Analysis of Acetylcholinesterase Activity in Cell Membrane Microarrays of Brain Areas as a Screening Tool to Identify Tissue Specific Inhibitors

Acetylcholinesterase (AChE) is responsible for hydrolyzing the acetylcholine neurotransmitter, bringing an end point to cholinergic neurotransmission. Thus, AChE is the primary target of a wide spectrum of compounds used as pesticides, nerve agents or therapeutic drugs for neurodegenerative diseases such as Alzheimer’s disease (AD). This enzyme is heterogeneously distributed in the brain showing different activity depending on the nervous region. Therefore, the aim of this work is to report a novel technology that enables the simultaneous determination of tissue specific AChE activity, as well as the analysis and screening of specific inhibitors, by using cell membrane microarrays. These microarrays were composed of cell membranes, isolated from 41 tissues, organs and brain areas, that were immobilized over a slide, maintaining the functionality of membrane proteins. To validate this platform, demonstrating its usefulness in drug discovery as a high throughput screening tool, a colorimetric protocol to detect the membrane-bound AChE activity was optimized. Thus, rat cortical and striatal AChE activities were estimated in presence of increased concentrations of AChE inhibitors, and the donepezil effect was assessed simultaneously in 41 tissues and organs, demonstrating the major potential of this microarray’s technology.The Spanish Ministry of Economy and Competitiveness (Innpacto program: IPT-2011-1205-010000), and the Basque Government Department of Economic Development, sustainability and environment (Etorgai program: ER-2011/00015, Bikaintek program: 48-AF-W2-2019-7)

Acute Stress-Induced Changes in the Lipid Composition of Cow’s Milk in Healthy and Pathological Animals

Producers of milk and dairy products have been faced with the challenge of responding to European society’s demand for guaranteed animal welfare production. In recent years, measures have been taken to improve animal welfare conditions on farms and evaluation systems have been developed to certify them, such as the Welfare Quality® protocol. Among the markers used for this purpose, acute phase proteins stand out, with haptoglobin being one of the most relevant. However, the diagnostic power of these tools is limited and more sensitive and specific technologies are required to monitor animal health status. Different factors such as diet, stress, and diseases modify the metabolism of the animals, altering the composition of the milk in terms of oligosaccharides, proteins, and lipids. Thus, in order to study oxidative-stress-associated lipids, a collection of well-characterized milk samples, both by veterinary diagnosis and by content of the acute stress biomarker haptoglobin, was analyzed by mass spectrometry and artificial intelligence. Two lipid species (sphingomyelin and phosphatidylcholine) were identified as potential biomarkers of health status in dairy cows. Both lipids allow for the discrimination of milk from sick animals and also milk from those with stress. Moreover, lipidomics revealed specific lipid profiles depending on the origin of the samples and the degree of freedom of the animals on the farm. These data provide evidence for specific lipid changes in stressed animals and open up the possibility that haptoglobin could also affect lipid metabolism in cow’s milk.This work is partially supported by the Basque Government, Bikaintek #010-B2/2021, and the Spanish Innovation Agency, AIE, Doctorados Industriales, #DIN2020-011349

Characterization of the Antitumor Potential of Extracts of Cannabis sativa Strains with High CBD Content in Human Neuroblastoma

Cannabis has been used for decades as a palliative therapy in the treatment of cancer. This is because of its beneficial effects on the pain and nausea that patients can experience as a result of chemo/radiotherapy. Tetrahydrocannabinol and cannabidiol are the main compounds present in Cannabis sativa, and both exert their actions through a receptor-mediated mechanism and through a non-receptor-mediated mechanism, which modulates the formation of reactive oxygen species. These oxidative stress conditions might trigger lipidic changes, which would compromise cell membrane stability and viability. In this sense, numerous pieces of evidence describe a potential antitumor effect of cannabinoid compounds in different types of cancer, although controversial results limit their implementation. In order to further investigate the possible mechanism involved in the antitumoral effects of cannabinoids, three extracts isolated from Cannabis sativa strains with high cannabidiol content were analyzed. Cell mortality, cytochrome c oxidase activity and the lipid composition of SH-SY5Y cells were determined in the absence and presence of specific cannabinoid ligands, with and without antioxidant pre-treatment. The cell mortality induced by the extracts in this study appeared to be related to the inhibition of the cytochrome c oxidase activity and to the THC concentration. This effect on cell viability was similar to that observed with the cannabinoid agonist WIN55,212-2. The effect was partially blocked by the selective CB1 antagonist AM281, and the antioxidant α-tocopherol. Moreover, certain membrane lipids were affected by the extracts, which demonstrated the importance of oxidative stress in the potential antitumoral effects of cannabinoids.This work has been partially supported by a grant from the Ministry of Economy and Competitiveness (DIN2019-010902 and DIN2020-011349) and the Basque Government Department of Economic Development, Sustainability and Environment (Bikaintek program: 005-B2/2021)

Integrity of the Mod(mdg4)-67.2 BTB Domain Is Critical to Insulator Function in Drosophila melanogaster

The Drosophila gypsy insulator contains binding sites for the Suppressor of Hairy-wing [Su(Hw)] protein. Enhancer and silencer blocking require Su(Hw) recruitment of Mod(mdg4)-67.2, a BTB/POZ domain protein that interacts with Su(Hw) through a carboxyl-terminal acidic domain. Here we conducted mutational analyses of the Mod(mdg4)-67.2 BTB domain. We demonstrate that this domain is essential for insulator function, in part through direction of protein dimerization. Our studies revealed the presence of a second domain (DD) that contributes to Mod(mdg4)-67.2 dimerization when the function of the BTB domain is compromised. Additionally, we demonstrate that mutations in amino acids of the charged pocket in the BTB domain that retain dimerization of the mutated protein cause a loss of insulator function. In these cases, the mutant proteins failed to localize to chromosomes, suggesting a role for the BTB domain in chromosome association. Interestingly, replacement of the Mod(mdg4)-67.2 BTB domain with the GAF BTB domain produced a nonfunctional protein. Taken together, these data suggest that the Mod(mdg4)-67.2 BTB domain confers novel activities to gypsy insulator function