Association of clusterin with the BRI2-derived amyloid molecules ABri and ADan

Familial British and Danish dementias (FBD and FDD) share striking neuropathological similarities with Alzheimer's disease (AD), including intraneuronal neurofibrillary tangles as well as parenchymal and vascular amyloid deposits. Multiple amyloid associated proteins with still controversial role in amyloidogenesis colocalize with the structurally different amyloid peptides ABri in FBD, ADan in FDD, and Aβ in AD. Genetic variants and plasma levels of one of these associated proteins, clusterin, have been identified as risk factors for AD. Clusterin is known to bind soluble Aβ in biological fluids, facilitate its brain clearance, and prevent its aggregation. The current work identifies clusterin as the major ABri- and ADan-binding protein and provides insight into the biochemical mechanisms leading to the association of clusterin with ABri and ADan deposits. Mirroring findings in AD, the studies corroborate clusterin co-localization with cerebral parenchymal and vascular amyloid deposits in both disorders. Ligand affinity chromatography with downstream Western blot and amino acid sequence analyses unequivocally identified clusterin as the major ABri- and ADan-binding plasma protein. ELISA highlighted a specific saturable binding of clusterin to ABri and ADan with low nanomolar Kd values within the same range as those previously demonstrated for the clusterin-Aβ interaction. Consistent with its chaperone activity, thioflavin T binding assays clearly showed a modulatory effect of clusterin on ABri and ADan aggregation/fibrillization properties. Our findings, together with the known multifunctional activity of clusterin and its modulatory activity on the complex cellular pathways leading to oxidative stress, mitochondrial dysfunction, and the induction of cell death mechanisms – all known pathogenic features of these protein folding disorders – suggests the likelihood of a more complex role and a translational potential for the apolipoprotein in the amelioration/prevention of these pathogenic mechanisms

Ternary combination of irinotecan, fluorouracil-folinic acid and oxaliplatin: results on human colon cancer cell lines

A marked antitumour efficacy is currently obtained by oxaliplatin (LOHP)–fluorouracil (FU)–folinic acid (FA) combination and by CPT11–FU–FA combination. Logically, the triple association LOHP, CPT11 and FUFA will be soon tested in cancer patients. The aim of the present study was to compare two schedules combining SN38 (the active metabolite of CPT11, irinotecan) with FU–FA and LOHP. The two schedules differed by the SN38 position. The relative contribution of each drug in the resulting global cytotoxicity was evaluated. Two human colon cancer cell lines were used (WIDR and SW620 both p53 mutated). LOHP plus FA were applied for 2 h, just before a 48 h FU exposure. The SN38 sequence was applied for 24 h, starting either 48 h before LOHP-FA (schedule A), or just after LOHP-FA exposure (schedule B). Cytotoxicity was assessed by the 3-(4,5-demethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) test and drug interactions were analysed according to the Chou and Talalay method, based on the computation of a combination index (CI). The SN38 position significantly induces a shift from additivity-antagonism when SN38 was applied after LOHP, towards additivity-synergism when SN38 was applied first (P = 0.03). The relative contribution (RC) of each drug in the overall cytotoxicity of the triple combination was defined as the drug concentration giving 50% cell lethality (IC 50) of the double association without that drug divided by the IC 50 of the triple association. Whatever the SN38 position, the larger contribution was made by LOHP (median RC = 2.4) and the smaller by SN38 (median RC = 1.1). In addition, the contribution of FUFA was improved when SN38 was applied first (median RC = 2.2) as compared to the opposite schedule (median RC = 1.2). Results were in agreement between the two explored cell lines. The present data should be taken into account when establishing the rationale of future trials combining CPT11, LOHP and FU–FA. © 2001 Cancer Research Campaign http://www.bjcancer.co

Combining eutectic solvents and pressurized liquid extraction coupled in-line with solid-phase extraction to recover, purify and stabilize anthocyanins from Brazilian berry waste

Pressurized techniques are straightforward for high-scale applications and highly controllable, which seems an excellent strategy for recovering unstable natural compounds. In this work, the main advance was the development of a platform based on the pressurized liquid extraction coupled in-line with a solid-phase extraction step (PLE-SPE) combined with the use of eutectic mixtures as solvents to promote an efficient extraction and purification of natural pigments from food wastes. Eutectic mixtures, conventionally known as (deep) eutectic solvents – (D)ES, are combinations of two or more substances with a lower melting point than any of their components. (D)ES are often referred as “green solvents” because they can potentially be more environmentally friendly than other solvents, especially volatile organic solvents (VOSs). Overall, (D)ES have the potential to contribute to the achievement of several of the SDGs (especially 3, 13, and 14) through their positive impacts on health, environment, and sustainable production and consumption practices. Thus, in this work, (D)ES were used as solvents to valorize Brazilian berry waste (Plinia cauliflora). Anthocyanins are the biomass's main compounds of commercial interest, mainly for food and cosmetic applications. However, there are several technological issues regarding color control due to their high sensitivity to light, heat, oxygen, and pH variations. Thus, the data achieved in this work highlighted the high efficiency and low environmental footprint of the PLE-SPE-(D)ES platform developed. The success of the downstream process here developed was proved by the high extraction efficiency and the purity level of the anthocyanins obtained. Besides, thermal stability analysis was evaluated, demonstrating that (D)ES are not only solvents but also stabilizing agents, improving the shelf-life of the extracted colorants.publishe

New proposal for production of bioactive compounds by supercritical technology integrated to a sugarcane biorefinery

The construction of a supercritical fluid extraction (SFE) plant inside or in close proximity to a sugarcane biorefinery producing first and second generation ethanol demonstrated to be very promising, increasing the economic potential of the SFE process in up to 57 %, since the SFE plant could use directly the ethanol, CO2, heat, and electricity already available, with lower prices. In this study, Brazilian ginseng roots were used as model bioactive compounds source and first the statistical influence of the extraction conditions including pressure (10-20 MPa), temperature (323-363 K), and CO2/ethanol proportion ratio (90:10, 50:50, and 0:100 %, w/w) on the beta-ecdysone content in the extracts was experimentally evaluated and compared with literature results. SFE process evaluated experimentally at the present study showed higher selective extraction for beta-ecdysone from Brazilian ginseng roots, providing an extract with up to 2.16 times higher beta-ecdysone than the results obtained in previous studies. Thermal integration of the SFE process diminished energy requirements of the process, resulting in a reduction of cold utility requirement of 87 % and a final electricity demand of 7.5 kWh/g of beta-ecdysone in the extract. In a situation in which the Brazilian ginseng roots price was increased to 4.71 USD/g, only the SFE integrated with the biorefinery solution would be economically feasible. Finally, the selling of the ginseng roots leftover could be an interesting answer to increase the economical attractiveness of the integrated SFE process to the biorefinery