Sesquiterpene lactones from Vernonia cinerascens sch. bip. and their in vitro antitrypanosomal activity

In the endeavor to obtain new antitrypanosomal agents, particularly sesquiterpene lactones, from Kenyan plants of the family Asteraceae, Vernonia cinerascens Sch. Bip. was investigated. Bioactivity-guided fractionation and isolation in conjunction with LC/MS-based dereplication has led to the identification of vernodalol (1) and isolation of vernodalin (2), 11β,13-dihydrovernodalin (3), 11β,13-dihydrovernolide (4), vernolide (5), 11β,13-dihydrohydroxyvernolide (6), hydroxyvernolide (7), and a new germacrolide type sesquiterpene lactone vernocinerascolide (8) from the dichloromethane extract of V. cinerascens leaves. Compounds 3-8 were characterized by extensive analysis of their 1D and 2D NMR spectroscopic and HR/MS spectrometric data. All the compounds were evaluated for their in vitro biological activity against bloodstream forms of Trypanosoma brucei rhodesiense and for cytotoxicity against the mammalian cell line L6. Vernodalin (2) was the most active compound with an IC50 value of 0.16 µM and a selectivity index of 35. Its closely related congener 11β,13-dihydrovernodalin (3) registered an IC50 value of 1.1 µM and a selectivity index of 4.2

Hygroline derivatives from Schizanthus tricolor and their anti-trypanosomatid and antiplasmodial activities

Chemical investigation of the alkaloid extract of the aerial parts of Schizanthus tricolor led to the targeted isolation of 26 hygroline derivatives of which 20 were fully characterized. They have not yet been described in the literature and their structures were established by 1D and 2D NMR, UV and IR spectroscopy, and HRESIMS. The configuration was determined by Gauge-Independent Atomic Orbital NMR chemical shift calculations supported by the advanced statistical method DP4 plus, vibrational circular dichroism, and measurement of optical rotation. Their anti-trypanosomatid, antiplasmodial and cytotoxic activities were measured. Several compounds exhibited low micromolar activity against Plasmodium falciparum. None of the identified molecules was cytotoxic

Antiprotozoal activity of dehydroabietic acid derivatives against Leishmania donovani and Trypanosoma cruzi - SPECIAL ISSUE "NEW TALENT: EUROPE"

Derivatives of dehydroabietic acid bearing different amino acids scaffolds have potent antiprotozoal activity against Leishmania donovani and Trypanosoma cruzi, with good to high selectivity, and can therefore be regarded as good models for further development into new drugs to fight leishmaniasis and Chagas disease. Several of the tested compounds were able to kill parasites residing inside cells, with IC50 values ranging from 2.3 to 9 mu M (L. donovani) and 1.4 to 5.8 mu M (T. cruzi), reflecting their ability to fight these infections at the relevant stage responsible for disease. One of the compounds, bearing a 3-pyridyl-Dalanine side chain, was 1.5-fold more potent against T. cruzi amastigotes residing in L6 cells than the reference compound benznidazole.Peer reviewe

Pan-Arctic distributions of continental runoff in the Arctic Ocean

Continental runoff is a major source of freshwater, nutrients and terrigenous material to the Arctic Ocean. As such, it influences water column stratification, light attenuation, surface heating, gas exchange, biological productivity and carbon sequestration. Increasing river discharge and thawing permafrost suggest that the impacts of continental runoff on these processes are changing. Here, a new optical proxy was developed and implemented with remote sensing to determine the first pan-Arctic distribution of terrigenous dissolved organic matter (tDOM) and continental runoff in the surface Arctic Ocean. Retrospective analyses revealed connections between the routing of North American runoff and the recent freshening of the Canada Basin, and indicated a correspondence between climate-driven changes in river discharge and tDOM inventories in the Kara Sea. By facilitating the real-time, synoptic monitoring of tDOM and freshwater runoff in surface polar waters, this novel approach will help understand the manifestations of climate change in this remote region

Discovering the RNA-binding proteome of plant leaves with an improved RNA interactome capture method

