FRET-Based Detection of M1 Muscarinic Acetylcholine Receptor Activation by Orthosteric and Allosteric Agonists

Muscarinic acetylcholine receptors (mAChRs) are 7-transmembrane, G protein-coupled receptors that regulate a variety of physiological processes and represent potentially important targets for therapeutic intervention. mAChRs can be stimulated by full and partial orthosteric and allosteric agonists, however the relative abilities of such ligands to induce conformational changes in the receptor remain unclear. To gain further insight into the actions of mAChR agonists, we have developed a fluorescently tagged M(1) mAChR that reports ligand-induced conformational changes in real-time by changes in Förster resonance energy transfer (FRET).Variants of CFP and YFP were inserted into the third intracellular loop and at the end of the C-terminus of the mouse M(1) mAChR, respectively. The optimized FRET receptor construct (M(1)-cam5) was expressed stably in HEK293 cells.The variant CFP/YFP-receptor chimera expressed predominantly at the plasma membrane of HEK293 cells and displayed ligand-binding affinities comparable with those of the wild-type receptor. It also retained an ability to interact with Gα(q/11) proteins and to stimulate phosphoinositide turnover, ERK1/2 phosphorylation and undergo agonist-dependent internalization. Addition of the full agonist methacholine caused a reversible decrease in M(1) FRET (F(EYFP)/F(ECFP)) that was prevented by atropine pre-addition and showed concentration-dependent amplitude and kinetics. Partial orthosteric agonists, arecoline and pilocarpine, as well as allosteric agonists, AC-42 and 77-LH-28-1, also caused atropine-sensitive decreases in the FRET signal, which were smaller in amplitude and significantly slower in onset compared to those evoked by methacholine.The M(1) FRET-based receptor chimera reports that allosteric and orthosteric agonists induce similar conformational changes in the third intracellular loop and/or C-terminus, and should prove to be a valuable molecular reagent for pharmacological and structural investigations of M(1) mAChR activation

Preparation of Microcrystals of Piroxicam Monohydrate by Antisolvent Precipitation via Microfabricated Metallic Membranes with Ordered Pore Arrays

Microcrystals of piroxicam (PRX) monohydrate with a narrow size distribution were prepared from acetone/PRX solutions by antisolvent crystallization via metallic membranes with ordered pore arrays. Crystallization was achieved by controlled addition of the feed solution through the membrane pores into a well-stirred antisolvent. A complete transformation of an anhydrous form I into a monohydrate form of PRX was confirmed by Raman spectroscopy and differential scanning calorimetry. The size of the crystals was 7–34 μm and was controlled by the PRX concentration in the feed solution (15–25 g L¯¹), antisolvent/solvent volume ratio (5–30), and type of antisolvent (Milli-Q water or 0.1–0.5 wt % aqueous solutions of hydroxypropyl methyl cellulose (HPMC), poly(vinyl alcohol) or Pluronic P-123). The smallest crystals were obtained by injecting 25 g L¯¹ PRX solution through a stainless-steel membrane with a pore size of 10 μm into a 0.06 wt % HPMC solution stirred at 1500 rpm using an antisolvent/solvent ratio of 20. HPMC provided better steric stabilization of microcrystals against agglomeration than poly(vinyl alcohol) and Pluronic P-123, due to hydrogen bonding interactions with PRX and water. A continuous production of large PRX monohydrate microcrystals with a volume-weighted mean diameter above 75 μm was achieved in a continuous stirred membrane crystallizer. Rapid pouring of Milli-Q water into the feed solution resulted in a mixture of highly polydispersed prism-shaped and needle-shaped crystals

Microfluidic systems for the analysis of the viscoelastic fluid flow phenomena in porous media

In this study, two microfluidic devices are proposed as simplified 1-D microfluidic analogues of a porous medium. The objectives are twofold: firstly to assess the usefulness of the microchannels to mimic the porous medium in a controlled and simplified manner, and secondly to obtain a better insight about the flow characteristics of viscoelastic fluids flowing through a packed bed. For these purposes, flow visualizations and pressure drop measurements are conducted with Newtonian and viscoelastic fluids. The 1-D microfluidic analogues of porous medium consisted of microchannels with a sequence of contractions/ expansions disposed in symmetric and asymmetric arrangements. The real porous medium is in reality, a complex combination of the two arrangements of particles simulated with the microchannels, which can be considered as limiting ideal configurations. The results show that both configurations are able to mimic well the pressure drop variation with flow rate for Newtonian fluids. However, due to the intrinsic differences in the deformation rate profiles associated with each microgeometry, the symmetric configuration is more suitable for studying the flow of viscoelastic fluids at low De values, while the asymmetric configuration provides better results at high De values. In this way, both microgeometries seem to be complementary and could be interesting tools to obtain a better insight about the flow of viscoelastic fluids through a porous medium. Such model systems could be very interesting to use in polymer-flood processes for enhanced oil recovery, for instance, as a tool for selecting the most suitable viscoelastic fluid to be used in a specific formation. The selection of the fluid properties of a detergent for cleaning oil contaminated soil, sand, and in general, any porous material, is another possible application

