A Chemical Proteomics Approach for the Search of Pharmacological Targets of the Antimalarial Clinical Candidate Albitiazolium in Plasmodium falciparum Using Photocrosslinking and Click Chemistry

Plasmodium falciparum is responsible for severe malaria which is one of the most prevalent and deadly infectious diseases in the world. The antimalarial therapeutic arsenal is hampered by the onset of resistance to all known pharmacological classes of compounds, so new drugs with novel mechanisms of action are critically needed. Albitiazolium is a clinical antimalarial candidate from a series of choline analogs designed to inhibit plasmodial phospholipid metabolism. Here we developed an original chemical proteomic approach to identify parasite proteins targeted by albitiazolium during their native interaction in living parasites. We designed a bifunctional albitiazolium-derived compound (photoactivable and clickable) to covalently crosslink drug–interacting parasite proteins in situ followed by their isolation via click chemistry reactions. Mass spectrometry analysis of drug–interacting proteins and subsequent clustering on gene ontology terms revealed parasite proteins involved in lipid metabolic activities and, interestingly, also in lipid binding, transport, and vesicular transport functions. In accordance with this, the albitiazolium-derivative was localized in the endoplasmic reticulum and trans-Golgi network of P. falciparum. Importantly, during competitive assays with albitiazolium, the binding of choline/ethanolamine phosphotransferase (the enzyme involved in the last step of phosphatidylcholine synthesis) was substantially displaced, thus confirming the efficiency of this strategy for searching albitiazolium targets.     

Salt Content Impacts Food Preferences and Intake among Children

Decreasing dietary sodium intake, which can be achieved by reducing salt content in food, is recommended. Salt contributes to the taste of foods and makes them more enjoyable. Whether a food is liked or disliked is an important determinant of food intake, especially among children. However, the role of salt in children''s food acceptance has received little attention. The impact of salt content on children''s hedonic rating and intake of two foods was investigated in children. Using a within-subject crossover design, we recruited 75 children (8–11 years) to participate in five lunches in their school cafeteria. The target foods were green beans and pasta. The added salt content was 0, 0.6 or 1.2 g/100 g. The children''s intake (g) of all lunch items was measured. The children provided their hedonic rating of the food, a preference ranking and a saltiness ranking in the laboratory. Children could rank the foods according to salt content, and they preferred the two saltier options. A food-specific effect of salt content on intake was observed. Compared to the intermediate level (0.6 g salt/100 g), not adding salt decreased green bean intake (−21%; p = 0.002), and increasing the salt content increased pasta intake (+24%; p<0.0001). Structural Equation Modeling was used to model the relative weights of the determinants of intake. It showed that the primary driver of food intake was the child''s hunger; the second most important factor was the child''s hedonic rating of the food, regardless of its salt content, and the last factor was the child''s preference for the particular salt content of the food. In conclusion, salt content has a positive and food-specific effect on intake; it impacted food preferences and intake differently in children. Taking into account children''s preferences for salt instead of their intake may lead to excessive added salt.     

Using sensitivity analysis to identify keystone species and keystone links in size‐based food webs

Human‐induced alterations in the birth and mortality rates of species and in the strength of interactions within and between species can lead to changes in the structure and resilience of ecological communities. Recent research points to the importance of considering the distribution of body sizes of species when exploring the response of communities to such perturbations. Here, we present a new size‐based approach for assessing the sensitivity and elasticity of community structure (species equilibrium abundances) and resilience (rate of return to equilibrium) to changes in the intrinsic growth rate of species and in the strengths of species interactions. We apply this approach on two natural systems, the pelagic communities of the Baltic Sea and Lake Vättern, to illustrate how it can be used to identify potential keystone species and keystone links. We find that the keystone status of a species is closely linked to its body size. The analysis also suggests that communities are structurally and dynamically more sensitive to changes in the effects of prey on their consumers than in the effects of consumers on their prey. Moreover, we discuss how community sensitivity analysis can be used to study and compare the fragility of communities with different body size distributions by measuring the mean sensitivity or elasticity over all species or all interaction links in a community. We believe that the community sensitivity analysis developed here holds some promise for identifying species and links that are critical for the structural and dynamic robustness of ecological communities.     

Characterization of protein spin labeling by maleimide: evidence for nitroxide reduction

A quantitative determination of maleimide spin label (MAL) binding in oxi and met hemoglobin (Hb) and bovine serum albumin are investigated using double integration to the ESR signal. This determination permitted the observation that a considerable fraction of MAL is reduced, losing its paramagnetism. Experiments using the same spin label with myoglobin and Hb with blocked-SH groups, where reduction was not observed, indicate the involvement of SH groups in the process. The 4-hydroxy-2,2,6,6-tetramethylpiperidino-1-oxyl spin label (which is not able to bind in the SH group) is reduced too, but the dependence on the molar ratio is different in comparison with the MAL case. In both cases the reduction percentage depends on the molar ratio spin label to protein and to the protein concentration. In order to obtain the total SH groups labeled (two in the Hb case) it is necessary to use an excessive amount of label (around 18:1) in the 0.5 mM Hb concentration.     

Reverse enzyme synthesis in microemulsion-based organo-gels.

