Arrhythmogenesis

Phospholamban (PLN), a key modulator of Ca²⁺-homeostasis, inhibits sarcoplasmic reticulum (SR) calcium-ATPase (SERCA2a) and regulates cardiac contractility. The human PLN mutation R14del has been identified in arrhythmogenic cardiomyopathy patients worldwide and is currently extensively investigated. In search of the molecular mechanisms mediating the pathological phenotype, we examined PLN-R14del associations to known PLN-interacting partners. We determined that PLN-R14del interactions to key Ca²⁺-handling proteins SERCA2a and HS-1-associated protein X-1 (HAX-1) were enhanced, indicating the super-inhibition of SERCA2a’s Ca²⁺-affinity. Additionally, histidine-rich calcium binding protein (HRC) binding to SERCA2a was increased, suggesting the inhibition of SERCA2a maximal velocity. As phosphorylation relieves the inhibitory effect of PLN on SERCA2a activity, we examined the impact of phosphorylation on the PLN-R14del/SERCA2a interaction. Contrary to PLN-WT, phosphorylation did not affect PLN-R14del binding to SERCA2a, due to a lack of Ser-16 phosphorylation in PLN-R14del. No changes were observed in the subcellular distribution of PLN-R14del or its co-localization to SERCA2a. However, in silico predictions suggest structural perturbations in PLN-R14del that could impact its binding and function. Our findings reveal for the first time that by increased binding to SERCA2a and HAX-1, PLN-R14del acts as an enhanced inhibitor of SERCA2a, causing a cascade of molecular events contributing to impaired Ca²⁺-homeostasis and arrhythmogenesis. Relieving SERCA2a super-inhibition could offer a promising therapeutic approach for PLN-R14del patients.     

Sensory and chemical profiles of three European olive varieties (Olea europea L); an approach for the characterisation and authentication of the extracted oils

Sensory data (1–16) as described by a trained panel, several characteristics (acidity, peroxide value, extinction coefficients at 232 and 270 nm, stability and phenolic compounds) (17–30) and chemical compositional data (fatty acids, sterols and triterpene diols, aliphatic and triterpene alcohols, and triglycerides) (31–67) were obtained for virgin olive oils of three European olive varieties, Coratina (Italy), Picual (Spain) and Koroneiki (Greece), at a certain stage of maturity—when half of the olives displayed partial or total purple colour—for two consecutive years of harvest, 1995–96 and 1996–97. The most remarkable characteristics for the classification of the oils were extracted using multivariate statistical analysis (correlation, hierarchical clustering and canonical discriminant analysis). Both sensory and/or chemical and quality characteristics were found to have significant potential for the authentication of the virgin olive oil varieties under investigation. © 2000 Society of Chemical Industry     

New Affordable Methods for Large-Scale Isolation of Major Olive Secoiridoids and Systematic Comparative Study of Their Antiproliferative/Cytotoxic Effect on Multiple Cancer Cell Lines of Different Cancer Origins

Olive oil phenols (OOPs) are associated with the prevention of many human cancers. Some of these have been shown to inhibit cell proliferation and induce apoptosis. However, no systematic comparative study exists for all the investigated compounds under the same conditions, due to difficulties in their isolation or synthesis. Herein are presented innovative methods for large-scale selective extraction of six major secoiridoids from olive oil or leaves enabling their detailed investigation. The cytotoxic/antiproliferative bioactivity of these six compounds was evaluated on sixteen human cancer cell lines originating from eight different tissues. Cell viability with half-maximal effective concentrations (EC₅₀) was evaluated after 72 h treatments. Antiproliferative and pro-apoptotic effects were also assessed for the most bioactive compounds (EC₅₀ ≤ 50 μM). Oleocanthal (1) showed the strongest antiproliferative/cytotoxic activity in most cancer cell lines (EC₅₀: 9–20 μM). The relative effectiveness of the six OOPs was: oleocanthal (1) > oleuropein aglycone (3a,b) > ligstroside aglycone (4a,b) > oleacein (2) > oleomissional (6a,b,c) > oleocanthalic acid (7). This is the first detailed study comparing the bioactivity of six OOPs in such a wide array of cancer cell lines, providing a reference for their relative antiproliferative/cytotoxic effect in the investigated cancers.     

