The yeast Pichia pastoris is a cost-effective and easily scalable system for recombinant protein production. In this work we compared the conformation of the receptor binding domain (RBD) from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Spike protein expressed in P. pastoris and in the well established HEK-293T mammalian cell system. RBD obtained from both yeast and mammalian cells was properly folded, as indicated by UV-absorption, circular dichroism and tryptophan fluorescence. They also had similar stability, as indicated by temperature-induced unfolding (observed Tm were 50 °C and 52 °C for RBD produced in P. pastoris and HEK-293T cells, respectively). Moreover, the stability of both variants was similarly reduced when the ionic strength was increased, in agreement with a computational analysis predicting that a set of ionic interactions may stabilize RBD structure. Further characterization by high-performance liquid chromatography, size-exclusion chromatography and mass spectrometry revealed a higher heterogeneity of RBD expressed in P. pastoris relative to that produced in HEK-293T cells, which disappeared after enzymatic removal of glycans. The production of RBD in P. pastoris was scaled-up in a bioreactor, with yields above 45 mg/L of 90% pure protein, thus potentially allowing large scale immunizations to produce neutralizing antibodies, as well as the large scale production of serological tests for SARS-CoV-2.
We present the development of the first procedure for hair cortisol measurement through an automated method. Hair samples were obtained from 286 individuals. After cortisol extraction, samples were measured in a Siemens Immulite 2000 (Gwynedd, UK) automated chemoluminiscent immunoassay analyzer. Normal reference values were obtained from hair cortisol levels measured in 213 healthy individuals with low levels of stress. Hair cortisol concentration median was 55 pg/mg hair (2.5–97.5 percentile (40–128)) in healthy individuals with low levels of stress and 250 pg/mg hair (range 182–520) in stressed individuals. No significant differences were observed in hair cortisol levels between subjects with and without dye (40 (40–107) and 40 (40–155) pg/mg hair, respectively; p = 0.128). The novel procedure presented here shows an adequate analytical performance.
Anxiety disorders, depression and pain are highly prevalent pathologies. Their pharmacotherapy is associated with unwanted side effects; hence there is a clinical need to develop more effective drugs with fewer adverse reactions.Chalcones are one of the major classes of naturally occurring compounds. Chalcones and their derivatives have a huge importance in medicinal chemistry, displaying a wide range of pharmacological activities including anti-inflammatory, antimicrobial, antioxidant, cytotoxic and antitumor actions.The aim of this work was to evaluate chalcone effects on different targets involved in these pathologies. We have synthesized a series of simple chalcone derivatives taking common structural requirements described in literature related to their anxiolytic-like, antidepressant-like and/or antinociceptive properties into account.Furthermore, their potential in vitro effects towards different targets involved in these pathologies were evaluated. We have obtained twenty chalcones with moderate to high yields and assessed their ability to bind distinctive receptors, from rat brain homogenates, by displacement of labelled specific ligands: [3H] FNZ (binding site of benzodiazepines/GABAA), [3H] 8-OH-DPAT (serotonin 5-HT1A) and [3H] DAMGO (μ-opioid). Those compounds that showed the better in vitro activities were evaluated in mice using different behavioural tasks. In vivo results showed that 5′-methyl-2′-hydroxychalcone (9) exerted anxiolytic-like effects in mice in the plus maze test. While chalcone nuclei (1) revealed antidepressant-like activities in the tail suspension test. In addition, the novel 5′-methyl-2′-hydroxy-3′-nitrochalcone (12) exhibited antinociceptive activity in acute chemical and thermal nociception tests (writhing and hot plate tests). In conclusion, chalcones are thus promising compounds for the development of novel drugs with central nervous system (CNS) actions.
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