RNA-binding proteins (RBPs) play a crucial role in regulating RNA function and fate. However, the full complement of RBPs has only recently begun to be uncovered through proteome-wide approaches such as RNA interactome capture (RIC). RIC has been applied to various cell lines and organisms, including plants, greatly expanding the repertoire of RBPs. However, several technical challenges have limited the efficacy of RIC when applied to plant tissues. Here, we report an improved version of RIC that overcomes the difficulties imposed by leaf tissue. Using this improved RIC method in Arabidopsis leaves, we identified 717 RBPs, generating a deep RNA-binding proteome for leaf tissues. While 75% of these RBPs can be linked to RNA biology, the remaining 25% were previously not known to interact with RNA. Interestingly, we observed that a large number of proteins related to photosynthesis associate with RNA in vivo, including proteins from the four major photosynthetic supercomplexes. As has previously been reported for mammals, a large proportion of leaf RBPs lack known RNA-binding domains, suggesting unconventional modes of RNA binding. We anticipate that this improved RIC method will provide critical insights into RNA metabolism in plants, including how cellular RBPs respond to environmental, physiological and pathological cues

Non-invasive monitoring of drug action: A new live in vitro assay design for Chagas' disease drug discovery.

New assay designs are needed to improve the predictive value of the Trypanosoma cruzi in vitro tests used as part of the Chagas' disease drug development pipeline. Here, we employed a green fluorescent protein (eGFP)-expressing parasite line and live high-content imaging to monitor the growth of T. cruzi amastigotes in mouse embryonic fibroblasts. A novel assay design allowed us to follow parasite numbers over 6 days, in four-hour intervals, while occupying the microscope for only 24 hours per biological replicate. Dose-response curves were calculated for each time point after addition of test compounds, revealing how EC50 values first decreased over the time of drug exposure, and then leveled off. However, we observed that parasite numbers could vary, even in the untreated controls, and at different sites in the same well, which caused variability in the EC50 values. To overcome this, we established that fold change in parasite number per hour is a more robust and informative measure of drug activity. This was calculated based on an exponential growth model for every biological sample. The net fold change per hour is the result of parasite replication, differentiation, and death. The calculation of this fold change enabled us to determine the tipping point of drug action, i.e. the time point when the death rate of the parasites exceeded the growth rate and the fold change dropped below 1, depending on the drug concentration and exposure time. This revealed specific pharmacodynamic profiles of the benchmark drugs benznidazole and posaconazole

Preparation of sesquiterpene lactone-loaded pla nanoparticles and evaluation of their antitrypanosomal activity

Human African trypanosomiasis (HAT), also commonly known as sleeping sickness, is a neglected tropical disease affecting millions of people in poorly developed regions in sub-Saharan Africa. There is no satisfactory treatment for this infection. The investment necessary to bring new drugs to the market is a big deterrent to drug development, considering that the affected communities form a non-lucrative sector. However, natural products and many sesquiterpene lactones (STLs) in particular are very strong trypanocides. Research and applications of nano-drug delivery systems such as nanoparticles (NPs) have undergone unprecedented growth in the recent past. This is mainly due to the advantages offered by these systems, such as targeted delivery of the drug to the place of action (cell, parasite, etc), sustained release of the drug, increased bioavailability, reduced drug dosage, and reduction of undesired side effects, among others. In this study, the STLs α-santonin, arglabin, schkuhrin II, vernolepin, and eucannabinolide, all trypanocides, were loaded into polylactic acid (PLA) NPs through an emulsification-diffusion method. The NPs were stable, homogenous, and spherical in shape with a rounded knotty depression like a navel orange. The average particle sizes were 202.3, 220.3, 219.5, 216.9, and 226.4 nm for α-santonin, arglabin, schkuhrin II, vernolepin, and eucannabinolide, respectively. The NPs had encapsulation efficiencies of 94.6, 78.1, 76.8, 60.7, and 78.9% for α-santonin, arglabin, schkuhrin II, vernolepin, and eucannabinolide, respectively. The NPs loaded with arglabin, vernolepin, and eucannabinolide exhibited considerable antitrypanosomal activity against; Trypanosoma brucei rhodesiense (Tbr); with free drug equivalent IC; 50; values of 3.67, 1.11 and 3.32 µM, respectively. None of the NP formulations displayed cytotoxicity towards mammalian cells (rat skeletal myoblast cell line L6). These results provide new insights into the possibility of incorporating STLs into nanoparticles, which may provide new options for their formulation in order to develop new drugs against HAT