N-Acetylcysteine Administration Attenuates Sensorimotor Impairments Following Neonatal Hypoxic-Ischemic Brain Injury in Rats

Hypoxic ischemic (HI) brain injury that occurs during neonatal period has been correlated with severe neuronal damage, behavioral deficits and infant mortality. Previous evidence indicates that N-acetylcysteine (NAC), a compound with antioxidant action, exerts a potential neuroprotective effect in various neurological disorders including injury induced by brain ischemia. The aim of the present study was to investigate the role of NAC as a potential therapeutic agent in a rat model of neonatal HI brain injury and explore its long-term behavioral effects. To this end, NAC (50 mg/kg/dose, i.p.) was administered prior to and instantly after HI, in order to evaluate hippocampal and cerebral cortex damage as well as long-term functional outcome. Immunohistochemistry was used to detect inducible nitric oxide synthase (iNOS) expression. The results revealed that NAC significantly alleviated sensorimotor deficits and this effect was maintained up to adulthood. These improvements in functional outcome were associated with a significant decrease in the severity of brain damage. Moreover, NAC decreased the short-term expression of iNOS, a finding implying that iNOS activity may be suppressed and that through this action NAC may exert its therapeutic action against neonatal HI brain injury.     

New N-(2-carboxybenzyl)chitosan composite scaffolds containing nanoTiO2 or bioactive glass with enhanced cell proliferation for bone-tissue engineering applications

In the present study N-(2-carboxbenzyl)chitosan (CBCS) 3D macroporous hybrid scaffolds with interconnected pore system, containing 0.5, 2.5, and 5?wt% TiO₂ nanoparticles (nTiO₂) and 2.5?wt% Bioglass 45S5 (BG) have been synthesized using freeze-drying technique. Compressive strength values verified that the modification of chitosan combined with the presence of inorganic fillers can attribute significant mechanical stiffness to the scaffold. The in vitro biomineralization test confirmed that all samples were bioinert as mineral deposits were detected with X-ray diffractometry after incubation in SBF. Cytotoxicity and biocompatibility of all scaffolds were tested by using and Wharton’s jelly–derived mesenchymal stem cells (WJ-MSCs) and human embryonic kidney 293 (HEK 293) cell line. Metabolic activity, proliferation, migration, and attachment to the scaffolds were examined. Cells appeared to attach around the superficial pores and migrate in them. Cells also maintained their morphology, proliferated, and migrated across the scaffolds and showed consistent and proved compatibility.     

The effects of changes in the traffic scene during overtaking

Overtaking maneuvers were studied in real traffic, by comparing cases where a change in the opposite traffic occurred during the overtaking maneuver i.e., appearance of an oncoming car, with cases where no change occurred during the maneuver i.e., either an already apparent oncoming car or no oncoming car. In total 45 naturally occurring cases of overtaking were analysed. By examining the time headways (TH) between the overtaking car and the other cars involved, at the end of the maneuver, a significant correlation was found between the TH to opposite traffic and the TH rear to the overtaken car. In cases where a change in the opposite traffic occurred and the proximity to opposite traffic was below a critical TH (<4 s) the appearance of a new-oncoming car caused the overtakers to actively press, both longitudinally and laterally, close to the overtaken car. In cases of overtaking where no change in the opposite traffic occurred, the TH rear to the overtaking vehicle was unaffected by the TH to the opposite traffic. Interestingly, in comparing cases where an oncoming car appeared during overtaking with cases where an oncoming car was apparent from the start of the maneuver both the TH to the opposite traffic and the duration of the maneuver were not found to be significantly different. It is suggested that drivers were probably expecting to be confronted with an oncoming car during the overtaking. However, the decreased available time to disambiguate this situation leads the overtaking driver to limit the rear safety margin of the vehicle being overtaken. The appropriateness of this practice, in terms of safety, remains questionable.