Behavioral differences in an over‐invasion scenario: marbled vs. spiny‐cheek crayfish

New species often invade ecosystems already dominated by previous invaders. Ornamental freshwater crayfish, particularly parthenogenetic marbled crayfish (Procambarus virginalis), increasingly establish in European water bodies where they interact with resident native and non‐native species. Behavioral traits and behavioral syndromes can influence the outcome of these species interactions. The behavior of non‐native crayfish is often studied in notorious invaders but rarely in new and emerging species, although those provide the best opportunity for management. Activity, aggressiveness, and boldness have repeatedly been associated with invasion success and species displacement. Further, crayfish can adapt their behavior after they have established in the new range. We investigated whether marbled crayfish can displace the widely established spiny‐cheek crayfish (Orconectes limosus). Specifically, we compared their behavioral traits and evaluated whether these traits differ, using marbled crayfish populations from aquaria and the field and spiny‐cheek crayfish from the field. We staged agonistic encounters, measured activity levels, and recorded the response to a simulated threat of both species and both origins (field and aquarium) in laboratory trials. We found that in agonistic encounters, marbled crayfish were on average more aggressive than spiny‐cheek crayfish, even against larger opponents. Aggressiveness and activity were positively correlated, which is indicative for an aggression syndrome. Marbled crayfish from the field were less active than those from aquaria, but there was no difference in aggressiveness. Marbled crayfish often froze in response to a simulated threat, whereas spiny‐cheek crayfish reacted either offensively or defensively. These results from the laboratory illustrate potentially important behavioral mechanisms behind crayfish over‐invasions and show behavioral plasticity in a species where all known individuals are genetically identical. To better understand the invasion process in nature, the species’ reproductive biology and interactions with other members of the community should be considered. We conclude that the recent success of marbled crayfish in establishing new populations could be influenced by their behavioral flexibility and their potential to competitively persist in the presence of established invasive crayfish

Emotional behavior in aquatic organisms? Lessons from crayfish and zebrafish

Experimental animal models are a valuable tool to study the neurobiology of emotional behavior and mechanisms underlying human affective disorders. Mounting evidence suggests that various aquatic organisms, including both vertebrate (e.g., zebrafish) and invertebrate (e.g., crayfish) species, may be relevant to study animal emotional response and its deficits. Ideally, model organisms of disease should possess considerable genetic and physiological homology to mammals, display robust behavioral and physiological responses to stress, and should be sensitive to a wide range of drugs known to modulate stress and affective behaviors. Here, we summarize recent findings in the field of zebrafish- and crayfish-based tests of stress, anxiety, aggressiveness and social preference, and discuss further perspectives of using these novel model organisms in translational biological psychiatry. Outlining the remaining questions in this field, we also emphasize the need in further development and a wider use of crayfish and zebrafish models to study the pathogenesis of affective disorders. © 2019 Wiley Periodicals, Inc.MCS is currently supported by National Funds through FCT ‐ Foundation for Science and Technology. AVK is supported by the Russian Science Foundation grant 19‐15‐00053. KAD is supported by the Fellowship of the President of Russia and SPSU Rector Productivity Fellowship for PhD Students. CM is supported by CNPq/Brazil under Edital Universal 2016 (400726/2016‐5). PMA and FB are supported by the strategic plan of MARE ‐ Marine and Environmental Sciences Centre (UID/MAR/04292/2019)

The activity of signal crayfish (Pacifastacus leniusculus) in relation to thermal and hydraulic dynamics of an alluvial stream, UK

Signal crayfish (Pacifastacus leniusculus) are an invasive species of global significance because of their detrimental impacts on freshwater environments and native organisms. The movement of signal crayfish was continuously monitored for 150-days through a 20-m reach of an alluvial stream in the UK. Passive integrated transponder-tags were attached to crayfish, allowing their location to be monitored relative to 16 antennae which were buried beneath the river bed. The activity of crayfish was related to water depth and temperature, which were continuously monitored within the instrumented reach. Crayfish were highly nocturnal, with less than 6% of movements recorded during daylight hours. Activity declined from September and was minimal in November when water temperature was low and flow depth was high. However, relations between environmental parameters and crayfish activity had poor explanatory power which may partly reflect biological processes not accounted for in this study. Water depth and temperature had a limiting relationship with crayfish activity, quantified using quantile regression. The results extend existing data on signal crayfish nocturnalism and demonstrate that, although signal crayfish can tolerate a range of flows, activity becomes limited as water temperature declines seasonally and when water depth remains high in autumn and winter months