Lipase from three different sources has been immobilised in microemulsion-based gels (MBGs) with retention of catalytic activity. Such lipase-containing MBGs prove to be novel solid-phase catalysts for use in apolar organic solvents such as n-heptane. Using these systems, preparative-scale synthesis of a wide variety of esters under mild conditions was possible with products easily isolated and obtained in high yield. Stereoselective esterification of octan-2-ol was observed for all three lipases with Chromobacterium viscosum (CV) lipase yielding product with an enantiomeric excess of 92%. Repeated usage of a CV lipase-containing MBG resulted in a visually unchanged gel whose activity was 75% of the initial value after 30 days. The sectioned MBGs were well suited for use in column flow reactors and were also found to be effective esterification catalysts at temperatures as low as -20 degrees C.     

Mouse model systems of autism spectrum disorder: Replicability and informatics signature

Phenotyping mouse model systems of human disease has proven to be a difficult task, with frequent poor inter‐ and intra‐laboratory replicability, particularly in behavioral domains such as social and cognitive function. However, establishing robust animal model systems with strong construct validity is of fundamental importance as they are central tools for understanding disease pathophysiology and developing therapeutics. To complete our studies of mouse model systems relevant to autism spectrum disorder (ASD), we present a replication of the main findings from our two published studies of five genetic mouse model systems of ASD. To assess the intra‐laboratory robustness of previous results, we chose the two model systems that showed the greatest phenotypic differences, the Shank3/F and Cntnap2, and repeated assessments of general health, activity and social behavior. We additionally explored all five model systems in the same framework, comparing all results obtained in this three‐yearlong effort using informatics techniques to assess commonalities and differences. Our results showed high intra‐laboratory replicability of results, even for those with effect sizes that were not particularly large, suggesting that discrepancies in the literature may be dependent on subtle but pivotal differences in testing conditions, housing enrichment, or background strains and less so on the variability of the behavioral phenotypes. The overall informatics analysis suggests that in our behavioral assays we can separate the set of tested mouse model system into two main classes that in some aspects lie on opposite ends of the behavioral spectrum, supporting the view that autism is not a unitary concept.     

Involvement of adenosine A1 and A2A receptors on guanosine-mediated anti-tremor effects in reserpinized mice

Parkinson’s disease (PD) signs and symptoms regularly include tremor. Interestingly, the nucleoside guanosine (GUO) has already proven to be effective in reducing reserpine-induced tremulous jaw movements (TJMs) in rodent models, thus becoming a promising antiparkinsonian drug. Here, we aimed at revealing the mechanism behind GUO antiparkinsonian efficacy by assessing the role of adenosine A₁ and A_2A receptors (A₁R and A_2AR) on GUO-mediated anti-tremor effects in the reserpinized mouse model of PD. Reserpinized mice showed elevated reactive oxygen species (ROS) production and cellular membrane damage in striatal slices assessed ex vivo and GUO treatment reversed ROS production. Interestingly, while the simultaneous administration of sub-effective doses of GUO (5 mg/kg) and SCH58261 (0.01 mg/kg), an A_2AR antagonist, precluded reserpine-induced TJMs, these were ineffective on reverting ROS production in ex vivo experiments. Importantly, GUO was able to reduce TJM and ROS production in reserpinized mouse lacking the A_2AR, thus suggesting an A_2AR-independent mechanism of GUO-mediated effects. Conversely, the administration of DPCPX (0.75 mg/kg), an A₁R antagonist, completely abolished both GUO-mediated anti-tremor effects and blockade of ROS production. Overall, these results indicated that GUO anti-tremor and antioxidant effects in reserpinized mice were A₁R dependent but A_2AR independent, thus suggesting a differential participation of adenosine receptors in GUO-mediated effects.

     

Reactive oxygen species play a role in P2X7 receptor-mediated IL-6 production in spinal astrocytes

Purinergic Signalling - Astrocytes mediate a remarkable variety of cellular functions, including gliotransmitter release. Under pathological conditions, high concentrations of the purinergic...     

Extracellular vesicle‐mediated crosstalk between NPCE cells and TM cells result in modulation of Wnt signalling pathway and ECM remodelling

Primary open‐angle glaucoma is a leading cause of irreversible blindness, often associated with increased intraocular pressure. Extracellular vesicles (EVs) carry a specific composition of proteins, lipids and nucleotides have been considered as essential mediators of cell‐cell communication. Their potential impact for crosstalk between tissues responsible for aqueous humour production and out‐flow is largely unknown. The study objective was to investigate the effects of EVs derived from non‐pigmented ciliary epithelium (NPCE) primary cells on the expression of Wnt proteins in a human primary trabecular meshwork (TM) cells and define the mechanism underlying exosome‐mediated regulation that signalling pathway. Consistent with the results in TM cell line, EVs released by both primary NPCE cells and NPCE cell line showed diminished pGSK3β phosphorylation and decreased cytosolic levels of β‐catenin in primary TM cells. At the molecular level, we showed that NPCE exosome treatment downregulated the expression of positive GSKβ regulator‐AKT protein but increased the levels of GSKβ negative regulator‐PP2A protein in TM cells. NPCE exosome protein analysis revealed 584 miRNAs and 182 proteins involved in the regulation of TM cellular processes, including WNT/β‐catenin signalling pathway, cell adhesion and extracellular matrix deposition. We found that negative modulator of Wnt signalling miR‐29b was abundant in NPCE exosomal samples and treatment of TM cells with NPCE EVs significantly decreased COL3A1 expression. Suggesting that miR‐29b can be responsible for decreased levels of WNT/β‐catenin pathway. Overall, this study highlights a potential role of EVs derived from NPCE cells in modulating ECM proteins and TM canonical Wnt signalling.