The Conformational Equilibrium of the Neuropeptide Y2 Receptor in Bilayer Membranes

Dynamic structural transitions within the seven-transmembrane bundle represent the mechanism by which G-protein-coupled receptors convert an extracellular chemical signal into an intracellular biological function. Here, the conformational dynamics of the neuropeptide Y receptor type 2 (Y2R) during activation was investigated. The apo, full agonist-, and arrestin-bound states of Y2R were prepared by cell-free expression, functional refolding, and reconstitution into lipid membranes. To study conformational transitions between these states, all six tryptophans of Y2R were(13)C-labeled. NMR-signal assignment was achieved by dynamic-nuclear-polarization enhancement and the individual functional states of the receptor were characterized by monitoring(13)C NMR chemical shifts. Activation of Y2R is mediated by molecular switches involving the toggle switch residue Trp281(6.48)of the highly conserved SWLP motif and Trp327(7.55)adjacent to the NPxxY motif. Furthermore, a conformationally preserved "cysteine lock"-Trp116(23.50)was identified

Adenosine Kinase of T. b. rhodesiense Identified as the Putative Target of 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine Using Chemical Proteomics

Human African trypanosomiasis (HAT), a devastating and fatal parasitic disease endemic in sub-Saharan Africa, urgently needs novel targets and efficacious chemotherapeutic agents. Recently, we discovered that 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine exhibits specific antitrypanosomal activity toward T. b. rhodesiense, the causative agent of the acute form of HAT. Here we applied a chemical proteomics approach to find the cellular target of this compound. Adenosine kinase, a key enzyme of the parasite purine salvage pathway, was isolated and identified as compound binding partner. Direct binding assays using recombinant protein, and tests on an adenosine kinase knock-down mutant of the parasite produced by RNA interference confirmed TbrAK as the putative target. Kinetic analyses showed that the title compound is an activator of adenosine kinase and that the observed hyperactivation of TbrAK is due to the abolishment of the intrinsic substrate-inhibition. Whereas hyperactivation as a mechanism of action is well known from drugs targeting cell signaling, this is a novel and hitherto unexplored concept for compounds targeting metabolic enzymes, suggesting that hyperactivation of TbrAK may represent a novel therapeutic strategy for the development of trypanocides

EEG Data Quality: Determinants and Impact in a Multicenter Study of Children, Adolescents, and Adults with Attention-Deficit/Hyperactivity Disorder (ADHD)

Electroencephalography (EEG) represents a widely established method for assessing altered and typically developing brain function. However, systematic studies on EEG data quality, its correlates, and consequences are scarce. To address this research gap, the current study focused on the percentage of artifact-free segments after standard EEG pre-processing as a data quality index. We analyzed participant-related and methodological influences, and validity by replicating landmark EEG effects. Further, effects of data quality on spectral power analyses beyond participant-related characteristics were explored. EEG data from a multicenter ADHD-cohort (age range 6 to 45 years), and a non-ADHD school-age control group were analyzed (ntotal = 305). Resting-state data during eyes open, and eyes closed conditions, and task-related data during a cued Continuous Performance Task (CPT) were collected. After pre-processing, general linear models, and stepwise regression models were fitted to the data. We found that EEG data quality was strongly related to demographic characteristics, but not to methodological factors. We were able to replicate maturational, task, and ADHD effects reported in the EEG literature, establishing a link with EEG-landmark effects. Furthermore, we showed that poor data quality significantly increases spectral power beyond effects of maturation and symptom severity. Taken together, the current results indicate that with a careful design and systematic quality control, informative large-scale multicenter trials characterizing neurophysiological mechanisms in neurodevelopmental disorders across the lifespan are feasible. Nevertheless, results are restricted to the limitations reported. Future work will clarify predictive value